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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">01581</article-id><article-id pub-id-type="doi">10.7554/eLife.01581</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Cell biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Genomics and evolutionary biology</subject></subj-group></article-categories><title-group><article-title>Endogenous RNA interference is driven by copy number</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-7992"><name><surname>Cruz</surname><given-names>Cristina</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-7960"><name><surname>Houseley</surname><given-names>Jonathan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Epigenetics Programme</institution>, <institution>The Babraham Institute</institution>, <addr-line><named-content content-type="city">Cambridge</named-content></addr-line>, <country>United Kingdom</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Proudfoot</surname><given-names>Nick J</given-names></name><role>Reviewing editor</role><aff><institution>University of Oxford</institution>, <country>United Kingdom</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>jon.houseley@babraham.ac.uk</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>11</day><month>02</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e01581</elocation-id><history><date date-type="received"><day>23</day><month>09</month><year>2013</year></date><date date-type="accepted"><day>29</day><month>12</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Cruz and Houseley</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Cruz and Houseley</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife01581.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01581.001</object-id><p>A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate RNA interference (RNAi) responses. Here we have used a synthetic RNAi system to show that gene copy number is a key factor controlling RNAi for transcripts from endogenous loci, since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci. Selectivity towards transcripts from high-copy DNA is therefore an emergent property of a minimal RNAi system. The ability of RNAi to selectively degrade transcripts from high-copy loci would allow suppression of newly emerging transposable elements, but such a surveillance system requires transcription. We show that low-level genome-wide pervasive transcription is sufficient to instigate RNAi, and propose that pervasive transcription is part of a defense mechanism capable of directing a sequence-independent RNAi response against transposable elements amplifying within the genome.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.001">http://dx.doi.org/10.7554/eLife.01581.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01581.002</object-id><title>eLife digest</title><p>Genes contain the codes that are needed to make the proteins used by cells. This code is transcribed to make a messenger RNA molecule that is then translated to make a protein. However, other types of RNA called non-coding RNA molecules can disrupt this process by binding to messenger RNA molecules, with matching sequences, before translation begins. This phenomenon, which is known as RNA interference, involves enzymes called Dicer and Argonaute.</p><p>Many cells contain large numbers of non-coding RNA molecules—so called because they are not translated to produce proteins—and many of these are capable of starting the process of RNA interference. However, most do not, and the reasons for this are not understood. Now, work by Cruz and Houseley has provided new insight into this phenomenon by showing that it is related to the number of copies of the gene encoding such RNAs in the genome.</p><p>Yeast cells normally do not have the genes for RNA interference, but Cruz and Houseley used genetically engineered yeast cells containing Dicer and Argonaute. Although most of the messenger RNA molecules in these cells showed no change, the expression of some genes with high ‘copy numbers’ was reduced. Further experiments that involved adding more and more copies of other genes showed that RNA interference could selectively target messenger RNA molecules produced from genes with an increased copy number—particularly if the copies of the genes were clustered in one location in the genome.</p><p>RNA interference is also used to defend against DNA sequences that invade and multiply within a genome, such as viruses and other ‘genetic parasites’. As such, the effect observed by Cruz and Houseley could explain why entire genomes are often continuously copied to RNA at low levels. This activity would allow the monitoring of the genome for the invasion of any genetic parasites that had multiplied to high numbers. Following on from this work, the next challenge will be to understand how gene copy number and location are balanced to achieve a selective RNA interference system.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.002">http://dx.doi.org/10.7554/eLife.01581.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>RNA interference</kwd><kwd>non-coding RNA</kwd><kwd>pervasive transcription</kwd><kwd>copy number</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>S. cerevisiae</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>Wellcome Trust</institution></institution-wrap></funding-source><award-id>088335</award-id><principal-award-recipient><name><surname>Cruz</surname><given-names>Cristina</given-names></name><name><surname>Houseley</surname><given-names>Jonathan</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>The ability to target transcripts from high-copy regions of the genome is an emergent property of a minimal RNA interference system.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Over the past decade, our understanding of the complexity of the eukaryotic transcriptome has been revolutionized. Genome-wide sequencing studies in many organisms have revealed that protein-coding mRNAs are augmented by a multitude of non-protein coding RNAs (ncRNAs), many produced from regions of the genome traditionally considered to be transcriptionally silent (<xref ref-type="bibr" rid="bib97">The ENCODE Project Consortium, 2012</xref>; <xref ref-type="bibr" rid="bib5">Bertone et al., 2004</xref>; <xref ref-type="bibr" rid="bib17">Cheng et al., 2005</xref>; <xref ref-type="bibr" rid="bib19">David et al., 2006</xref>; <xref ref-type="bibr" rid="bib6">Birney et al., 2007</xref>). Functional data for the vast majority of ncRNAs are currently lacking, with only a few examples characterized in any detail; however, the diversity of mechanisms by which these act suggests that ncRNAs have a rich and varied biology that is largely still to be sampled.</p><p>Long ncRNAs which overlap protein-coding genes have the potential to modulate the expression of their cognate coding RNA. Early characterized examples in yeast were thought to work by directly disrupting transcription factor or polymerase binding to the promoter of the coding RNA (<xref ref-type="bibr" rid="bib53">Martens et al., 2004</xref>; <xref ref-type="bibr" rid="bib37">Hongay et al., 2006</xref>); however, more recent data implicate specific chromatin structure changes in repression (<xref ref-type="bibr" rid="bib27">Gelfand et al., 2011</xref>; <xref ref-type="bibr" rid="bib32">Hainer et al., 2011</xref>), and many other cases of ncRNAs that alter chromatin modifications have been described (<xref ref-type="bibr" rid="bib11">Camblong et al., 2007</xref>; <xref ref-type="bibr" rid="bib4">Berretta et al., 2008</xref>; <xref ref-type="bibr" rid="bib40">Houseley et al., 2008</xref>; <xref ref-type="bibr" rid="bib62">Pinskaya et al., 2009</xref>; <xref ref-type="bibr" rid="bib82">van Werven et al., 2012</xref>). Chromatin modifications are not necessarily repressive, and ncRNAs that enhance expression of their overlapping coding gene have also been described (<xref ref-type="bibr" rid="bib78">Uhler et al., 2007</xref>; <xref ref-type="bibr" rid="bib35">Hirota et al., 2008</xref>). In these examples, chromatin modifications are deposited during transcription, and therefore the act of transcription rather than the ncRNA itself is important. This is not always the case, and in higher eukaryotes multiple <italic>cis</italic>-acting ncRNAs have also been characterized, particularly as functional agents in imprinting. For example, <italic>Air</italic> and <italic>Kcnq1ot1</italic> act in <italic>cis</italic> to deposit repressive chromatin marks and DNA methylation, but these ncRNAs interact with chromatin modifiers and allele specificity is achieved by restriction of the ncRNA to the vicinity of the transcription site, although the importance of the transcription itself remains controversial (<xref ref-type="bibr" rid="bib55">Nagano et al., 2008</xref>; <xref ref-type="bibr" rid="bib61">Pandey et al., 2008</xref>; <xref ref-type="bibr" rid="bib66">Redrup et al., 2009</xref>; <xref ref-type="bibr" rid="bib49">Latos et al., 2012</xref>).</p><p>Genomes are also replete with low abundance and unstable RNA. The vast majority of ncRNAs in budding yeast are unstable (<xref ref-type="bibr" rid="bib56">Neil et al., 2009</xref>; <xref ref-type="bibr" rid="bib80">van Dijk et al., 2011</xref>), limiting the potential action of the RNAs themselves, although the transcription of such RNAs can still alter gene expression (reviewed in <xref ref-type="bibr" rid="bib38">Houseley, 2012</xref>). Such unstable RNAs are also widespread in higher eukaryotes, probably with similar functional roles (<xref ref-type="bibr" rid="bib16">Chekanova et al., 2007</xref>; <xref ref-type="bibr" rid="bib63">Preker et al., 2008</xref>). More mysterious is the pervasive transcription that permeates eukaryotic genomes; the ENCODE project found that almost all the human genome is transcribed at some point, but the products of this transcription are vanishingly rare (<xref ref-type="bibr" rid="bib17">Cheng et al., 2005</xref>; <xref ref-type="bibr" rid="bib6">Birney et al., 2007</xref>; <xref ref-type="bibr" rid="bib43">Kapranov et al., 2007</xref>; <xref ref-type="bibr" rid="bib29">Goodman et al., 2012</xref>). It appears that regions of the genome which are not actively transcribed for other reasons undergo pervasive transcription; however, it is not known whether this pervasive transcription simply represents transcriptional noise or whether the transcription or RNAs themselves have important but as yet undiscovered functions.</p><p>Systems in which a ncRNA is transcribed antisense to a sense protein-coding RNA are common and have strong regulatory potential (<xref ref-type="fig" rid="fig1">Figure 1A</xref>) (<xref ref-type="bibr" rid="bib90">Derrien et al., 2012</xref>; <xref ref-type="bibr" rid="bib13">Carninci et al., 2005</xref>; <xref ref-type="bibr" rid="bib85">Xu et al., 2009</xref>). It has been suggested that, since antisense ncRNAs are perfectly complementary to their cognate mRNA, the two species could form double stranded RNA (dsRNA) that would be a substrate for the RNA interference system (RNAi). During a basic RNAi response, dsRNA is cleaved by the endonuclease Dicer into short interfering RNA (siRNA), of which one strand is then loaded onto an Argonaute protein. The Argonaute–siRNA complex can anneal to complementary sequences in target RNAs, which are then cleaved by the endonuclease activity of Argonaute. RNAi was originally discovered in <italic>Caenorhabditis elegans</italic> and rapidly linked to the phenomenon of post-transcriptional gene silencing in plants; however, almost all eukaryotes contain Dicer and Argonaute orthologues and therefore have some form of RNAi system (<xref ref-type="bibr" rid="bib33">Hamilton and Baulcombe, 1999</xref>; <xref ref-type="bibr" rid="bib91">Fire, 1998</xref>; <xref ref-type="bibr" rid="bib93">Hannon, 2002</xref>). RNAi probably evolved to protect cells against dsRNA viruses, a role which is maintained in plants, insects, and lower eukaryotes (<xref ref-type="bibr" rid="bib20">Ding, 2010</xref>) and has recently been described in mammalian cells (<xref ref-type="bibr" rid="bib51">Li et al., 2013</xref>; <xref ref-type="bibr" rid="bib52">Maillard et al., 2013</xref>). RNAi also forms a potent defense against transposons, and high-copy transposon-derived sequences are excellent targets for RNAi, giving rise to copious siRNAs in most eukaryotes including mammals (<xref ref-type="bibr" rid="bib88">Yang and Kazazian, 2006</xref>; <xref ref-type="bibr" rid="bib72">Slotkin and Martienssen, 2007</xref>; <xref ref-type="bibr" rid="bib3">Babiarz et al., 2008</xref>). In addition to degrading transposon-derived and viral RNA, siRNAs can mediate transcriptional repression of target RNAs through chromatin modifications and DNA methylation, although this activity is seemingly much stronger in lower eukaryotes and plants than in mammals (<xref ref-type="bibr" rid="bib54">Martienssen et al., 2005</xref>; <xref ref-type="bibr" rid="bib50">Lejeune et al., 2010</xref>; <xref ref-type="bibr" rid="bib89">Zhang and Zhu, 2011</xref>). However, the source of the dsRNA that is processed into siRNA is not always obvious, nor is the mechanism by which cells differentiate host and transposon-derived sequences. siRNA-mediated repression is complemented by PIWI-interacting RNAs (piRNAs), which bind to the Argonaut-related PIWI-domain proteins and enforce transposon repression in the germline of eukaryotes from worms to mammals (<xref ref-type="bibr" rid="bib71">Siomi et al., 2011</xref>). piRNAs are derived from specific genomic clusters, but it is unclear how the transcripts from these clusters are selected for processing into primary piRNAs and many of the processing enzymes remain to be identified (reviewed in <xref ref-type="bibr" rid="bib42">Ishizu et al., 2012</xref>).<fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01581.003</object-id><label>Figure 1.</label><caption><title>Frequency of annotated antisense non-protein coding RNAs (ncRNAs) and effects on mRNA abundance.</title><p>(<bold>A)</bold> Schematic of an example sense mRNA-antisense (ncRNA) system. (<bold>B</bold>) Number of annotated open reading frames (ORFs) with antisense transcripts. Positions of CUTs, SUTs, and XUTs were collated with expressed ORFs (<xref ref-type="bibr" rid="bib85">Xu et al., 2009</xref>; <xref ref-type="bibr" rid="bib80">van Dijk et al., 2011</xref>), SUTs were later re-classified as XUTs were removed. Overlaps between ORFs expressed in glucose media (total 5171, <xref ref-type="bibr" rid="bib85">Xu et al., 2009</xref>) and other RNAs were calculated and summed for increasing minimum overlaps of 50–500 bp. ORF–ORF overlaps and ORF–ncRNA overlaps were analyzed separately as ORF–ORF overlaps are consistently smaller. Detailed figures are given in <xref ref-type="table" rid="tbl1">Table 1</xref>. (<bold>C</bold>) Abundance of short interfering RNAs (siRNAs) in RNA interference (RNAi)+ strain produced from expressed ORFs with and without an annotated overlapping antisense ncRNA, based on read counts from published high-throughput sequencing data (<xref ref-type="bibr" rid="bib22">Drinnenberg et al., 2011</xref>). Minimum antisense overlap with ORF was set at 250 bp; only ORFs with >100 reads in the wild-type poly(A)+ library were assessed to remove noise. Stated p value calculated by Student’s <italic>t</italic> test. (<bold>D</bold>) Abundance of mRNA in RNAi+ cells relative to wild-type; data source and categories as in <bold>C</bold>, differences were not significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.003">http://dx.doi.org/10.7554/eLife.01581.003</ext-link></p></caption><graphic xlink:href="elife01581f001"/></fig></p><p>Various studies have looked for endogenous sense-antisense RNA pairs that instigate RNAi responses. Efficient generation of siRNAs from endogenous sense-antisense systems (endo-siRNA) has been observed in plants under stress (<xref ref-type="bibr" rid="bib7">Borsani et al., 2005</xref>; <xref ref-type="bibr" rid="bib45">Katiyar-Agarwal et al., 2006</xref>), and mammalian oocytes generate endo-siRNAs that can mediate mRNA knockdown (<xref ref-type="bibr" rid="bib77">Tam et al., 2008</xref>; <xref ref-type="bibr" rid="bib84">Watanabe et al., 2008</xref>). However, although endo-siRNAs have been detected outside the germline in mammals, they are surprisingly under-represented where sense and antisense RNAs are co-expressed (<xref ref-type="bibr" rid="bib24">Faghihi and Wahlestedt, 2006</xref>; <xref ref-type="bibr" rid="bib59">Okamura et al., 2008</xref>; <xref ref-type="bibr" rid="bib12">Carlile et al., 2009</xref>), and overall there is a positive correlation between antisense and sense RNA expression in mammalian genomes, which is inconsistent with RNAi (<xref ref-type="bibr" rid="bib90">Derrien et al., 2012</xref>; <xref ref-type="bibr" rid="bib44">Katayama et al., 2005</xref>). This raises questions about whether endogenous sense-antisense systems do in fact form dsRNA in vivo and, if so, whether all dsRNA is equivalently accessible to Dicer.</p><p>The tight integration of the RNAi system into the physiology of most eukaryotic cells makes it very difficult to disentangle direct and indirect effects of mutating RNAi components (reviewed in <xref ref-type="bibr" rid="bib46">Ketting, 2011</xref>). To elucidate factors important for the induction of RNAi by endogenous sense-antisense systems, we therefore used a recently described synthetic system in which RNAi is reconstituted in <italic>Saccharomyces cerevisiae</italic> by the introduction of Argonaute and Dicer from the related yeast <italic>S. castellii</italic> (<xref ref-type="bibr" rid="bib23">Drinnenberg et al., 2009</xref>). <italic>S. cerevisiae</italic> is highly unusual in lacking an endogenous RNAi system, allowing maintenance of the symbiotic dsRNA Killer virus (<xref ref-type="bibr" rid="bib22">Drinnenberg et al., 2011</xref>). The reconstituted system is functional, since RNAi+ <italic>S. cerevisiae</italic> efficiently degrades exogenous hairpin RNAs and endogenous Ty retrotransposon transcripts; however, no clear mRNA expression changes are detectable in these cells (<xref ref-type="bibr" rid="bib23">Drinnenberg et al., 2009</xref>, <xref ref-type="bibr" rid="bib22">2011</xref>)<italic>.</italic></p></sec><sec id="s2" sec-type="results"><title>Results</title><p>Antisense ncRNAs exist for many <italic>S. cerevisiae</italic> genes; combining published datasets we found that 15–30% of yeast open reading frames (ORFs) have an annotated antisense ncRNA depending on the minimum size of overlap considered (<xref ref-type="bibr" rid="bib85">Xu et al., 2009</xref>; <xref ref-type="bibr" rid="bib80">van Dijk et al., 2011</xref>), not including overlapping convergent ORFs (<xref ref-type="fig" rid="fig1">Figure 1B</xref> and <xref ref-type="table" rid="tbl1">Table 1</xref>). In RNAi+ cells, these ORFs produce more siRNA than ORFs lacking an antisense (<xref ref-type="fig" rid="fig1">Figure 1C</xref>), showing that they are transcribed into dsRNA that is targeted by the RNAi machinery. However, these ORFs do not show reduced mRNA levels in RNAi+ cells consistent with published data, suggesting that insufficient siRNAs are produced to elicit a detectable mRNA knockdown (<xref ref-type="bibr" rid="bib90">Derrien et al., 2012</xref>; <xref ref-type="bibr" rid="bib44">Katayama et al., 2005</xref>; <xref ref-type="bibr" rid="bib22">Drinnenberg et al., 2011</xref>) (<xref ref-type="fig" rid="fig1">Figure 1D</xref>).<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01581.004</object-id><label>Table 1.</label><caption><p>Stability of antisense ncRNAs</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.004">http://dx.doi.org/10.7554/eLife.01581.004</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="2">Overlap size (bp)</th><th colspan="4">Overlap type</th><th colspan="2">Totals</th></tr><tr><th>ORF–ORF</th><th>ORF–XUT</th><th>ORF–CUT</th><th>ORF–SUT</th><th>ORF–ncRNA</th><th>ORF–unstable ncRNA</th></tr></thead><tbody><tr><td align="char" char=".">50</td><td align="char" char=".">700</td><td align="char" char=".">1066</td><td align="char" char=".">575</td><td align="char" char=".">522</td><td align="char" char=".">1596</td><td>1448 (91%)</td></tr><tr><td align="char" char=".">100</td><td align="char" char=".">449</td><td align="char" char=".">1008</td><td align="char" char=".">543</td><td align="char" char=".">475</td><td align="char" char=".">1507</td><td>1367 (91%)</td></tr><tr><td align="char" char=".">150</td><td align="char" char=".">216</td><td align="char" char=".">967</td><td align="char" char=".">508</td><td align="char" char=".">425</td><td align="char" char=".">1423</td><td>1306 (92%)</td></tr><tr><td align="char" char=".">200</td><td align="char" char=".">136</td><td align="char" char=".">931</td><td align="char" char=".">478</td><td align="char" char=".">403</td><td align="char" char=".">1358</td><td>1249 (92%)</td></tr><tr><td align="char" char=".">250</td><td align="char" char=".">96</td><td align="char" char=".">893</td><td align="char" char=".">434</td><td align="char" char=".">380</td><td align="char" char=".">1287</td><td>1181 (92%)</td></tr><tr><td align="char" char=".">300</td><td align="char" char=".">69</td><td align="char" char=".">812</td><td align="char" char=".">391</td><td align="char" char=".">363</td><td align="char" char=".">1189</td><td>1085 (91%)</td></tr><tr><td align="char" char=".">350</td><td align="char" char=".">62</td><td align="char" char=".">759</td><td align="char" char=".">335</td><td align="char" char=".">351</td><td align="char" char=".">1086</td><td>990 (91%)</td></tr><tr><td align="char" char=".">400</td><td align="char" char=".">54</td><td align="char" char=".">694</td><td align="char" char=".">292</td><td align="char" char=".">334</td><td align="char" char=".">992</td><td>899 (91%)</td></tr><tr><td align="char" char=".">450</td><td align="char" char=".">51</td><td align="char" char=".">637</td><td align="char" char=".">244</td><td align="char" char=".">322</td><td align="char" char=".">904</td><td>811 (90%)</td></tr><tr><td align="char" char=".">500</td><td align="char" char=".">48</td><td align="char" char=".">591</td><td align="char" char=".">204</td><td align="char" char=".">302</td><td align="char" char=".">828</td><td>741 (89%)</td></tr></tbody></table><table-wrap-foot><fn><p>Number of ORFs overlapping with ORFs and various classes of ncRNAs, with various minimum size cut-offs for the overlapping region.</p></fn><fn><p>Totals are given for ORFs overlapping with ncRNAs and with unstable ncRNAs, including a percentage of overlapping ncRNAs that are unstable.</p></fn><fn><p>ncRNA: non-protein coding RNA; ORF: optical reading frame; XUT: Xrn1-sensitive unstable transcript (degraded in cytoplasm); CUT: cryptic unstable transcript (degraded by nuclear exosome); SUT: stable unannotated transcript (not known to be degraded).</p></fn></table-wrap-foot></table-wrap></p><p>We first asked whether any endo-siRNA pairs are degraded by RNAi in this reconstituted system. RNAi+ cells produce abundant siRNAs from sub-telomeric Y′ elements and from the ribosomal DNA (rDNA) intergenic spacers (<xref ref-type="bibr" rid="bib22">Drinnenberg et al., 2011</xref> and <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>) and, despite transcriptional repression by the histone deacetylase Sir2, both regions transcribe sense and antisense ncRNAs that could hybridize to form dsRNA (<xref ref-type="bibr" rid="bib2">Aparicio et al., 1991</xref>; <xref ref-type="bibr" rid="bib86">Yamada et al., 1998</xref>; <xref ref-type="bibr" rid="bib48">Kobayashi and Ganley, 2005</xref>; <xref ref-type="bibr" rid="bib39">Houseley et al., 2007</xref>) (<xref ref-type="fig" rid="fig2">Figure 2A,D</xref>). Northern blots revealed full-length ncRNAs from both strands of the Y′ elements in wild-type cells (<xref ref-type="fig" rid="fig2">Figure 2B</xref> lanes 1,5 marked with arrows); these were largely absent in the RNAi+ strain, being replaced by heterogeneous degradation products and readily detectable siRNAs (<xref ref-type="fig" rid="fig2">Figure 2B,C</xref>). Despite weak transcriptional repression in this genetic background, ncRNAs and siRNAs were more abundant in <italic>sir2</italic>Δ mutants reinforcing the precursor–product relationship (<xref ref-type="fig" rid="fig2">Figure 2B,C</xref>). Equivalent results were seen for the rDNA intergenic spacer region (<xref ref-type="fig" rid="fig2">Figure 2D–F</xref>). These data show that endo-siRNA pairs can form RNAi substrates and undergo efficient degradation by a minimal RNAi system.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01581.005</object-id><label>Figure 2.</label><caption><title>High-copy endogenous sense-antisense pairs instigate efficient RNA interference (RNAi).</title><p>(<bold>A</bold>) Schematic diagram of sub-telomeric Y′ elements. (<bold>B</bold>) Northern analysis of Y′ element non-protein coding RNAs (ncRNAs) comparing wild-type and RNAi+ strains in <italic>SIR2</italic> and <italic>sir2</italic>Δ backgrounds. 18S ribosomal RNA is shown as a loading control. Arrows indicate full-length RNA species. (<bold>C</bold>) Northern analysis of Y′ element-derived short interfering RNAs (siRNAs) from cells in <bold>B</bold>, tRNAs are shown as a loading control. (<bold>D</bold>–<bold>F</bold>) Equivalent analysis of rDNA intergenic spacer ncRNAs.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.005">http://dx.doi.org/10.7554/eLife.01581.005</ext-link></p></caption><graphic xlink:href="elife01581f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.006</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Correlation of short interfering RNAs (siRNAs) with RNA abundance and silencing. Published small RNA sequencing data (<xref ref-type="bibr" rid="bib22">Drinnenberg et al., 2011</xref>) were re-mapped to the complete <italic>Saccharomyces cerevisiae</italic> genome including non-unique sequences and reads were then summed in 50 bp bins on each strand.</title><p>(<bold>A</bold>) Density of small RNAs in sub-telomeric Y′ elements in wild-type and RNA interference (RNAi)+ strains compared with published data for silencing efficiency of inserted transgenes (<xref ref-type="bibr" rid="bib65">Pryde and Louis, 1999</xref>). Separate traces are shown for siRNAs mapping to sense and antisense sequences above and below the x axis, respectively. siRNA density is inversely correlated with transcriptional silencing, as would be expected if siRNAs are produced from sub-telomeric non-protein coding RNAs (ncRNAs). (<bold>B</bold>) siRNA density in a single rDNA repeat. Many small RNA fragments are produced from the 35S ribosomal RNA gene, as previously observed in <italic>Schizosaccharomyces pombe</italic> (<xref ref-type="bibr" rid="bib9">Buhler et al., 2008</xref>); however, a discrete cluster of siRNAs is produced from the overlapping region between the IGS1-F and IGS2-R ncRNAs in the intergenic spacer region (highlighted).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.006">http://dx.doi.org/10.7554/eLife.01581.006</ext-link></p></caption><graphic xlink:href="elife01581fs001"/></fig></fig-group></p><p>One distinguishing feature of these regions is high copy number; to determine whether copy number amplification can drive RNAi, we examined <italic>MAL32</italic> which has a clearly defined antisense RNA that is co-expressed with the sense mRNA (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). <italic>MAL32</italic> is effectively present at two copies in the haploid genome as the orthologous gene <italic>MAL12</italic> shares 99.5% nucleotide identity, reducing potential transcriptional repression. As for approximately 90% of yeast antisense RNAs (<xref ref-type="table" rid="tbl1">Table 1</xref>), <italic>MAL32</italic> antisense RNA is highly unstable and is only detectable in strains lacking the nuclear exosome co-factor Trf4 (reviewed in <xref ref-type="bibr" rid="bib41">Houseley and Tollervey, 2009</xref>) (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>), but endogenous <italic>MAL32</italic> mRNA was not down-regulated in the RNAi+ strain even in <italic>trf4</italic>Δ cells (<xref ref-type="fig" rid="fig3">Figure 3B</xref> and <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2A</xref>). When expressed from a high-copy plasmid, however, <italic>MAL32</italic> mRNA was significantly down-regulated by RNAi (<xref ref-type="fig" rid="fig3">Figure 3C</xref>) with concurrent production of siRNA (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). The <italic>MAL32</italic> antisense RNA was only detected in these experiments as a smear of degradation products and was not noticeably altered by RNAi, probably because nuclear degradation acts faster than RNAi on this substrate. To confirm that the knockdown of <italic>MAL32</italic> mRNA was not an indirect effect of the strain background or an undirected Argonaute cleavage, we reconstituted the RNAi system in the BY4741 background using separate plasmids expressing Dicer and Argonaute. dsRNA from the <italic>MAL32</italic> locus was detectable in these cells and was removed by Dicer; however, a significant knockdown of the mRNA was only observed in cells expressing both Dicer and Argonaute, confirming that the knockdown represents a genuine RNAi response (<xref ref-type="fig" rid="fig3s3">Figure 3—figure supplement 3</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01581.007</object-id><label>Figure 3.</label><caption><title>Copy number amplification of coding genes can instigate RNA interference (RNAi).</title><p>(<bold>A</bold>) Schematic of <italic>MAL32</italic> locus. (<bold>B</bold>) <italic>MAL32</italic> mRNA and antisense non-protein coding RNA (ncRNA) comparing wild-type and RNAi+ strains in wild-type and <italic>trf4</italic>Δ backgrounds; cells grown on YP raffinose (extended image and quantification shown in <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2A</xref>). (<bold>C</bold>) mRNA and antisense ncRNA from <italic>MAL32</italic> cloned onto a high-copy plasmid in wild-type and RNAi+ strains. Lanes 3,4 show empty vector control. Antisense panel shows degradation products, no full-length antisense is detectable due to Trf4 activity. (<bold>D</bold>) Short interfering RNA (siRNA) analysis of cells from <bold>C</bold>. (<bold>E</bold>) Schematic of <italic>GAL4</italic> locus. (<bold>F</bold>) <italic>GAL4</italic> mRNA and antisense ncRNA in wild-type and RNAi+ strains; cells grown in YP galactose (extended image and quantification shown in <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2B</xref>). (<bold>G</bold>) mRNA and antisense ncRNA from <italic>GAL4</italic> locus cloned onto a high-copy plasmid in wild-type and RNAi + strains. Lanes 5,6 show empty vector, signal is from genomic <italic>GAL4</italic>; note that cells used here are diploids to mitigate defects in galactose response (see ‘Materials and methods’). Lanes 3,4 show a previously described <italic>GAL4</italic> antisense mutant (<xref ref-type="bibr" rid="bib26">Geisler et al., 2012</xref>); this removes detectable antisense RNA for genomic <italic>GAL4</italic>, but the mutant sequence still expresses an antisense ncRNA when cloned on the high-copy plasmid (see <xref ref-type="fig" rid="fig3s4">Figure 3—figure supplement 4</xref>). (<bold>H</bold>) siRNA analysis of cells in <bold>G</bold>. For quantification, n = 4 biological replicates, error bars represent ± 1se, *p<0.05, ***p<0.01 by Student’s <italic>t</italic> test, y axes in arbitrary units.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.007">http://dx.doi.org/10.7554/eLife.01581.007</ext-link></p></caption><graphic xlink:href="elife01581f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.008</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Characterization of the <italic>MAL32</italic> sense/antisense system.</title><p>Left: diagram of the MAL cluster near telomere at the right hand end of Chr. II. Sites enriched for H3K4 Me3 are indicated for log phase cells grown in glucose (<xref ref-type="bibr" rid="bib47">Kirmizis et al., 2007</xref>), including an enrichment site at the 3′ end of the <italic>MAL32</italic> open reading frame (ORF) corresponding to the antisense non-protein coding RNA (ncRNA) promoter. The <italic>Saccharomyces cerevisiae</italic> genome contains two MAL clusters; the other is located at the right hand end of Chr. VII and contains the <italic>MAL32</italic> paralogue, <italic>MAL12</italic>, which differs by only a few single-nucleotide polymorphisms in the ORF (99.5% nucleotide identity). Right: northern blot showing <italic>MAL32</italic> antisense and mRNA expression in glucose and raffinose. The antisense ncRNA, a major 2 kb transcript with additional 2.7 kb and 4.7 kb species, is ubiquitously expressed but completely degraded by Trf4 in wild-type cells. 5′ RACE analysis in <italic>trf4</italic>Δ cells (not shown) reveals that the antisense is produced from both <italic>MAL32</italic> and <italic>MAL12</italic> genes. The mRNA is repressed in glucose, but in raffinose is produced from both loci as revealed by the reduced but detectable mRNA expression in the <italic>mal12</italic>Δ strain.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.008">http://dx.doi.org/10.7554/eLife.01581.008</ext-link></p></caption><graphic xlink:href="elife01581fs002"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.009</object-id><label>Figure 3—figure supplement 2.</label><caption><title>Images and quantification of RNA interference (RNAi) degradation patterns.</title><p>(<bold>A</bold>) Degradation pattern of <italic>MAL32</italic> mRNA and antisense non-protein coding RNA (ncRNA) in cells grown on YPRaf. Note the degradation of <italic>MAL32</italic> mRNA in the <italic>trf4</italic>Δ sample (lane 3), which is abolished in the RNAi+ strain. This result was seen in multiple experiments and appears to be an indirect effect of RNAi. The large increase in <italic>MAL32</italic> mRNA in the <italic>trf4</italic>Δ RNAi+ strain compared with <italic>trf4</italic>Δ results from this loss of RNA degradation. All antisense RNA species are included in the quantification; note that RNAi reduces the level of the full-length antisense RNA but slightly increases some truncated species. (<bold>B</bold>) Degradation pattern of <italic>GAL4</italic> mRNA and antisense ncRNA in cells grown on YPGal. Note that degradation products are clearly visible in RNAi+ <italic>xrn1</italic>Δ sample even though quantification does not show a significant change in full-length RNAs (compare heterogeneous species of 0.1–3 kb in lanes 4,8 with other lanes). For quantification, n = 4 biological replicates, error bars are ±1se, ***p<0.01 by Student’s <italic>t</italic> test, y axes in arbitrary units.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.009">http://dx.doi.org/10.7554/eLife.01581.009</ext-link></p></caption><graphic xlink:href="elife01581fs003"/></fig><fig id="fig3s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.010</object-id><label>Figure 3—figure supplement 3.</label><caption><title>Verification of RNA interference (RNAi) knockdown.</title><p>Dicer and Argonaute were cloned out of the RNAi+ strain onto single-copy plasmids and transformed into the BY4741 background along with the <italic>MAL32</italic> high-copy plasmid. Cells were grown on raffinose as in <xref ref-type="fig" rid="fig3">Figure 3C</xref>. (<bold>A</bold>) 3 µg RNase A-treated RNA was loaded in each lane to detect double stranded RNA (dsRNA), which is largely resistant to degradation by RNase A. (<bold>B</bold>) <italic>MAL32</italic> mRNA levels, with L-A viral RNA as a positive control. For quantification, n = 3 biological replicates, error bars represent ±1se, ***p<0.01 by Student’s <italic>t</italic> test.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.010">http://dx.doi.org/10.7554/eLife.01581.010</ext-link></p></caption><graphic xlink:href="elife01581fs004"/></fig><fig id="fig3s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.011</object-id><label>Figure 3—figure supplement 4.</label><caption><title>Expression of <italic>GAL4</italic> antisense RNA in mutant construct.</title><p>Left: structure of the 3′ end of wild-type and mutant <italic>GAL4</italic> constructs showing primer locations for 5′ RACE. Right: 5′ RACE for capped RNA present in wild-type cells with the <italic>GAL4</italic>, <italic>GAL4</italic>-3HA, or empty plasmids (RNA samples as in <xref ref-type="fig" rid="fig3">Figure 3G</xref> lanes 1,3,5). The <italic>GAL4</italic> primer detects both wild-type and mutant species, and notably detects a product in the <italic>GAL4</italic>-3HA construct which is not reported to express an antisense RNA. This product is specific for 3HA as a product is also detected only in this construct with the 3HA primer at the predicted size.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.011">http://dx.doi.org/10.7554/eLife.01581.011</ext-link></p></caption><graphic xlink:href="elife01581fs005"/></fig><fig id="fig3s5" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.012</object-id><label>Figure 3—figure supplement 5.</label><caption><title>Plasmid copy number in high-copy plasmid strains.</title><p>(<bold>A</bold>) Southern blot of wild-type and RNA interference (RNAi)+ <italic>ura3</italic>Δ cells containing high-copy plasmids for <italic>MAL32</italic> and <italic>GAL4</italic>, with a W303 <italic>URA3</italic> wild-type as a quantification control. DNA was extracted from cells grown to stationary phase in selective media, separated on a 1% gel, and probed for <italic>URA3</italic> with <italic>GAL7</italic> as a loading control. Note that the <italic>GAL7</italic> signal is stronger in p<italic>GAL4</italic> containing strains as these are diploids as described in ‘Materials and methods’. Plasmid copy numbers were calculated relative to the W303 <italic>URA3</italic> signal. (<bold>B</bold>) Plasmid analysis of <italic>MAL32</italic> high-copy plasmid in log-phase cells showing that plasmid copy numbers are similar between wild-type and RNAi+ cells in growth conditions used for RNA isolation. A proportion of the total plasmid is always isolated during RNA preparation and is detected as a slow migrating RNase A-insensitive band. For RNase treatment, the sample shown in lanes 3/9 was treated with RNase A or no enzyme followed by purification with phenol:chloroform and ethanol precipitation. A short exposure of the full-length RNA is shown for the sense probe as this signal is over-exposed when the plasmid signal is visible. (<bold>C</bold>) Short interfering RNA (siRNA) reads per 100 bp for 2µ plasmid and chromosome I, showing that the endogenous 2µ plasmid produces high levels of siRNA from all genes compared with chromosomal genes; the three clear peaks correspond to FLP1, REF1, and RAF1. The x axes are not to the same scale, data from (<xref ref-type="bibr" rid="bib22">Drinnenberg et al., 2011</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.012">http://dx.doi.org/10.7554/eLife.01581.012</ext-link></p></caption><graphic xlink:href="elife01581fs006"/></fig><fig id="fig3s6" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.013</object-id><label>Figure 3—figure supplement 6.</label><caption><title>Confirmation of RNA interference (RNAi) action by immunoprecipitation and qRT-PCR.</title><p>Cells were grown under the same conditions as in <xref ref-type="fig" rid="fig3">Figure 3C,G</xref>, then lysed and immunoprecipitated with either no antibody or monoclonal antibody J2 against double stranded RNA (dsRNA). Quantitative RT-PCR was used to quantify <italic>MAL32</italic> or <italic>GAL4</italic>, L-A (a dsRNA produced by Killer virus) and <italic>ACT1</italic> in each fraction. Signals were normalized to <italic>ACT1</italic>, which should produce minimal dsRNA under normal conditions. For quantification, n = 3 biological replicates, error bars represent ±1se, *p<0.05, ***p<0.01 by Student’s <italic>t</italic> test, data were log transformed for <italic>t</italic> test to avoid issues caused by the very large difference in dsRNA quantification between wt and RNAi+ cells, y axes in arbitrary units. The significant differences in the no antibody controls match the difference in the total lysates and reflect non-specific RNA binding.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.013">http://dx.doi.org/10.7554/eLife.01581.013</ext-link></p></caption><graphic xlink:href="elife01581fs007"/></fig></fig-group></p><p>We then tested <italic>GAL4</italic>, which is a single-copy gene with a co-expressed antisense that is degraded in the cytoplasm by Xrn1 (<xref ref-type="bibr" rid="bib26">Geisler et al., 2012</xref>) (<xref ref-type="fig" rid="fig3">Figure 3E</xref>). Cells lacking Xrn1 show increased levels of antisense and, unexpectedly, sense RNA, but, as for <italic>MAL32</italic>, we did not detect a significant decrease in full-length RNA in RNAi+ <italic>xrn1</italic>Δ cells (<xref ref-type="fig" rid="fig3">Figure 3F</xref> and <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2B</xref>). However, amplification of the locus by cloning on a high-copy plasmid leads to significant degradation of the sense RNA along with the antisense RNA by RNAi (<xref ref-type="fig" rid="fig3">Figure 3G,H</xref>). Surprisingly, this occurred even in a known mutant that lacks antisense expression (<xref ref-type="bibr" rid="bib26">Geisler et al., 2012</xref>), but 5′ RACE (Rapid Amplification of 5′ Complementary DNA Ends) experiments revealed that antisense RNA is still produced by this mutant when expressed from a high-copy plasmid, even if it is too heterogeneous for detection by northern blot (<xref ref-type="fig" rid="fig3s4">Figure 3—figure supplement 4</xref>). Taken together, these experiments on <italic>MAL32</italic> and <italic>GAL4</italic> demonstrate that increasing gene copy number can make the products of a normal gene susceptible to RNAi.</p><p>One potential confounding factor in these experiments is the copy number of the high-copy plasmids; if the copy number drops in RNAi+ cells, this would provide a trivial explanation for the observed knockdowns. However, Southern blotting revealed that RNAi+ cells contained approximately twofold more plasmid than the controls, which would tend to decrease rather than increase the apparent effect of RNAi (<xref ref-type="fig" rid="fig3s5">Figure 3—figure supplement 5A,B</xref>). It is likely that RNAi degrades the mRNA for the plasmid-encoded selectable marker and 2µ maintenance genes (2µ genes produce copious siRNA, <xref ref-type="fig" rid="fig3s5">Figure 3—figure supplement 5C</xref>), and the plasmid copy number rises to compensate for this. We also wanted to use a different method to confirm the northern blot results. We therefore lysed wild-type and RNAi+ cells containing the <italic>MAL32</italic> and <italic>GAL4</italic> plasmids, precipitated dsRNA using a specific monoclonal antibody (<xref ref-type="bibr" rid="bib68">Schonborn et al., 1991</xref>; <xref ref-type="bibr" rid="bib31">Gullerova and Proudfoot, 2012</xref>), and assayed total and dsRNA levels by quantitative RT-PCR. <italic>MAL32</italic> mRNA was knocked down approximately 75%, as observed by northern blot analysis, while dsRNA was reduced 11-fold, consistent with specific removal of dsRNA by Dicer. <italic>GAL4</italic> mRNA knockdown was measured at 80% in this assay, again with an 11-fold reduction in dsRNA in the RNAi+ strain (<xref ref-type="fig" rid="fig3s6">Figure 3—figure supplement 6</xref>).</p><p>Increasing gene copy number also increases RNA production. To separate the contributions of RNA abundance and copy number, we analyzed existing genome-wide data (<xref ref-type="bibr" rid="bib94">Hobson et al., 2012</xref>; <xref ref-type="bibr" rid="bib22">Drinnenberg et al., 2011</xref>). If siRNA formation depends only on precursor RNA abundance, a positive correlation should be observed between total RNA abundance and siRNA abundance, and there should be no difference between distributions of single-copy loci and multi-copy loci. We observed little evidence for such a positive correlation (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>); however, plots of siRNA versus total RNA abundance for single-copy and multi-copy loci showed strikingly different distributions, with multi-copy loci clearly biased towards higher siRNA production (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). To quantify this difference, loci were segregated into eight groups of increasing total RNA abundance and siRNA abundance was assessed for single-copy and multi-copy loci in each group (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). siRNA production was significantly higher from multi-copy loci than from single-copy loci in all except the lowest category of RNA abundance. This result was robust to changes in the threshold between low and high copy, and was still observed in a comparison of low to medium copy number, showing that high-copy Ty1 retrotransposons were not distorting the analysis (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>). A normalization step is required in these analyses to deal with mapping of sequence reads to multi-copy loci (discussed in detail in ‘Materials and methods’); however, the same differences were observed with no normalization or a different normalization scheme (<xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3</xref>). These surprising results show that multi-copy loci produce more siRNA than single-copy loci with equivalent RNA abundance. If this observation is real, the siRNA:total RNA ratio should be predictive of copy number, an important test since this comparison requires no copy number normalization. As predicted, the 1% of genome with the highest siRNA:total RNA ratio is massively enriched for multi-copy loci (<xref ref-type="fig" rid="fig4">Figure 4C</xref>), and when this ratio was plotted across a chromosome, an obvious correlation was observed between regions of high-copy number and regions with high siRNA:total RNA abundance (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). These analyses clearly demonstrate that selectivity towards the products of multi-copy loci is an emergent property of a minimal RNAi system.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01581.014</object-id><label>Figure 4.</label><caption><title>Multi-copy loci are preferentially targeted by RNA interference (RNAi).</title><p>(<bold>A</bold>) Short interfering RNA (siRNA) (<xref ref-type="bibr" rid="bib22">Drinnenberg et al., 2011</xref>) and total RNA (<xref ref-type="bibr" rid="bib94">Hobson et al., 2012</xref>) abundance for loci with copy number <2 (left, single-copy) or ≥2 (right, multi-copy). (<bold>B</bold>) Quantification of data from <bold>A</bold> binned into categories of increasing total RNA level, with p values for pairwise comparisons of siRNA abundance in single-copy and multi-copy datasets using the Wilcoxon Rank Sum test. (<bold>C</bold>) Copy number distribution of the 1% of loci with the highest siRNA:total RNA ratio compared with other loci; difference is significant by Wilcoxon Rank Sum test, p<2.2 × 10<sup>−16</sup>, loci scoring below noise threshold (0–2 category in <bold>B</bold>) were removed. n values for tests in <bold>B</bold> and <bold>C</bold> are given in <xref ref-type="table" rid="tbl2">Table 2</xref>. (<bold>D</bold>) Comparison of copy number with siRNA:total RNA ratio across chromosome I.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.014">http://dx.doi.org/10.7554/eLife.01581.014</ext-link></p></caption><graphic xlink:href="elife01581f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.015</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Short interfering RNA (siRNA) versus total RNA abundance.</title><p>Plot of genome-wide siRNA abundance (<xref ref-type="bibr" rid="bib22">Drinnenberg et al., 2011</xref>) versus total RNA abundance (<xref ref-type="bibr" rid="bib94">Hobson et al., 2012</xref>). Abundance scores represent log-transformed sums of total read counts for each 100 bp segment of the yeast genome.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.015">http://dx.doi.org/10.7554/eLife.01581.015</ext-link></p></caption><graphic xlink:href="elife01581fs008"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.016</object-id><label>Figure 4—figure supplement 2.</label><caption><title>Increased short interfering RNA (siRNA) production from multi-copy sequences is robust to different cut-offs.</title><p>The dataset shown in <xref ref-type="fig" rid="fig4">Figure 4B</xref> was re-analyzed with varying parameters; significant differences between multi-copy and single-copy loci are observed in all cases. p values shown were calculated by Wilcoxon Rank Sum test, n values are given in <xref ref-type="table" rid="tbl2">Table 2</xref>. (<bold>A</bold>) Very high-copy sequences (>5 copies) were removed from the analysis to avoid the influence of a small number of very high-copy sequences. (<bold>B</bold>) The datasets were split into <5 and ≥5 copies to ensure that the observed difference was not an artefact of the arbitrary two-copy cut-off used in <xref ref-type="fig" rid="fig4">Figure 4B</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.016">http://dx.doi.org/10.7554/eLife.01581.016</ext-link></p></caption><graphic xlink:href="elife01581fs009"/></fig><fig id="fig4s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.017</object-id><label>Figure 4—figure supplement 3.</label><caption><title>Increased short interfering RNA (siRNA) production from multi-copy sequences is not due to copy number normalization.</title><p>(<bold>A</bold>) No normalization for copy number was applied to total RNA abundance levels. (<bold>B</bold>) Both total RNA and siRNA abundance were multiplied by copy number for each analyzed locus. See ‘Materials and methods’ section for information on these normalization methods.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.017">http://dx.doi.org/10.7554/eLife.01581.017</ext-link></p></caption><graphic xlink:href="elife01581fs010"/></fig></fig-group></p><p>We then directly tested the effect of copy number at the <italic>MAL32</italic> locus. We constructed strains in which <italic>MAL32</italic> sense and antisense RNAs were expressed at similar levels from multi-copy or single-copy loci by over-expressing single-copy sense and antisense (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). In this system, both sense and antisense RNAs were produced at higher levels from the single-copy system (<xref ref-type="fig" rid="fig5">Figure 5B</xref> compare lanes 1 and 3) but more siRNAs were produced from the multi-copy system (<xref ref-type="fig" rid="fig5">Figure 5C</xref> compare lanes 2 and 4). The over-expression of both RNAs from the single-copy <italic>MAL32</italic> locus led to the production of easily detectable siRNA, as would be expected; however, this result directly demonstrates that gene copy number influences the formation of siRNA above and beyond the effect of total RNA abundance. The increased siRNA production in these cells is most likely due to enhanced dsRNA formation in the multi-copy system. To confirm this, we quantified <italic>MAL32</italic> RNA in wild-type cells that is resistant to the single-strand specific nuclease RNase A, and observed significantly more RNase-resistant material in cells expressing <italic>MAL32</italic> from the multi-copy system than the single-copy system (<xref ref-type="fig" rid="fig5">Figure 5D</xref>). This experiment shows that a multi-copy locus produces more dsRNA than an equivalently expressed single-copy locus in wild-type cells without the RNAi system, explaining the increased siRNA formation in RNAi+ cells.<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01581.018</object-id><label>Figure 5.</label><caption><title>Single gene analysis of copy number effect on RNA interference (RNAi).</title><p>(<bold>A</bold>) Schematic of single-copy and multi-copy <italic>MAL32</italic> system. (<bold>B</bold>) Northern analysis of <italic>MAL32</italic> RNA from single-copy and multi-copy systems. All visible species are included in antisense quantification. (<bold>C</bold>) <italic>MAL32</italic> short interfering RNA (siRNA) abundance from cells in <bold>B</bold>. (<bold>D</bold>) RNase A sensitivity of <italic>MAL32</italic> in wild-type cells expressing multi-copy and single-copy <italic>MAL32</italic>. Cells were lysed on ice, treated with RNase A as indicated and analyzed by northern blot. 25S and <italic>L-A</italic> (a double stranded RNA) are shown as controls for loading and RNase specificity. n = 3 biological replicates, error bars ±1se, *p<0.05, ***p<0.01 by Student’s <italic>t</italic> test, y axes in arbitrary units.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.018">http://dx.doi.org/10.7554/eLife.01581.018</ext-link></p></caption><graphic xlink:href="elife01581f005"/></fig></p><p>The influence of copy number suggested that dsRNA formation and potentially siRNA production may occur in the nucleus. We initially tested this by immunofluorescence against Dicer and dsRNA in mixed populations of wild-type and RNAi+ cells (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>). Dicer was present in cytoplasmic foci, but also showed a diffuse cytoplasmic staining (compare the indicated wild-type and RNAi+ cells) and, under wide-field imaging, appeared to be present in the nucleus. However, super-resolution images of the same cells showed nuclear exclusion of Dicer; therefore, if Dicer is present in the nucleus, it is at low levels. dsRNA staining in these cells revealed many cytoplasmic foci, presumably Killer virus dsRNAs that are known to be incompletely cleared by RNAi (<xref ref-type="bibr" rid="bib22">Drinnenberg et al., 2011</xref>), but did not show unambiguous nuclear staining.</p><p>As an alternative, we asked whether the spatial configuration of gene copies within the nucleus could affect siRNA formation; such an effect would provide strong evidence for the formation of dsRNA in the nucleus. In systems which undergo efficient RNAi such as the rDNA and 2µ plasmids, all gene copies are clustered together in a small sub-nuclear volume. To test the importance of this clustering, we used the sense-antisense system at the <italic>TRP1</italic> locus which produces detectable siRNA even when present in the genome in only two tandem copies (data not shown). We generated strains with three tandem copies of <italic>TRP1</italic> on a single plasmid (Clustered, Cls) or three unlinked copies (Dispersed, Dsp) (<xref ref-type="fig" rid="fig6">Figure 6A</xref>), and expressed Dicer without Argonaute to allow siRNA formation but minimize the effect of RNAi on total RNA levels. Quantification of total RNA showed that both systems produced similar amounts of sense and antisense RNA molecules (<xref ref-type="fig" rid="fig6">Figure 6B</xref>), although this experiment was complicated by read-through transcripts of antisense <italic>TRP1</italic> from the clustered system (<xref ref-type="fig" rid="fig6">Figure 6B</xref> lanes 1,2), a behavior that was not prevented by insertion of transcriptional stop cassettes between the repeats. Nonetheless, the clustered system produced fourfold more <italic>TRP1</italic> siRNA than the dispersed system (<xref ref-type="fig" rid="fig6">Figure 6C</xref>), showing that close nuclear juxtaposition of transcriptional loci enhances dsRNA formation.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01581.019</object-id><label>Figure 6.</label><caption><title>Clustered loci show higher efficiency of short interfering RNA (siRNA) formation.</title><p>(<bold>A</bold>) Comparison of the systems used. Three copies of <italic>TRP1</italic> were placed either in tandem on a single-copy plasmid (Clustered, Cls) or a single copy was left in the genome at the <italic>TRP1</italic> locus and two further copies were placed on different single-copy plasmids (Dispersed, Dsp). Dicer was expressed from a single-copy plasmid. (<bold>B</bold>) Sense and antisense RNA expression in clustered and dispersed systems with and without Dicer. Quantification shows that Dicer alone has little effect on total RNA levels. Read-through species visible in lanes 1,2 are included in the quantification; values have been normalized for the different number of probe binding sites in the read-through RNAs. In the absence of this normalization (i.e., counting the number of binding sites rather than the number of molecules), the clustered antisense is approximately twofold more abundant than the dispersed antisense, which is insufficient to explain the difference in siRNA formation. (<bold>C</bold>) siRNA produced from <italic>TRP1</italic> in clustered and dispersed systems. n = 3 biological replicates, error bars ±1se, ***p<0.01 by Student’s <italic>t</italic> test, y axes in arbitrary units.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.019">http://dx.doi.org/10.7554/eLife.01581.019</ext-link></p></caption><graphic xlink:href="elife01581f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.020</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Analysis of Dicer and double stranded RNA (dsRNA) localization.</title><p>Wild-type and RNA interference (RNAi)+ cells carrying a green fluorescent protein (GFP)-tagged Dicer were grown separately in yeast extract peptone dextrose, then mixed in equal proportions before fixation. Cells were stained with anti-GFP for Dicer (green) and an antibody to dsRNA (red). Images were acquired on a Nikon N-SIM microscope; the wide-field images and the processed super resolution images are shown for comparison. Three wild-type cells (white arrows) and three RNAi+ cells (purple arrows) are indicated. Examination of multiple cells in multiple stacks did not reveal any convincing evidence of Dicer or dsRNA foci in the nucleus, and the Dicer staining overlapping the nucleus in the wide-field images was not visible after processing, nor was it detectable by confocal imaging.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.020">http://dx.doi.org/10.7554/eLife.01581.020</ext-link></p></caption><graphic xlink:href="elife01581fs011"/></fig></fig-group></p><p>While testing the effects of copy number amplification on siRNA production, we noticed that even low abundance sense-antisense ncRNA pairs (selected from a published dataset, <xref ref-type="bibr" rid="bib85">Xu et al., 2009</xref>) underwent efficient RNAi when amplified to high copy number. For both the SUT176 and SUT430 systems (<xref ref-type="fig" rid="fig7">Figure 7A</xref>), the sense and antisense RNAs are barely detectable by northern blot and are clearly not targeted for degradation by RNAi (<xref ref-type="fig" rid="fig7">Figure 7B</xref>). However, after cloning on high-copy plasmids, the full-length RNAs became highly susceptible to RNAi and produced copious siRNAs (<xref ref-type="fig" rid="fig7">Figure 7C,D</xref>). This raised the interesting prospect that low abundance pervasive transcription would be sufficient to trigger efficient RNAi responses from sequences that undergo copy number amplification.<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.01581.021</object-id><label>Figure 7.</label><caption><title>RNA interference (RNAi) against transcripts from amplified low-expression systems.</title><p>(<bold>A</bold>) Schematic diagrams of SUT176 and SUT430 loci. (<bold>B</bold>) Northern analysis of SUT176 and SUT430 transcripts from single-copy genomic loci in wild-type and RNAi+ cells. Ty1 RNA is a positive control for RNAi, <italic>ACT1</italic> is a loading control. (<bold>C</bold>) Analysis of SUT176 and SUT430 non-protein coding RNAs (ncRNAs) expressed from high-copy plasmids in wild-type and RNAi+ cells. Amplified regions are indicated in <bold>A</bold>. (<bold>D</bold>) Short interfering RNA analysis of cells in <bold>C</bold>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.021">http://dx.doi.org/10.7554/eLife.01581.021</ext-link></p></caption><graphic xlink:href="elife01581f007"/></fig></p><p>Clear examples of pervasive transcription are not well defined in any organism because, by definition, the products of pervasive transcription are almost undetectable. We therefore chose to examine the GAL gene cluster (<xref ref-type="fig" rid="fig8">Figure 8A</xref>), which is tightly transcriptionally repressed in cells grown in glucose. Under these conditions, antisense ncRNAs are produced from the <italic>GAL10</italic> ORF with a known steady-state abundance of one RNA molecule per 14 cells (<xref ref-type="bibr" rid="bib40">Houseley et al., 2008</xref>; <xref ref-type="bibr" rid="bib62">Pinskaya et al., 2009</xref>) (arrows in <xref ref-type="fig" rid="fig8">Figure 8B</xref> lane 1). Transcription of these ncRNAs is abrogated in a previously described Reb1 binding site mutant (RBSΔ), leaving almost no detectable RNAs from this locus (<xref ref-type="fig" rid="fig8">Figure 8B</xref> lane 3). For reasons that remain unclear, the GAL cluster is slightly de-repressed in the RNAi+ strain (<xref ref-type="fig" rid="fig8">Figure 8B</xref> compare lanes 1,2); nonetheless, the RBSΔ RNAi+ strain (<xref ref-type="fig" rid="fig8">Figure 8B</xref> lane 4) only produces very low level heterogeneous transcripts from <italic>GAL10</italic>, suggesting that it forms a good model of pervasive transcription. Cloning either wild-type or RBSΔ GAL clusters onto high-copy plasmids substantially increased the levels of detectable ncRNA as expected (<xref ref-type="fig" rid="fig8">Figure 8B</xref> lanes 5,7), and these ncRNAs were processed into easily detectable siRNAs (<xref ref-type="fig" rid="fig8">Figure 8C</xref> lanes 6,8). Therefore, ncRNAs produced at the level of pervasive transcription are sufficient to mediate extensive siRNA production when the copy number of the transcribing locus is increased.<fig-group><fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.01581.022</object-id><label>Figure 8.</label><caption><title>RNA interference (RNAi) against pervasive transcripts from the repressed GAL cluster.</title><p>(<bold>A</bold>) Schematic representation of the GAL cluster. (<bold>B</bold>) Non-strand-specific northern blot of non-protein coding RNAs (ncRNAs) produced from the GAL locus present at single-copy (lanes 1–4) or high-copy (lanes 5–8), showing wild-type and RBSΔ mutant. Arrow indicates <italic>GAL10</italic> antisense RNA. Strand-specific northern blots for the same RNA are shown in <xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1</xref>. (<bold>C</bold>) <italic>GAL10</italic> short interfering RNA (siRNA) from the same cells as in <bold>B</bold>. (<bold>D</bold>) Expression of <italic>GAL10</italic> mRNA from a single-copy genomic locus under the control of a Cu<sup>2+</sup>-responsive promoter in wild-type and RNAi+ strains carrying an empty vector (lanes 1,2), high-copy wild-type GAL cluster (lanes 3,4), or high-copy RBSΔ GAL cluster (lanes 5,6). n = 3 biological replicates, error bars ±1se, ***p<0.01 by Student’s <italic>t</italic> test, y axes in arbitrary units.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.022">http://dx.doi.org/10.7554/eLife.01581.022</ext-link></p></caption><graphic xlink:href="elife01581f008"/></fig><fig id="fig8s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01581.023</object-id><label>Figure 8—figure supplement 1.</label><caption><title>Strand-specific analysis of <italic>GAL10</italic> non-protein coding RNAs (ncRNAs).</title><p>RNA samples from <xref ref-type="fig" rid="fig8">Figure 8B</xref> were separated on two gels and probed with strand-specific probes sense and antisense to the <italic>GAL10</italic> optical reading frame (ORF). Known <italic>GAL10</italic> antisense ncRNA species (<xref ref-type="bibr" rid="bib40">Houseley et al., 2008</xref>; <xref ref-type="bibr" rid="bib62">Pinskaya et al., 2009</xref>) are visible in lanes 9 and 10, and are more prevalent in the RNA interference (RNAi)+ strain for unknown reasons. These species are highly expressed from the high-copy plasmid (lanes 13,14), along with an additional high molecular weight RNA. No sense RNAs were detected from the single-copy wild-type <italic>GAL10</italic> locus (lanes 1,2), although heterogeneous RNA species of 0.8–1.5 kb are observed in the RNAi+ strain carrying the single-copy RBSΔ <italic>GAL10</italic> plasmid (lane 4). These sense RNAs are more clearly visible from the high-copy RBSΔ <italic>GAL10</italic> plasmid in wild-type cells (lane 7), and are faintly visible in the wild-type <italic>GAL10</italic> plasmid (lane 5).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.023">http://dx.doi.org/10.7554/eLife.01581.023</ext-link></p></caption><graphic xlink:href="elife01581fs012"/></fig></fig-group></p><p>The siRNAs produced from the high-copy <italic>GAL10</italic> locus are clearly sufficient to degrade the <italic>GAL10</italic> ncRNAs in the RNAi+ background (<xref ref-type="fig" rid="fig8">Figure 8B</xref> compare lanes 5,7 with lanes 6,8); however, a classical RNAi response should be able to degrade RNA expressed from a separate locus. To test this we introduced the high-copy GAL cluster plasmids into a strain in which the single-copy genomic <italic>GAL10</italic> ORF is expressed at high levels from a Cu<sup>2+</sup>-dependent promoter, allowing expression of the <italic>GAL10</italic> mRNA from the single-copy locus while the GAL clusters present on the high-copy plasmids remain fully repressed. As observed for the <italic>GAL10</italic> ncRNAs, the <italic>GAL10</italic> mRNA was expressed at higher levels in the RNAi+ strain than in the wild-type (<xref ref-type="fig" rid="fig8">Figure 8D</xref> compare lanes 1,2) but, nonetheless, both wild-type and RBSΔ high-copy GAL cluster plasmids caused highly significant >50% knockdowns of the <italic>GAL10</italic> mRNA compared with the empty vector control (<xref ref-type="fig" rid="fig8">Figure 8D</xref> lanes 2,4,6). This was not an indirect effect of the high-copy GAL clusters alone as, in the wild-type background, <italic>GAL10</italic> mRNA levels were slightly increased by the presence of the GAL plasmids (<xref ref-type="fig" rid="fig8">Figure 8D</xref> lanes 1,3,5). These data demonstrate that pervasive transcription of a high-copy locus is sufficient to instigate an effective RNAi response that can mediate the degradation of a target mRNA in trans.</p></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>The ability of the RNAi system to selectively target the products of high-copy sequences such as transposons provides a remarkably efficient genome defense, as well as an effective way to differentiate heterochromatic regions, which are often repetitive, from gene-rich euchromatin. Here we have demonstrated that RNAi has an innate preference for the products of high-copy sequences, probably because RNA from high-copy sequences forms dsRNA more efficiently. It has long been known that cells can recognize and silence high-copy DNA, which would form a basic defense against uncontrolled amplification of transposable elements (reviewed in <xref ref-type="bibr" rid="bib95 bib91">Hsieh and Fire, 2000</xref>). This of course requires a mechanism to count copy number, or at least differentiate high- and low-copy regions, which has remained mysterious. Our data show that RNAi provides such a mechanism by selectively targeting the products of high-copy loci.</p><p>The production of siRNA from high-copy DNA, which would be the basis of such a counting mechanism, absolutely requires that all DNA is transcribed; if this does not occur, transposable elements that remain transcriptionally silent would be invisible to the system. Pervasive transcription, the general background of very low level RNA produced across the genome, ensures that the vast majority of the genome is transcribed, and therefore that no region remains completely silent (<xref ref-type="bibr" rid="bib17">Cheng et al., 2005</xref>; <xref ref-type="bibr" rid="bib6">Birney et al., 2007</xref>; <xref ref-type="bibr" rid="bib43">Kapranov et al., 2007</xref>; <xref ref-type="bibr" rid="bib29">Goodman et al., 2012</xref>). The extent to which mammalian genomes are pervasively transcribed has been controversial; however, many of the questions revolve around whether the pervasive transcripts represent defined functional products or whether much of the detected RNA represents random transcriptional noise (<xref ref-type="bibr" rid="bib79">van Bakel et al., 2010</xref>; <xref ref-type="bibr" rid="bib18">Clark et al., 2011</xref>). For a general surveillance function, it does not really matter whether pervasive transcription is formed of many discrete transcripts or occurs at random since either process should be sufficient to generate dsRNA. If a large proportion of pervasive transcription does represent random noise, this would be actively advantageous; random transcription would be sequence independent, and therefore transposable elements could not become fully silent by mutating individual promoter sequences. We suggest that the primary function of pervasive transcription lies in ensuring the whole genome is transcribed to allow identification and suppression of transposable elements; this does not conflict with the idea that some proportion of these transcripts may have additional functions.</p><p>We propose that hybridization kinetics explains the dependence of RNAi on copy number (shown in <xref ref-type="fig" rid="fig9">Figure 9</xref>). The rate of formation of dsRNA from single stranded sense and antisense RNA is proportional to the concentration of each strand of RNA, and so is inversely proportional to the square of the reaction volume. Technically, the reaction volume is the non-excluded volume of the cell; however, this assumes a uniform distribution of RNA throughout the cell. In reality the RNA is far from evenly distributed, so some small volumes may have very high concentrations of RNA and, within these volumes, the rate of dsRNA formation will be dramatically higher than in the bulk of the cell. Single-copy loci cannot simultaneously transcribe sense and antisense RNA (<xref ref-type="bibr" rid="bib94">Hobson et al., 2012</xref>) so, although such a locus can give rise to a mixed population of sense and antisense RNA in the cytoplasm over time, in the vicinity of the transcription site only one sense of RNA should ever be present, assuming efficient RNA export. Annealing of sense and antisense RNA must therefore occur in the relatively large non-excluded cytoplasmic volume of the cell, which will be inefficient except for very highly expressed RNA. In contrast, at multi-copy loci the simultaneous production of sense and antisense RNA from many closely spaced sites can lead to high concentrations of sense and antisense RNA around the transcription site, causing efficient duplex formation and hence efficient RNAi.<fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.01581.024</object-id><label>Figure 9.</label><caption><title>Proposed mechanism for RNA interference (RNAi) on high-copy loci.</title><p>The rate of double stranded RNA (dsRNA) formation, the required first step in RNAi, is highly dependent on local RNA concentration. Single-copy loci cannot simultaneously transcribe sense and antisense RNA, allowing RNA export to occur before the strands meet and requiring hybridization to occur in the relatively large cytoplasmic volume. In contrast, sense and antisense RNA can be simultaneously transcribed from different parts of a multi-copy locus and, therefore, if the copies are closely juxtaposed in the genome or in 3D space, the local concentration of sense and antisense RNA around the transcription sites should be high, favoring dsRNA formation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.024">http://dx.doi.org/10.7554/eLife.01581.024</ext-link></p></caption><graphic xlink:href="elife01581f009"/></fig></p><p>This mechanism predicts that dsRNA formation should occur in the nucleus, but we were not able to detect Dicer in the nucleus by immunofluorescence. This reflects the situation in higher eukaryotes where Dicer is largely cytoplasmic, but recent experiments in Drosophila and mammalian cells have detected small quantities of nuclear Dicer, particularly associated with chromatin (<xref ref-type="bibr" rid="bib70">Sinkkonen et al., 2010</xref>; <xref ref-type="bibr" rid="bib15">Cernilogar et al., 2011</xref>; <xref ref-type="bibr" rid="bib30">Gullerova et al., 2011</xref>; <xref ref-type="bibr" rid="bib21">Doyle et al., 2013</xref>). Low but functional levels of Dicer may therefore be present in the nucleus of RNAi+ cells and able to generate siRNA. Alternatively, the dsRNA may be exported and processed in the cytoplasm. To our knowledge, there is no clear evidence for or against export of dsRNA by normal pathways; certainly these would not be too large or too structured to pass through nuclear pores compared, for example, with pre-ribosomes.</p><p>One notable prediction of this mechanism is that clustering of multi-copy transcription sites would be a particularly efficient way to increase the local density of sense and antisense RNA. All the systems we have described in this paper are clustered: rDNA repeats are arranged in tandem, telomeres are known to cluster together at various points in the cell cycle (<xref ref-type="bibr" rid="bib92">Gotta et al., 1996</xref>), and high-copy 2µ plasmids exist in a discrete focus that is vital for copy number maintenance (<xref ref-type="bibr" rid="bib98">Velmurugan et al., 2000</xref>; <xref ref-type="bibr" rid="bib99">Wong et al., 2002</xref>). The comparison of siRNA formation from clustered and dispersed <italic>TRP1</italic> loci provides experimental evidence for this effect since, for a given quantity of sense and antisense RNA, the clustered system produces more siRNA. Although the clustered system also produces read-through transcripts, these would not have a higher hybridization rate than the non-read-through RNAs as the hybridization rate depends on the frequency of collisions between molecules. Intriguingly, Tf2 retrotransposons in <italic>Schizosaccharomyces pombe</italic> are clustered by the action of centromere protein B (CENP-B), which also silences these elements through histone deacetylation (<xref ref-type="bibr" rid="bib10">Cam et al., 2008</xref>). This clustering would allow cells to produce siRNA against the Tf2 elements through pervasive transcription, although multiple mechanisms silence Tf2 retrotransposons (<xref ref-type="bibr" rid="bib87">Yamanaka et al., 2012</xref>), providing an extra defense against retrotransposon activation. Similarly, gypsy retrotransposons in Drosophila are known to cluster (<xref ref-type="bibr" rid="bib28">Gerasimova et al., 2000</xref>), which may again facilitate siRNA production. Hence, the clustering of transposable elements by factors such as CENP-B would facilitate their recognition by RNAi and allow for selective RNA degradation.</p><p>Mammalian germline cells are replete with small RNAs including endogenous siRNA (<xref ref-type="bibr" rid="bib83">Watanabe et al., 2006</xref>, <xref ref-type="bibr" rid="bib84">2008</xref>; <xref ref-type="bibr" rid="bib77">Tam et al., 2008</xref>; <xref ref-type="bibr" rid="bib73">Song et al., 2011</xref>), and siRNAs in sperm and oocytes show a pronounced bias towards high-copy sequences that would be effectively explained by the selectivity of the RNAi system towards the products of high-copy loci (<xref ref-type="bibr" rid="bib83">Watanabe et al., 2006</xref>; <xref ref-type="bibr" rid="bib73">Song et al., 2011</xref>). However, it remains unclear how some dsRNA precursors of siRNAs are generated, particularly for retrotransposons that are primarily expressed only on the sense strand. We suggest that pervasive transcription would provide sufficient antisense RNA for this role, just as we observed for high-copy GAL cluster sequences in yeast.</p><p>In comparison to the germline, the response of mammalian somatic cells to dsRNA is distinctly muted. dsRNA could be processed into siRNA, be altered by RNA editing (<xref ref-type="bibr" rid="bib36">Hogg et al., 2011</xref>), or could activate the interferon response leading to apoptosis (<xref ref-type="bibr" rid="bib25">Gantier and Williams, 2007</xref>). However, transgenic mice expressing a hairpin dsRNA construct produce minimal siRNAs, little edited RNA, and show no phenotype that might indicate cell death (<xref ref-type="bibr" rid="bib57">Nejepinska et al., 2012a</xref>). Nonetheless, siRNAs produced from LINE-1 retrotransposons have been detected in cell culture (<xref ref-type="bibr" rid="bib88">Yang and Kazazian, 2006</xref>), and high-copy transfected plasmids expressing a sense-antisense RNA pair do produce detectable siRNA in HEK293 cells (<xref ref-type="bibr" rid="bib58">Nejepinska et al., 2012b</xref>). This shows that a basic siRNA response with an apparent bias towards high-copy sequences is functional in mammalian somatic cells.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Yeast strains, plasmids and culture conditions</title><p>Yeast deletion strains (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>) were created by standard methods using the oligonucleotides in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>. Plasmids are described in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref> with construction details. Cells were grown on rich media (2% peptone, 1% yeast extract, 2% sugar) or synthetic media (0.69% yeast nitrogen base with ammonium sulfate, amino acids, 2% sugar) for plasmid assays. <italic>GAL10</italic> mRNA was induced with 20 µM CuSO<sub>4</sub> in <xref ref-type="fig" rid="fig8">Figure 8D</xref>. Media components were purchased from Formedium. Cells were grown to mid-log (OD 0.4–0.6) at 30°C for most experiments or at 25°C for experiments involving <italic>trf4</italic>Δ mutants. The W303 background strain used here has defects in galactose induction in synthetic media, so strains in <xref ref-type="fig" rid="fig3">Figure 3G,H</xref> were diploids of W303xBY4741 that show a normal galactose response.</p></sec><sec id="s4-2"><title>RNA extraction and northern analysis</title><p>RNA was extracted by three procedures described below. High molecular weight RNA was prepared using the hot phenol method for all experiments except <xref ref-type="fig" rid="fig7 fig3">Figures 7B, 3B, 3F</xref> and <xref ref-type="fig" rid="fig3s1 fig3s2">Figure 3—figure supplements 1,2</xref> where guanidine thiocyanate (GTC)-phenol preparations were used. 5–10 µg glyoxylated RNA was resolved on 1.2% gels as described (<xref ref-type="bibr" rid="bib67">Sambrook and Russell, 2001</xref>), transferred to Hybond N+ membrane (GE) and hybridized with probes listed in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref> using either Church Hyb (<xref ref-type="bibr" rid="bib67">Sambrook and Russell, 2001</xref>) or UltraHyb (Life Technologies). RNA probes were hybridized at 65°C and washed at 65°C using 0.1× SSC, 0.1% SDS, DNA probes in Church Hyb were hybridized at 65°C and washed at 65°C with 0.5× SSC, 0.1% SDS, DNA probes in UltraHyb were hybridized at 42°C and washed at 55°C using 0.2× SSC, 0.1% SDS. Small RNA enriched fractions were isolated using the mirVANA kit (Ambion). 4–10 µg small RNA was separated on 15% polyacrylamide/8 M urea gels containing 20 mM MOPS or 1× TBE, transferred in 20 mM MOPS or 0.5× TBE to Hybond N membrane (GE) and chemically cross-linked as described (<xref ref-type="bibr" rid="bib60">Pall and Hamilton, 2008</xref>). We observed no difference in cross-linking efficiency between MOPS and TBE gels, but resolution of TBE gels was superior in our hands. siRNAs were detected using random primed probes (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>) hybridized in UltraHyb Oligo (Life Technologies) at 42°C and washed with 2× SSC, 0.5% SDS at 42°C, U6 control oligonucleotide was labeled using T4 polynucleotide kinase and hybridized in Church Hyb under the same conditions.</p></sec><sec id="s4-3"><title>Hot phenol RNA preparation</title><p>10 × 10<sup>7</sup> cells in 15 ml tubes were re-suspended in 600 µl TES (10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS) and 600 µl phenol pH 7. The mixture was incubated at 65°C for 20 min with 30 s vortexing every 5 min, before briefly chilling on ice. Samples were centrifuged for 5 min and the upper phase extracted. This phase (5–600 µl) was extracted twice with phenol:chloroform (5:1) and once with chloroform before precipitation with 50 µl 3 M sodium acetate (NaOAc) pH 5.2 and 1.1 ml ethanol. The pellet was washed with 70% ethanol and re-suspended in 30 µl water.</p></sec><sec id="s4-4"><title>GTC-phenol RNA preparation</title><p>2 × 10<sup>7</sup> cells were lysed by 5 min vortexing at 4°C with 50 µl glass beads and 40 µl GTC-phenol (2.1 M GTC, 26.5 mM Na citrate pH7, 5.3 mM EDTA, 76 mM β-mercaptoethanol, 1.06% N-lauryl sarcosine, 50% phenol pH7). 600 µl GTC-phenol was added, mixed, and samples were heated at 65°C for 10 min, then placed on ice for 10 min. 160 µl 100 mM NaOAc pH 5.2 and 300 µl chloroform:isoamyl alcohol (24:1) were added, samples were vortexed and centrifuged at top speed for 5 min. The upper phase was extracted, precipitated with 1 ml ethanol, washed with 70% ethanol and the pellet re-suspended in 6 µl water. 3 µl RNA was analyzed per lane.</p></sec><sec id="s4-5"><title>Small RNA purification</title><p>Small RNAs were isolated using a mirVANA kit (Life Technologies) with minor modifications. 35 × 10<sup>7</sup> cells were thoroughly re-suspended in 100 µl lysis/binding buffer, 200 µl glass beads were added, and the samples were vortexed for 5 min at 4°C. 500 µl lysis/binding buffer were added and the samples were mixed before proceeding with the isolation as per the manufacturer’s instructions, starting with addition of the miRNA homogenate additive. After isolation the samples were generally re-precipitated and re-suspended in 20 µl water.</p></sec><sec id="s4-6"><title>RNase A treatment</title><p>20 × 10<sup>7</sup> cells were harvested and split into two aliquots, then re-suspended in 600 µl 10 mM Tris pH 7.5, 10 mM EDTA on ice. Cells were lysed with glass beads (10 cycles of 30 s vortex, 60 s on ice), and 5 µg RNase A was added to one aliquot followed by 30 min incubation on ice. After centrifugation for 10 min at 4500×<italic>g</italic>, 600 µl lysate was extracted, SDS added to 0.5%, and RNA extracted by the hot phenol method as above. RNase A treated samples were re-suspended in 12 µl water.</p></sec><sec id="s4-7"><title>DNA isolation and Southern blotting</title><p>Cells from 2 ml saturated culture were washed with 50 mM EDTA, then spheroplasted with 250 µl 0.34 U/ml lyticase (Sigma L4025) in 1.2 M sorbitol, 50 mM EDTA, 10 mM DTT. After centrifuging at 1000<italic>×g</italic>, the cells were gently resuspended in 400 µl of 0.3% SDS, 50 mM EDTA, 100 µg/ml RNase A and incubated at 37°C for 30 min. 4 µl of 20 mg/ml proteinase K was added and the samples were mixed by inversion and heated to 65°C for 30 min. 160 µl 5 M potassium acetate was added after cooling to room temperature, the samples were mixed by inversion and then chilled on ice for 30 min. After 10 min centrifuging at 10,000×<italic>g</italic>, the supernatant was poured into a new tube containing 500 µl phenol:chloroform pH 8 and the samples were mixed on a wheel for 15 min. The samples were centrifuged for 10 min at 10,000×<italic>g</italic> and the upper phase was extracted using cut tips and precipitated with 400 µl isopropanol. Pellets were washed with 70% ethanol, air-dried and left overnight at 4°C to dissolve in 30 µl TE. After gentle mixing, 10 µl of each sample was digested with 40 U of EcoRV, ethanol precipitated and separated on a 25 cm 1% TBE gel at 90 V overnight. The gel was washed in 0.25 N HCl for 15 min, 0.5 N NaOH for 45 min, and twice in 1.5 M NaCl 0.5 M Tris pH 7.5 for 20 min before transfer to HyBond N+ membrane in 6× SSC. The membrane was probed for <italic>URA3</italic> and <italic>GAL7</italic> in Church Hyb at 65°C and washed with 0.5× SSC, 0.1% SDS at 65°C.</p></sec><sec id="s4-8"><title>Immunofluorescence</title><p>Cells were grown to OD 0.5 in YPD and the cultures mixed as required, 4 ml per coverslip. The cells were fixed with 440 µl 37% formaldehyde (Merck, microscopy grade) for 15 min at room temperature, then centrifuged for 2 min at 4600 rpm. The cells were washed three times with 1 ml of buffer B (0.1 M sodium phosphate pH 7.5, 1.2 M sorbitol), then re-suspended in 100 µl buffer B containing 3 µl 17 U/µl lyticase (Sigma L2524) and 10 mM DTT for 15 min. The cells were centrifuged for 2 min at 1000×<italic>g</italic>, then washed with 1 ml buffer B. The cells were re-suspended in 40 µl buffer B, applied to a poly-L-lysine coated coverslip (Zeiss 18 × 18 × 0.170 ± 0.005 mm) and left for 20 min before washing twice with buffer B. Coverslips were treated with −20°C methanol for 6 min, then dipped in −20°C acetone for 10 s, followed by two washes with PBS. Coverslips were blocked for 30 min with 5% milk 0.3% Triton-X100 in PBS, washed with PBS, then incubated overnight at 4°C with primary antibodies in 50 µl 1% BSA 0.3% Triton-X100 in PBS. Coverslips were washed three times with PBS and incubated for 30 min at room temperature with secondary antibodies 1:1000 in same buffer as primaries. After washing three times with PBS, the samples were dehydrated with 70%, 90%, and 100% ethanol and mounted in Pro-long Gold with DAPI (Life Technologies). Antibodies were rabbit anti-GFP (Life Technologies A11122) at 1:500 and mouse anti-dsRNA J2 (ESC 10010200) at 1:1000. Images were acquired using a Nikon N-SIM microscope comprising a Nikon Ti-E microscope, Nikon 100× 1.49 NA lens, Nikon SIM illumination module, and Andor iXon 897 EM-CCD camera. SIM data were acquired in ‘3D-SIM’ mode using five phases and three rotations. DAPI, Alexa Fluor 488, and Alexa Fluor 594 dyes were excited using 405, 488, and 561 nm laser light, respectively. Super-resolution images were reconstructed using Nikon Elements software. Equivalent wide-field images were reconstructed in FIJI (ImageJ, NIH) by summing the phase shifts from one grid rotation.</p></sec><sec id="s4-9"><title>Imaging and analysis</title><p>Gels and phosphor screens were imaged using FLA 3000 (Fuji) or FLA 7000 (GE) systems. Quantification was performed using AIDA (Fuji) or ImageQuant (GE), depending on the scanner used. Images were prepared for publication with ImageJ by smoothing and minimal contrast enhancement. Images from the FLA3000 had a Gamma Shift of 3 applied.</p></sec><sec id="s4-10"><title>5′ RACE</title><p>5′ RACE was performed with an ExactSTART Eukaryotic mRNA 5′- and 3′-RACE Kit (Epicentre) as per manufacturer’s instructions, except that reverse transcription was primed from random hexamers.</p></sec><sec id="s4-11"><title>RNA immunoprecipitation</title><p>50 ml of cells at 0.7 × 10<sup>7</sup> cells/ml were harvested, washed, and frozen on nitrogen. Cells were thawed, washed twice in 1 ml lysis buffer (50 mM HEPES pH7.5, 50 mM KCl, 5 mM MgCl<sub>2</sub>, 1 mM DTT, 1× complete protease inhibitors (Roche)) and transferred to 2 ml tubes, then re-suspended in 300 µl lysis buffer. 300 µl zirconium beads were added and cells were lysed by vortexing 10 × 30 s with 30 s on ice between cycles. The lysate was clarified by centrifuging twice for 5 min at 14,000 rpm, a 12 µl aliquot was removed for total RNA and the remaining lysate was split in half and 2.5 µl mouse anti-dsRNA J2 (ESC 10010200) added to one aliquot. Antibody was bound for 2 hr at 4°C, then 20 µl GammaBind beads (GE) pre-blocked overnight with 1% BSA were added and incubation continued on a wheel for 2 hr at 4°C. The beads were washed 5× for 10 min with 1 ml wash buffer (10 mM Tris pH 7.5, 120 mM NaCl, 5 mM MgCl<sub>2</sub>, 0.1% NP-40, 1 mM DTT). RNA was extracted from the beads and total lysate samples using Tri-reagent (Sigma) according to the manufacturer’s instructions. 1 µg total lysate and whole immunoprecipitation samples were treated with RQ1 DNase (Promega), purified by phenol:chloroform and ethanol precipitation, then reverse transcribed from random hexamers using Superscript III (Life Technologies). Quantitative PCR was performed using Maxima SYBR qPCR mix (Fermentas).</p></sec><sec id="s4-12"><title>Bioinformatics</title><sec id="s4-12-1"><title>Antisense analysis</title><p>Locations of XUTs (<xref ref-type="bibr" rid="bib80">van Dijk et al., 2011</xref>) were merged with CUT and SUT locations (<xref ref-type="bibr" rid="bib85">Xu et al., 2009</xref>), along with expression validated ORFs (<xref ref-type="bibr" rid="bib85">Xu et al., 2009</xref>), and overlap between ORFs and other features calculated using an R script.<table-wrap id="tbl2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01581.025</object-id><label>Table 2.</label><caption><p>n values for statistical tests</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.025">http://dx.doi.org/10.7554/eLife.01581.025</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Figure</th><th>Category</th><th>n</th><th>Category</th><th>n</th></tr></thead><tbody><tr><td rowspan="9">4B</td><td>0–2 single-copy</td><td>19,892</td><td>0–2 multi-copy</td><td>1128</td></tr><tr><td>2–4 single-copy</td><td>36,474</td><td>2–4 multi-copy</td><td>1111</td></tr><tr><td>4–6 single-copy</td><td>34,921</td><td>4–6 multi-copy</td><td>1177</td></tr><tr><td>6–8 single-copy</td><td>16,208</td><td>6–8 multi-copy</td><td>1356</td></tr><tr><td>8–10 single-copy</td><td>4060</td><td>8–10 multi-copy</td><td>1424</td></tr><tr><td>10–12 single-copy</td><td>922</td><td>10–12 multi-copy</td><td>1505</td></tr><tr><td>12–14 single-copy</td><td>270</td><td>12–14 multi-copy</td><td>887</td></tr><tr><td>14–16 single-copy</td><td>56</td><td>14–16 multi-copy</td><td>199</td></tr><tr><td/><td/><td/><td/></tr><tr><td rowspan="7">4C</td><td>Bulk 1–2 copies</td><td>85,495</td><td>Top 1% 1–2 copies</td><td>252</td></tr><tr><td>Bulk 2–4 copies</td><td>1419</td><td>Top 1% 2–4 copies</td><td>95</td></tr><tr><td>Bulk 4–8 copies</td><td>544</td><td>Top 1% 4–8 copies</td><td>98</td></tr><tr><td>Bulk 8–16 copies</td><td>981</td><td>Top 1% 8–16 copies</td><td>238</td></tr><tr><td>Bulk 16–32 copies</td><td>1978</td><td>Top 1% 16–32 copies</td><td>212</td></tr><tr><td>Bulk >32 copies</td><td>695</td><td>Top 1% >32 copies</td><td>25</td></tr><tr><td/><td/><td/><td/></tr><tr><td rowspan="9">4—Supplement 1A</td><td>0–2 single-copy</td><td>19,892</td><td>0–2 multi-copy</td><td>836</td></tr><tr><td>2–4 single-copy</td><td>36,474</td><td>2–4 multi-copy</td><td>906</td></tr><tr><td>4–6 single-copy</td><td>34,921</td><td>4–6 multi-copy</td><td>724</td></tr><tr><td>6–8 single-copy</td><td>16,208</td><td>6–8 multi-copy</td><td>387</td></tr><tr><td>8–10 single-copy</td><td>4060</td><td>8–10 multi-copy</td><td>189</td></tr><tr><td>10–12 single-copy</td><td>922</td><td>10–12 multi-copy</td><td>167</td></tr><tr><td>12–14 single-copy</td><td>270</td><td>12–14 multi-copy</td><td>128</td></tr><tr><td>14–16 single-copy</td><td>56</td><td>14–16 multi-copy</td><td>29</td></tr><tr><td/><td/><td/><td/></tr><tr><td rowspan="9">4—Supplement 1B</td><td>0–2 low-copy</td><td>20,728</td><td>0–2 high-copy</td><td>292</td></tr><tr><td>2–4 low-copy</td><td>37,380</td><td>2–4 high-copy</td><td>205</td></tr><tr><td>4–6 low-copy</td><td>35,645</td><td>4–6 high-copy</td><td>453</td></tr><tr><td>6–8 low-copy</td><td>16,595</td><td>6–8 high-copy</td><td>969</td></tr><tr><td>8–10 low-copy</td><td>4249</td><td>8–10 high-copy</td><td>1235</td></tr><tr><td>10–12 low-copy</td><td>1089</td><td>10–12 high-copy</td><td>1338</td></tr><tr><td>12–14 low-copy</td><td>398</td><td>12–14 high-copy</td><td>759</td></tr><tr><td>14–16 low-copy</td><td>85</td><td>14–16 high-copy</td><td>170</td></tr><tr><td/><td/><td/><td/></tr><tr><td rowspan="9">4-Supplement 2A</td><td>0–2 single-copy</td><td>20,030</td><td>0–2 multi-copy</td><td>1999</td></tr><tr><td>2–4 single-copy</td><td>36,494</td><td>2–4 multi-copy</td><td>2104</td></tr><tr><td>4–6 single-copy</td><td>34,883</td><td>4–6 multi-copy</td><td>2098</td></tr><tr><td>6–8 single-copy</td><td>16,178</td><td>6–8 multi-copy</td><td>1668</td></tr><tr><td>8–10 single-copy</td><td>4078</td><td>8–10 multi-copy</td><td>644</td></tr><tr><td>10–12 single-copy</td><td>873</td><td>10–12 multi-copy</td><td>204</td></tr><tr><td>12–14 single-copy</td><td>215</td><td>12–14 multi-copy</td><td>66</td></tr><tr><td>14–16 single-copy</td><td>52</td><td>14–16 multi-copy</td><td>10</td></tr><tr><td/><td/><td/><td/></tr><tr><td rowspan="8">4-Supplement 2B</td><td>0–2 single-copy</td><td>19,892</td><td>0–2 multi-copy</td><td>1128</td></tr><tr><td>2–4 single-copy</td><td>36,474</td><td>2–4 multi-copy</td><td>1111</td></tr><tr><td>4–6 single-copy</td><td>34,921</td><td>4–6 multi-copy</td><td>1177</td></tr><tr><td>6–8 single-copy</td><td>16,208</td><td>6–8 multi-copy</td><td>1356</td></tr><tr><td>8–10 single-copy</td><td>4060</td><td>8–10 multi-copy</td><td>1424</td></tr><tr><td>10–12 single-copy</td><td>922</td><td>10–12 multi-copy</td><td>1505</td></tr><tr><td>12–14 single-copy</td><td>270</td><td>12–14 multi-copy</td><td>887</td></tr><tr><td>14–16 single-copy</td><td>56</td><td>14–16 multi-copy</td><td>199</td></tr></tbody></table></table-wrap></p></sec><sec id="s4-12-2"><title>siRNA analysis</title><p>Sequencing data for mRNA and siRNA fractions in RNAi strains (GEO accession GSE31300) (<xref ref-type="bibr" rid="bib22">Drinnenberg et al., 2011</xref>) and W303 total RNA (GEO accession GSE38383) (<xref ref-type="bibr" rid="bib94">Hobson et al., 2012</xref>) were quality and adapter trimmed using Trim Galore (v0.2.3; default options; <ext-link ext-link-type="uri" xlink:href="http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/">http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/</ext-link>) and aligned to the yeast genome (build SGD1.01) using Bowtie (<xref ref-type="bibr" rid="bib96">Langmead et al., 2009</xref>) (v0.12.7; default settings plus ‘--best’) allowing non-unique sequences to be assigned at random. For expressed gene analysis (<xref ref-type="fig" rid="fig1">Figure 1</xref>), reads overlapping each ORF were binned and only ORFs with >100 reads were used. For siRNA profiles (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>), reads were binned over 50 bp intervals using SeqMonk (<ext-link ext-link-type="uri" xlink:href="http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/">http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/</ext-link>). For siRNA versus total RNA expression (<xref ref-type="fig" rid="fig4">Figure 4</xref>), read counts were quantified in consecutive 100 bp bins across the genome using SeqMonk, bins with >10,0000 total RNA reads were excluded as were bins derived from 2µ sequence which is single copy in the genome sequence but high copy in reality, and a pseudocount of one read was added to total and siRNA read counts for each bin. Total RNA levels were multiplied by copy number to correct for the division of reads amongst copies that occurs during mapping, or alternate normalization was applied in <xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref> (see below for the reasoning underlying this copy number normalization methodology). The copy number for each bin was determined by splitting the complete genomic sequence into overlapping 20 bp segments at 1 bp intervals and re-mapping to the genome with reads allowed to align to all perfectly matching sequences, producing a measure of the number of genomic sequences matching each 100 bp bin. Little difference was seen if one mismatch was allowed (data not shown).</p></sec></sec><sec id="s4-13"><title>Read count normalization for multi-copy sequences</title><p>Multi-copy loci are problematic for standard high-throughput sequencing mapping pipelines and are commonly discarded. Reads mapping to a non-unique genome sequence are usually assigned at random to one copy in the genome, therefore the total reads are divided evenly amongst the copies and the apparent abundance of RNA matching each copy is effectively divided by the copy number. In order to assess total RNA abundance (as in <xref ref-type="fig" rid="fig4">Figure 4A,B</xref>), we multiplied all total RNA read counts by the copy number to obtain the real total RNA abundance. However, we decided that the siRNA abundance should be analyzed per producing locus because each copy in the genome was analyzed separately for comparison with single-copy loci (i.e., we quantified how many siRNA reads an individual copy of a multi-copy locus produced, not how many the combined copies produced). We therefore did not multiply the siRNA read counts by the copy number. Such normalizations clearly have the potential to introduce systematic biases, and we therefore repeated the analysis in <xref ref-type="fig" rid="fig4">Figure 4</xref> either with no copy number normalization or with both total and siRNA read counts multiplied by copy number (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2</xref>). We found that although the distributions changed somewhat, the majority of total RNA abundance categories had higher siRNA levels for multi-copy loci irrespective of the copy number normalization applied. We note that the alternative copy number analysis in <xref ref-type="fig" rid="fig4">Figure 4C,D</xref> did not require any normalization; indeed, any copy number normalization would simply cancel out in the calculation of the siRNA:total RNA ratio, therefore any systematic bias that might be introduced by copy number normalization would have no effect.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank Simon Andrews, Felix Krueger, Laura Biggins and Anne Segonds-Pichon for Bioinformatics support, Simon Walker for microscopy support, David Bartel for strains, Robin Allshire, Wolf Reik and Sarah Elderkin for comments, and Alex Murray and Tim Hore for critical reading. This work was supported by the Wellcome Trust [grant number 088335].</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>CC, Conception and design, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con2"><p>JH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.01581.026</object-id><label>Supplementary file 1.</label><caption><p>Yeast strains, primers, plasmids, and probes used in this study. <bold>Strains</bold>. Strains used in this study. <sup>a</sup>Diploid strains from mating W303α with BY4741. <bold>Primers</bold>. Primers used in this study organized by locus. <bold>Plasmids</bold>. Plasmids used in this study. <bold>Probes</bold>. Probes used in this study for northern and Southern blot analysis. Probe target is given in parentheses after probe name.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01581.026">http://dx.doi.org/10.7554/eLife.01581.026</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife01581s001.xlsx"/></supplementary-material><sec sec-type="datasets"><title>Major dataset</title><p>The following previously published datasets were used:</p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro1"><name><surname>Drinnenberg</surname><given-names>IA</given-names></name>, <name><surname>Fink</surname><given-names>GR</given-names></name>, <name><surname>Bartel</surname><given-names>DP</given-names></name>, <year>2011</year><x>, </x><source>Compatibility with Killer explains the Rise of RNAi-deficient Fungi</source><x>, </x><object-id pub-id-type="art-access-id">GSE31300</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31300">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31300</ext-link><x>, </x><comment>Publicly available at GEO (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/">http://www.ncbi.nlm.nih.gov/geo/</ext-link>).</comment></related-object></p><p><related-object content-type="generated-dataset" document-id="Dataset ID and/or url" document-id-type="dataset" document-type="data" id="dataro2"><name><surname>Hobson</surname><given-names>DJ</given-names></name>, <name><surname>Wei</surname><given-names>W</given-names></name>, <name><surname>Steinmetz</surname><given-names>LM</given-names></name>, <name><surname>Svejstrup</surname><given-names>JQ</given-names></name>, <year>2012</year><x>, </x><source>RNA polymerase II collision interrupts convergent transcription (RNA-seq)</source><x>, </x><object-id pub-id-type="art-access-id">GSE38383</object-id><x>; </x><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38383">http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38383</ext-link><x>, </x><comment>Publicly available at GEO (<ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/">http://www.ncbi.nlm.nih.gov/geo/</ext-link>).</comment></related-object></p></sec></sec><ref-list><title>References</title><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aparicio</surname><given-names>OM</given-names></name><name><surname>Billington</surname><given-names>BL</given-names></name><name><surname>Gottschling</surname><given-names>DE</given-names></name></person-group><year>1991</year><article-title>Modifiers of position effect are shared between telomeric and silent mating-type loci in <italic>S. cerevisiae</italic></article-title><source>Cell</source><volume>66</volume><fpage>1279</fpage><lpage>1287</lpage><pub-id pub-id-type="doi">10.1016/0092-8674(91)90049-5</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Babiarz</surname><given-names>JE</given-names></name><name><surname>Ruby</surname><given-names>JG</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Bartel</surname><given-names>DP</given-names></name><name><surname>Blelloch</surname><given-names>R</given-names></name></person-group><year>2008</year><article-title>Mouse ES cells express endogenous shRNAs, siRNAs, and other microprocessor-independent, Dicer-dependent small RNAs</article-title><source>Genes Development</source><volume>22</volume><fpage>2773</fpage><lpage>2785</lpage><pub-id pub-id-type="doi">10.1101/gad.1705308</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Berretta</surname><given-names>J</given-names></name><name><surname>Pinskaya</surname><given-names>M</given-names></name><name><surname>Morillon</surname><given-names>A</given-names></name></person-group><year>2008</year><article-title>A cryptic unstable transcript mediates transcriptional trans-silencing of the Ty1 retrotransposon in <italic>S. cerevisiae</italic></article-title><source>Genes Development</source><volume>22</volume><fpage>615</fpage><lpage>626</lpage><pub-id pub-id-type="doi">10.1101/gad.458008</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bertone</surname><given-names>P</given-names></name><name><surname>Stolc</surname><given-names>V</given-names></name><name><surname>Royce</surname><given-names>TE</given-names></name><name><surname>Rozowsky</surname><given-names>JS</given-names></name><name><surname>Urban</surname><given-names>AE</given-names></name><name><surname>Zhu</surname><given-names>X</given-names></name><name><surname>Rinn</surname><given-names>JL</given-names></name><name><surname>Tongprasit</surname><given-names>W</given-names></name><name><surname>Samanta</surname><given-names>M</given-names></name><name><surname>Weissman</surname><given-names>S</given-names></name><name><surname>Gerstein</surname><given-names>M</given-names></name><name><surname>Snyder</surname><given-names>M</given-names></name></person-group><year>2004</year><article-title>Global identification of human transcribed sequences with genome tiling arrays</article-title><source>Science</source><volume>306</volume><fpage>2242</fpage><lpage>2246</lpage><pub-id pub-id-type="doi">10.1126/science.1103388</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Birney</surname><given-names>E</given-names></name><name><surname>Stamatoyannopoulos</surname><given-names>JA</given-names></name><name><surname>Dutta</surname><given-names>A</given-names></name><name><surname>Guigo</surname><given-names>R</given-names></name><name><surname>Gingeras</surname><given-names>TR</given-names></name><name><surname>Margulies</surname><given-names>EH</given-names></name><name><surname>Weng</surname><given-names>Z</given-names></name><name><surname>Snyder</surname><given-names>M</given-names></name><name><surname>Dermitzakis</surname><given-names>ET</given-names></name><name><surname>Thurman</surname><given-names>RE</given-names></name><name><surname>Kuehn</surname><given-names>MS</given-names></name><name><surname>Taylor</surname><given-names>CM</given-names></name><name><surname>Neph</surname><given-names>S</given-names></name><name><surname>Koch</surname><given-names>CM</given-names></name><name><surname>Asthana</surname><given-names>S</given-names></name><name><surname>Malhotra</surname><given-names>A</given-names></name><name><surname>Adzhubei</surname><given-names>I</given-names></name><name><surname>Greenbaum</surname><given-names>JA</given-names></name><name><surname>Andrews</surname><given-names>RM</given-names></name><name><surname>Flicek</surname><given-names>P</given-names></name><name><surname>Boyle</surname><given-names>PJ</given-names></name><name><surname>Cao</surname><given-names>H</given-names></name><name><surname>Carter</surname><given-names>NP</given-names></name><name><surname>Clelland</surname><given-names>GK</given-names></name><name><surname>Davis</surname><given-names>S</given-names></name><name><surname>Day</surname><given-names>N</given-names></name><name><surname>Dhami</surname><given-names>P</given-names></name><name><surname>Dillon</surname><given-names>SC</given-names></name><name><surname>Dorschner</surname><given-names>MO</given-names></name><name><surname>Fiegler</surname><given-names>H</given-names></name><name><surname>Giresi</surname><given-names>PG</given-names></name><name><surname>Goldy</surname><given-names>J</given-names></name><name><surname>Hawrylycz</surname><given-names>M</given-names></name><name><surname>Haydock</surname><given-names>A</given-names></name><name><surname>Humbert</surname><given-names>R</given-names></name><name><surname>James</surname><given-names>KD</given-names></name><name><surname>Johnson</surname><given-names>BE</given-names></name><name><surname>Johnson</surname><given-names>EM</given-names></name><name><surname>Frum</surname><given-names>TT</given-names></name><name><surname>Rosenzweig</surname><given-names>ER</given-names></name><name><surname>Karnani</surname><given-names>N</given-names></name><name><surname>Lee</surname><given-names>K</given-names></name><name><surname>Lefebvre</surname><given-names>GC</given-names></name><name><surname>Navas</surname><given-names>PA</given-names></name><name><surname>Neri</surname><given-names>F</given-names></name><name><surname>Parker</surname><given-names>SC</given-names></name><name><surname>Sabo</surname><given-names>PJ</given-names></name><name><surname>Sandstrom</surname><given-names>R</given-names></name><name><surname>Shafer</surname><given-names>A</given-names></name><name><surname>Vetrie</surname><given-names>D</given-names></name><name><surname>Weaver</surname><given-names>M</given-names></name><name><surname>Wilcox</surname><given-names>S</given-names></name><name><surname>Yu</surname><given-names>M</given-names></name><name><surname>Collins</surname><given-names>FS</given-names></name><name><surname>Dekker</surname><given-names>J</given-names></name><name><surname>Lieb</surname><given-names>JD</given-names></name><name><surname>Tullius</surname><given-names>TD</given-names></name><name><surname>Crawford</surname><given-names>GE</given-names></name><name><surname>Sunyaev</surname><given-names>S</given-names></name><name><surname>Noble</surname><given-names>WS</given-names></name><name><surname>Dunham</surname><given-names>I</given-names></name><name><surname>Denoeud</surname><given-names>F</given-names></name><name><surname>Reymond</surname><given-names>A</given-names></name><name><surname>Kapranov</surname><given-names>P</given-names></name><name><surname>Rozowsky</surname><given-names>J</given-names></name><name><surname>Zheng</surname><given-names>D</given-names></name><name><surname>Castelo</surname><given-names>R</given-names></name><name><surname>Frankish</surname><given-names>A</given-names></name><name><surname>Harrow</surname><given-names>J</given-names></name><name><surname>Ghosh</surname><given-names>S</given-names></name><name><surname>Sandelin</surname><given-names>A</given-names></name><name><surname>Hofacker</surname><given-names>IL</given-names></name><name><surname>Baertsch</surname><given-names>R</given-names></name><name><surname>Keefe</surname><given-names>D</given-names></name><name><surname>Dike</surname><given-names>S</given-names></name><name><surname>Cheng</surname><given-names>J</given-names></name><name><surname>Hirsch</surname><given-names>HA</given-names></name><name><surname>Sekinger</surname><given-names>EA</given-names></name><name><surname>Lagarde</surname><given-names>J</given-names></name><name><surname>Abril</surname><given-names>JF</given-names></name><name><surname>Shahab</surname><given-names>A</given-names></name><name><surname>Flamm</surname><given-names>C</given-names></name><name><surname>Fried</surname><given-names>C</given-names></name><name><surname>Hackermüller</surname><given-names>J</given-names></name><name><surname>Hertel</surname><given-names>J</given-names></name><name><surname>Lindemeyer</surname><given-names>M</given-names></name><name><surname>Missal</surname><given-names>K</given-names></name><name><surname>Tanzer</surname><given-names>A</given-names></name><name><surname>Washietl</surname><given-names>S</given-names></name><name><surname>Korbel</surname><given-names>J</given-names></name><name><surname>Emanuelsson</surname><given-names>O</given-names></name><name><surname>Pedersen</surname><given-names>JS</given-names></name><name><surname>Holroyd</surname><given-names>N</given-names></name><name><surname>Taylor</surname><given-names>R</given-names></name><name><surname>Swarbreck</surname><given-names>D</given-names></name><name><surname>Matthews</surname><given-names>N</given-names></name><name><surname>Dickson</surname><given-names>MC</given-names></name><name><surname>Thomas</surname><given-names>DJ</given-names></name><name><surname>Weirauch</surname><given-names>MT</given-names></name><name><surname>Gilbert</surname><given-names>J</given-names></name><name><surname>Drenkow</surname><given-names>J</given-names></name><name><surname>Bell</surname><given-names>I</given-names></name><name><surname>Zhao</surname><given-names>X</given-names></name><name><surname>Srinivasan</surname><given-names>KG</given-names></name><name><surname>Sung</surname><given-names>WK</given-names></name><name><surname>Ooi</surname><given-names>HS</given-names></name><name><surname>Chiu</surname><given-names>KP</given-names></name><name><surname>Foissac</surname><given-names>S</given-names></name><name><surname>Alioto</surname><given-names>T</given-names></name><name><surname>Brent</surname><given-names>M</given-names></name><name><surname>Pachter</surname><given-names>L</given-names></name><name><surname>Tress</surname><given-names>ML</given-names></name><name><surname>Valencia</surname><given-names>A</given-names></name><name><surname>Choo</surname><given-names>SW</given-names></name><name><surname>Choo</surname><given-names>CY</given-names></name><name><surname>Ucla</surname><given-names>C</given-names></name><name><surname>Manzano</surname><given-names>C</given-names></name><name><surname>Wyss</surname><given-names>C</given-names></name><name><surname>Cheung</surname><given-names>E</given-names></name><name><surname>Clark</surname><given-names>TG</given-names></name><name><surname>Brown</surname><given-names>JB</given-names></name><name><surname>Ganesh</surname><given-names>M</given-names></name><name><surname>Patel</surname><given-names>S</given-names></name><name><surname>Tammana</surname><given-names>H</given-names></name><name><surname>Chrast</surname><given-names>J</given-names></name><name><surname>Henrichsen</surname><given-names>CN</given-names></name><name><surname>Kai</surname><given-names>C</given-names></name><name><surname>Kawai</surname><given-names>J</given-names></name><name><surname>Nagalakshmi</surname><given-names>U</given-names></name><name><surname>Wu</surname><given-names>J</given-names></name><name><surname>Lian</surname><given-names>Z</given-names></name><name><surname>Lian</surname><given-names>J</given-names></name><name><surname>Newburger</surname><given-names>P</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Bickel</surname><given-names>P</given-names></name><name><surname>Mattick</surname><given-names>JS</given-names></name><name><surname>Carninci</surname><given-names>P</given-names></name><name><surname>Hayashizaki</surname><given-names>Y</given-names></name><name><surname>Weissman</surname><given-names>S</given-names></name><name><surname>Hubbard</surname><given-names>T</given-names></name><name><surname>Myers</surname><given-names>RM</given-names></name><name><surname>Rogers</surname><given-names>J</given-names></name><name><surname>Stadler</surname><given-names>PF</given-names></name><name><surname>Lowe</surname><given-names>TM</given-names></name><name><surname>Wei</surname><given-names>CL</given-names></name><name><surname>Ruan</surname><given-names>Y</given-names></name><name><surname>Struhl</surname><given-names>K</given-names></name><name><surname>Gerstein</surname><given-names>M</given-names></name><name><surname>Antonarakis</surname><given-names>SE</given-names></name><name><surname>Fu</surname><given-names>Y</given-names></name><name><surname>Green</surname><given-names>ED</given-names></name><name><surname>Karaöz</surname><given-names>U</given-names></name><name><surname>Siepel</surname><given-names>A</given-names></name><name><surname>Taylor</surname><given-names>J</given-names></name><name><surname>Liefer</surname><given-names>LA</given-names></name><name><surname>Wetterstrand</surname><given-names>KA</given-names></name><name><surname>Good</surname><given-names>PJ</given-names></name><name><surname>Feingold</surname><given-names>EA</given-names></name><name><surname>Guyer</surname><given-names>MS</given-names></name><name><surname>Cooper</surname><given-names>GM</given-names></name><name><surname>Asimenos</surname><given-names>G</given-names></name><name><surname>Dewey</surname><given-names>CN</given-names></name><name><surname>Hou</surname><given-names>M</given-names></name><name><surname>Nikolaev</surname><given-names>S</given-names></name><name><surname>Montoya-Burgos</surname><given-names>JI</given-names></name><name><surname>Löytynoja</surname><given-names>A</given-names></name><name><surname>Whelan</surname><given-names>S</given-names></name><name><surname>Pardi</surname><given-names>F</given-names></name><name><surname>Massingham</surname><given-names>T</given-names></name><name><surname>Huang</surname><given-names>H</given-names></name><name><surname>Zhang</surname><given-names>NR</given-names></name><name><surname>Holmes</surname><given-names>I</given-names></name><name><surname>Mullikin</surname><given-names>JC</given-names></name><name><surname>Ureta-Vidal</surname><given-names>A</given-names></name><name><surname>Paten</surname><given-names>B</given-names></name><name><surname>Seringhaus</surname><given-names>M</given-names></name><name><surname>Church</surname><given-names>D</given-names></name><name><surname>Rosenbloom</surname><given-names>K</given-names></name><name><surname>Kent</surname><given-names>WJ</given-names></name><name><surname>Stone</surname><given-names>EA</given-names></name><name><surname>Batzoglou</surname><given-names>S</given-names></name><name><surname>Goldman</surname><given-names>N</given-names></name><name><surname>Hardison</surname><given-names>RC</given-names></name><name><surname>Haussler</surname><given-names>D</given-names></name><name><surname>Miller</surname><given-names>W</given-names></name><name><surname>Sidow</surname><given-names>A</given-names></name><name><surname>Trinklein</surname><given-names>ND</given-names></name><name><surname>Zhang</surname><given-names>ZD</given-names></name><name><surname>Barrera</surname><given-names>L</given-names></name><name><surname>Stuart</surname><given-names>R</given-names></name><name><surname>King</surname><given-names>DC</given-names></name><name><surname>Ameur</surname><given-names>A</given-names></name><name><surname>Enroth</surname><given-names>S</given-names></name><name><surname>Bieda</surname><given-names>MC</given-names></name><name><surname>Kim</surname><given-names>J</given-names></name><name><surname>Bhinge</surname><given-names>AA</given-names></name><name><surname>Jiang</surname><given-names>N</given-names></name><name><surname>Liu</surname><given-names>J</given-names></name><name><surname>Yao</surname><given-names>F</given-names></name><name><surname>Vega</surname><given-names>VB</given-names></name><name><surname>Lee</surname><given-names>CW</given-names></name><name><surname>Ng</surname><given-names>P</given-names></name><name><surname>Shahab</surname><given-names>A</given-names></name><name><surname>Yang</surname><given-names>A</given-names></name><name><surname>Moqtaderi</surname><given-names>Z</given-names></name><name><surname>Zhu</surname><given-names>Z</given-names></name><name><surname>Xu</surname><given-names>X</given-names></name><name><surname>Squazzo</surname><given-names>S</given-names></name><name><surname>Oberley</surname><given-names>MJ</given-names></name><name><surname>Inman</surname><given-names>D</given-names></name><name><surname>Singer</surname><given-names>MA</given-names></name><name><surname>Richmond</surname><given-names>TA</given-names></name><name><surname>Munn</surname><given-names>KJ</given-names></name><name><surname>Rada-Iglesias</surname><given-names>A</given-names></name><name><surname>Wallerman</surname><given-names>O</given-names></name><name><surname>Komorowski</surname><given-names>J</given-names></name><name><surname>Fowler</surname><given-names>JC</given-names></name><name><surname>Couttet</surname><given-names>P</given-names></name><name><surname>Bruce</surname><given-names>AW</given-names></name><name><surname>Dovey</surname><given-names>OM</given-names></name><name><surname>Ellis</surname><given-names>PD</given-names></name><name><surname>Langford</surname><given-names>CF</given-names></name><name><surname>Nix</surname><given-names>DA</given-names></name><name><surname>Euskirchen</surname><given-names>G</given-names></name><name><surname>Hartman</surname><given-names>S</given-names></name><name><surname>Urban</surname><given-names>AE</given-names></name><name><surname>Kraus</surname><given-names>P</given-names></name><name><surname>Van Calcar</surname><given-names>S</given-names></name><name><surname>Heintzman</surname><given-names>N</given-names></name><name><surname>Kim</surname><given-names>TH</given-names></name><name><surname>Wang</surname><given-names>K</given-names></name><name><surname>Qu</surname><given-names>C</given-names></name><name><surname>Hon</surname><given-names>G</given-names></name><name><surname>Luna</surname><given-names>R</given-names></name><name><surname>Glass</surname><given-names>CK</given-names></name><name><surname>Rosenfeld</surname><given-names>MG</given-names></name><name><surname>Aldred</surname><given-names>SF</given-names></name><name><surname>Cooper</surname><given-names>SJ</given-names></name><name><surname>Halees</surname><given-names>A</given-names></name><name><surname>Lin</surname><given-names>JM</given-names></name><name><surname>Shulha</surname><given-names>HP</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Xu</surname><given-names>M</given-names></name><name><surname>Haidar</surname><given-names>JN</given-names></name><name><surname>Yu</surname><given-names>Y</given-names></name><name><surname>Ruan</surname><given-names>Y</given-names></name><name><surname>Iyer</surname><given-names>VR</given-names></name><name><surname>Green</surname><given-names>RD</given-names></name><name><surname>Wadelius</surname><given-names>C</given-names></name><name><surname>Farnham</surname><given-names>PJ</given-names></name><name><surname>Ren</surname><given-names>B</given-names></name><name><surname>Harte</surname><given-names>RA</given-names></name><name><surname>Hinrichs</surname><given-names>AS</given-names></name><name><surname>Trumbower</surname><given-names>H</given-names></name><name><surname>Clawson</surname><given-names>H</given-names></name><name><surname>Hillman-Jackson</surname><given-names>J</given-names></name><name><surname>Zweig</surname><given-names>AS</given-names></name><name><surname>Smith</surname><given-names>K</given-names></name><name><surname>Thakkapallayil</surname><given-names>A</given-names></name><name><surname>Barber</surname><given-names>G</given-names></name><name><surname>Kuhn</surname><given-names>RM</given-names></name><name><surname>Karolchik</surname><given-names>D</given-names></name><name><surname>Armengol</surname><given-names>L</given-names></name><name><surname>Bird</surname><given-names>CP</given-names></name><name><surname>de Bakker</surname><given-names>PI</given-names></name><name><surname>Kern</surname><given-names>AD</given-names></name><name><surname>Lopez-Bigas</surname><given-names>N</given-names></name><name><surname>Martin</surname><given-names>JD</given-names></name><name><surname>Stranger</surname><given-names>BE</given-names></name><name><surname>Woodroffe</surname><given-names>A</given-names></name><name><surname>Davydov</surname><given-names>E</given-names></name><name><surname>Dimas</surname><given-names>A</given-names></name><name><surname>Eyras</surname><given-names>E</given-names></name><name><surname>Hallgrímsdóttir</surname><given-names>IB</given-names></name><name><surname>Huppert</surname><given-names>J</given-names></name><name><surname>Zody</surname><given-names>MC</given-names></name><name><surname>Abecasis</surname><given-names>GR</given-names></name><name><surname>Estivill</surname><given-names>X</given-names></name><name><surname>Bouffard</surname><given-names>GG</given-names></name><name><surname>Guan</surname><given-names>X</given-names></name><name><surname>Hansen</surname><given-names>NF</given-names></name><name><surname>Idol</surname><given-names>JR</given-names></name><name><surname>Maduro</surname><given-names>VV</given-names></name><name><surname>Maskeri</surname><given-names>B</given-names></name><name><surname>McDowell</surname><given-names>JC</given-names></name><name><surname>Park</surname><given-names>M</given-names></name><name><surname>Thomas</surname><given-names>PJ</given-names></name><name><surname>Young</surname><given-names>AC</given-names></name><name><surname>Blakesley</surname><given-names>RW</given-names></name><name><surname>Muzny</surname><given-names>DM</given-names></name><name><surname>Sodergren</surname><given-names>E</given-names></name><name><surname>Wheeler</surname><given-names>DA</given-names></name><name><surname>Worley</surname><given-names>KC</given-names></name><name><surname>Jiang</surname><given-names>H</given-names></name><name><surname>Weinstock</surname><given-names>GM</given-names></name><name><surname>Gibbs</surname><given-names>RA</given-names></name><name><surname>Graves</surname><given-names>T</given-names></name><name><surname>Fulton</surname><given-names>R</given-names></name><name><surname>Mardis</surname><given-names>ER</given-names></name><name><surname>Wilson</surname><given-names>RK</given-names></name><name><surname>Clamp</surname><given-names>M</given-names></name><name><surname>Cuff</surname><given-names>J</given-names></name><name><surname>Gnerre</surname><given-names>S</given-names></name><name><surname>Jaffe</surname><given-names>DB</given-names></name><name><surname>Chang</surname><given-names>JL</given-names></name><name><surname>Lindblad-Toh</surname><given-names>K</given-names></name><name><surname>Lander</surname><given-names>ES</given-names></name><name><surname>Koriabine</surname><given-names>M</given-names></name><name><surname>Nefedov</surname><given-names>M</given-names></name><name><surname>Osoegawa</surname><given-names>K</given-names></name><name><surname>Yoshinaga</surname><given-names>Y</given-names></name><name><surname>Zhu</surname><given-names>B</given-names></name><name><surname>de Jong</surname><given-names>PJ</given-names></name></person-group><year>2007</year><article-title>Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project</article-title><source>Nature</source><volume>447</volume><fpage>799</fpage><lpage>816</lpage><pub-id pub-id-type="doi">10.1038/nature05874</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Borsani</surname><given-names>O</given-names></name><name><surname>Zhu</surname><given-names>J</given-names></name><name><surname>Verslues</surname><given-names>PE</given-names></name><name><surname>Sunkar</surname><given-names>R</given-names></name><name><surname>Zhu</surname><given-names>JK</given-names></name></person-group><year>2005</year><article-title>Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis</article-title><source>Cell</source><volume>123</volume><fpage>1279</fpage><lpage>1291</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2005.11.035</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Buhler</surname><given-names>M</given-names></name><name><surname>Spies</surname><given-names>N</given-names></name><name><surname>Bartel</surname><given-names>DP</given-names></name><name><surname>Moazed</surname><given-names>D</given-names></name></person-group><year>2008</year><article-title>TRAMP-mediated RNA surveillance prevents spurious entry of RNAs into the Schizosaccharomyces pombe siRNA pathway</article-title><source>Nature Structure & Molecular Biology</source><volume>15</volume><fpage>1015</fpage><lpage>1023</lpage><pub-id pub-id-type="doi">10.1038/nsmb.1481</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cam</surname><given-names>HP</given-names></name><name><surname>Noma</surname><given-names>K</given-names></name><name><surname>Ebina</surname><given-names>H</given-names></name><name><surname>Levin</surname><given-names>HL</given-names></name><name><surname>Grewal</surname><given-names>SI</given-names></name></person-group><year>2008</year><article-title>Host genome surveillance for retrotransposons by transposon-derived proteins</article-title><source>Nature</source><volume>451</volume><fpage>431</fpage><lpage>436</lpage><pub-id pub-id-type="doi">10.1038/nature06499</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Camblong</surname><given-names>J</given-names></name><name><surname>Iglesias</surname><given-names>N</given-names></name><name><surname>Fickentscher</surname><given-names>C</given-names></name><name><surname>Dieppois</surname><given-names>G</given-names></name><name><surname>Stutz</surname><given-names>F</given-names></name></person-group><year>2007</year><article-title>Antisense RNA stabilization induces transcriptional gene silencing via histone deacetylation in <italic>S. cerevisiae</italic></article-title><source>Cell</source><volume>131</volume><fpage>706</fpage><lpage>717</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2007.09.014</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Carlile</surname><given-names>M</given-names></name><name><surname>Swan</surname><given-names>D</given-names></name><name><surname>Jackson</surname><given-names>K</given-names></name><name><surname>Preston-Fayers</surname><given-names>K</given-names></name><name><surname>Ballester</surname><given-names>B</given-names></name><name><surname>Flicek</surname><given-names>P</given-names></name><name><surname>Werner</surname><given-names>A</given-names></name></person-group><year>2009</year><article-title>Strand selective generation of endo-siRNAs from the Na/phosphate transporter gene Slc34a1 in murine tissues</article-title><source>Nucleic Acids Research</source><volume>37</volume><fpage>2274</fpage><lpage>2282</lpage><pub-id pub-id-type="doi">10.1093/nar/gkp088</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Carninci</surname><given-names>P</given-names></name><name><surname>Kasukawa</surname><given-names>T</given-names></name><name><surname>Katayama</surname><given-names>S</given-names></name><name><surname>Gough</surname><given-names>J</given-names></name><name><surname>Frith</surname><given-names>MC</given-names></name><name><surname>Maeda</surname><given-names>N</given-names></name><name><surname>Oyama</surname><given-names>R</given-names></name><name><surname>Ravasi</surname><given-names>T</given-names></name><name><surname>Lenhard</surname><given-names>B</given-names></name><name><surname>Wells</surname><given-names>C</given-names></name><name><surname>Kodzius</surname><given-names>R</given-names></name><name><surname>Shimokawa</surname><given-names>K</given-names></name><name><surname>Bajic</surname><given-names>VB</given-names></name><name><surname>Brenner</surname><given-names>SE</given-names></name><name><surname>Batalov</surname><given-names>S</given-names></name><name><surname>Forrest</surname><given-names>AR</given-names></name><name><surname>Zavolan</surname><given-names>M</given-names></name><name><surname>Davis</surname><given-names>MJ</given-names></name><name><surname>Wilming</surname><given-names>LG</given-names></name><name><surname>Aidinis</surname><given-names>V</given-names></name><name><surname>Allen</surname><given-names>JE</given-names></name><name><surname>Ambesi-Impiombato</surname><given-names>A</given-names></name><name><surname>Apweiler</surname><given-names>R</given-names></name><name><surname>Aturaliya</surname><given-names>RN</given-names></name><name><surname>Bailey</surname><given-names>TL</given-names></name><name><surname>Bansal</surname><given-names>M</given-names></name><name><surname>Baxter</surname><given-names>L</given-names></name><name><surname>Beisel</surname><given-names>KW</given-names></name><name><surname>Bersano</surname><given-names>T</given-names></name><name><surname>Bono</surname><given-names>H</given-names></name><name><surname>Chalk</surname><given-names>AM</given-names></name><name><surname>Chiu</surname><given-names>KP</given-names></name><name><surname>Choudhary</surname><given-names>V</given-names></name><name><surname>Christoffels</surname><given-names>A</given-names></name><name><surname>Clutterbuck</surname><given-names>DR</given-names></name><name><surname>Crowe</surname><given-names>ML</given-names></name><name><surname>Dalla</surname><given-names>E</given-names></name><name><surname>Dalrymple</surname><given-names>BP</given-names></name><name><surname>de Bono</surname><given-names>B</given-names></name><name><surname>Della Gatta</surname><given-names>G</given-names></name><name><surname>di Bernardo</surname><given-names>D</given-names></name><name><surname>Down</surname><given-names>T</given-names></name><name><surname>Engstrom</surname><given-names>P</given-names></name><name><surname>Fagiolini</surname><given-names>M</given-names></name><name><surname>Faulkner</surname><given-names>G</given-names></name><name><surname>Fletcher</surname><given-names>CF</given-names></name><name><surname>Fukushima</surname><given-names>T</given-names></name><name><surname>Furuno</surname><given-names>M</given-names></name><name><surname>Futaki</surname><given-names>S</given-names></name><name><surname>Gariboldi</surname><given-names>M</given-names></name><name><surname>Georgii-Hemming</surname><given-names>P</given-names></name><name><surname>Gingeras</surname><given-names>TR</given-names></name><name><surname>Gojobori</surname><given-names>T</given-names></name><name><surname>Green</surname><given-names>RE</given-names></name><name><surname>Gustincich</surname><given-names>S</given-names></name><name><surname>Harbers</surname><given-names>M</given-names></name><name><surname>Hayashi</surname><given-names>Y</given-names></name><name><surname>Hensch</surname><given-names>TK</given-names></name><name><surname>Hirokawa</surname><given-names>N</given-names></name><name><surname>Hill</surname><given-names>D</given-names></name><name><surname>Huminiecki</surname><given-names>L</given-names></name><name><surname>Iacono</surname><given-names>M</given-names></name><name><surname>Ikeo</surname><given-names>K</given-names></name><name><surname>Iwama</surname><given-names>A</given-names></name><name><surname>Ishikawa</surname><given-names>T</given-names></name><name><surname>Jakt</surname><given-names>M</given-names></name><name><surname>Kanapin</surname><given-names>A</given-names></name><name><surname>Katoh</surname><given-names>M</given-names></name><name><surname>Kawasawa</surname><given-names>Y</given-names></name><name><surname>Kelso</surname><given-names>J</given-names></name><name><surname>Kitamura</surname><given-names>H</given-names></name><name><surname>Kitano</surname><given-names>H</given-names></name><name><surname>Kollias</surname><given-names>G</given-names></name><name><surname>Krishnan</surname><given-names>SP</given-names></name><name><surname>Kruger</surname><given-names>A</given-names></name><name><surname>Kummerfeld</surname><given-names>SK</given-names></name><name><surname>Kurochkin</surname><given-names>IV</given-names></name><name><surname>Lareau</surname><given-names>LF</given-names></name><name><surname>Lazarevic</surname><given-names>D</given-names></name><name><surname>Lipovich</surname><given-names>L</given-names></name><name><surname>Liu</surname><given-names>J</given-names></name><name><surname>Liuni</surname><given-names>S</given-names></name><name><surname>McWilliam</surname><given-names>S</given-names></name><name><surname>Madan Babu</surname><given-names>M</given-names></name><name><surname>Madera</surname><given-names>M</given-names></name><name><surname>Marchionni</surname><given-names>L</given-names></name><name><surname>Matsuda</surname><given-names>H</given-names></name><name><surname>Matsuzawa</surname><given-names>S</given-names></name><name><surname>Miki</surname><given-names>H</given-names></name><name><surname>Mignone</surname><given-names>F</given-names></name><name><surname>Miyake</surname><given-names>S</given-names></name><name><surname>Morris</surname><given-names>K</given-names></name><name><surname>Mottagui-Tabar</surname><given-names>S</given-names></name><name><surname>Mulder</surname><given-names>N</given-names></name><name><surname>Nakano</surname><given-names>N</given-names></name><name><surname>Nakauchi</surname><given-names>H</given-names></name><name><surname>Ng</surname><given-names>P</given-names></name><name><surname>Nilsson</surname><given-names>R</given-names></name><name><surname>Nishiguchi</surname><given-names>S</given-names></name><name><surname>Nishikawa</surname><given-names>S</given-names></name><name><surname>Nori</surname><given-names>F</given-names></name><name><surname>Ohara</surname><given-names>O</given-names></name><name><surname>Okazaki</surname><given-names>Y</given-names></name><name><surname>Orlando</surname><given-names>V</given-names></name><name><surname>Pang</surname><given-names>KC</given-names></name><name><surname>Pavan</surname><given-names>WJ</given-names></name><name><surname>Pavesi</surname><given-names>G</given-names></name><name><surname>Pesole</surname><given-names>G</given-names></name><name><surname>Petrovsky</surname><given-names>N</given-names></name><name><surname>Piazza</surname><given-names>S</given-names></name><name><surname>Reed</surname><given-names>J</given-names></name><name><surname>Reid</surname><given-names>JF</given-names></name><name><surname>Ring</surname><given-names>BZ</given-names></name><name><surname>Ringwald</surname><given-names>M</given-names></name><name><surname>Rost</surname><given-names>B</given-names></name><name><surname>Ruan</surname><given-names>Y</given-names></name><name><surname>Salzberg</surname><given-names>SL</given-names></name><name><surname>Sandelin</surname><given-names>A</given-names></name><name><surname>Schneider</surname><given-names>C</given-names></name><name><surname>Schönbach</surname><given-names>C</given-names></name><name><surname>Sekiguchi</surname><given-names>K</given-names></name><name><surname>Semple</surname><given-names>CA</given-names></name><name><surname>Seno</surname><given-names>S</given-names></name><name><surname>Sessa</surname><given-names>L</given-names></name><name><surname>Sheng</surname><given-names>Y</given-names></name><name><surname>Shibata</surname><given-names>Y</given-names></name><name><surname>Shimada</surname><given-names>H</given-names></name><name><surname>Shimada</surname><given-names>K</given-names></name><name><surname>Silva</surname><given-names>D</given-names></name><name><surname>Sinclair</surname><given-names>B</given-names></name><name><surname>Sperling</surname><given-names>S</given-names></name><name><surname>Stupka</surname><given-names>E</given-names></name><name><surname>Sugiura</surname><given-names>K</given-names></name><name><surname>Sultana</surname><given-names>R</given-names></name><name><surname>Takenaka</surname><given-names>Y</given-names></name><name><surname>Taki</surname><given-names>K</given-names></name><name><surname>Tammoja</surname><given-names>K</given-names></name><name><surname>Tan</surname><given-names>SL</given-names></name><name><surname>Tang</surname><given-names>S</given-names></name><name><surname>Taylor</surname><given-names>MS</given-names></name><name><surname>Tegner</surname><given-names>J</given-names></name><name><surname>Teichmann</surname><given-names>SA</given-names></name><name><surname>Ueda</surname><given-names>HR</given-names></name><name><surname>van Nimwegen</surname><given-names>E</given-names></name><name><surname>Verardo</surname><given-names>R</given-names></name><name><surname>Wei</surname><given-names>CL</given-names></name><name><surname>Yagi</surname><given-names>K</given-names></name><name><surname>Yamanishi</surname><given-names>H</given-names></name><name><surname>Zabarovsky</surname><given-names>E</given-names></name><name><surname>Zhu</surname><given-names>S</given-names></name><name><surname>Zimmer</surname><given-names>A</given-names></name><name><surname>Hide</surname><given-names>W</given-names></name><name><surname>Bult</surname><given-names>C</given-names></name><name><surname>Grimmond</surname><given-names>SM</given-names></name><name><surname>Teasdale</surname><given-names>RD</given-names></name><name><surname>Liu</surname><given-names>ET</given-names></name><name><surname>Brusic</surname><given-names>V</given-names></name><name><surname>Quackenbush</surname><given-names>J</given-names></name><name><surname>Wahlestedt</surname><given-names>C</given-names></name><name><surname>Mattick</surname><given-names>JS</given-names></name><name><surname>Hume</surname><given-names>DA</given-names></name><name><surname>Kai</surname><given-names>C</given-names></name><name><surname>Sasaki</surname><given-names>D</given-names></name><name><surname>Tomaru</surname><given-names>Y</given-names></name><name><surname>Fukuda</surname><given-names>S</given-names></name><name><surname>Kanamori-Katayama</surname><given-names>M</given-names></name><name><surname>Suzuki</surname><given-names>M</given-names></name><name><surname>Aoki</surname><given-names>J</given-names></name><name><surname>Arakawa</surname><given-names>T</given-names></name><name><surname>Iida</surname><given-names>J</given-names></name><name><surname>Imamura</surname><given-names>K</given-names></name><name><surname>Itoh</surname><given-names>M</given-names></name><name><surname>Kato</surname><given-names>T</given-names></name><name><surname>Kawaji</surname><given-names>H</given-names></name><name><surname>Kawagashira</surname><given-names>N</given-names></name><name><surname>Kawashima</surname><given-names>T</given-names></name><name><surname>Kojima</surname><given-names>M</given-names></name><name><surname>Kondo</surname><given-names>S</given-names></name><name><surname>Konno</surname><given-names>H</given-names></name><name><surname>Nakano</surname><given-names>K</given-names></name><name><surname>Ninomiya</surname><given-names>N</given-names></name><name><surname>Nishio</surname><given-names>T</given-names></name><name><surname>Okada</surname><given-names>M</given-names></name><name><surname>Plessy</surname><given-names>C</given-names></name><name><surname>Shibata</surname><given-names>K</given-names></name><name><surname>Shiraki</surname><given-names>T</given-names></name><name><surname>Suzuki</surname><given-names>S</given-names></name><name><surname>Tagami</surname><given-names>M</given-names></name><name><surname>Waki</surname><given-names>K</given-names></name><name><surname>Watahiki</surname><given-names>A</given-names></name><name><surname>Okamura-Oho</surname><given-names>Y</given-names></name><name><surname>Suzuki</surname><given-names>H</given-names></name><name><surname>Kawai</surname><given-names>J</given-names></name><name><surname>Hayashizaki</surname><given-names>Y</given-names></name>, <collab>FANTOM Consortium</collab>, <collab>Genome Network Project Core Group</collab></person-group><year>2005</year><article-title>The transcriptional landscape of the mammalian genome</article-title><source>Science</source><volume>309</volume><fpage>1559</fpage><lpage>1563</lpage><pub-id pub-id-type="doi">10.1126/science.1112014</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cernilogar</surname><given-names>FM</given-names></name><name><surname>Onorati</surname><given-names>MC</given-names></name><name><surname>Kothe</surname><given-names>GO</given-names></name><name><surname>Burroughs</surname><given-names>AM</given-names></name><name><surname>Parsi</surname><given-names>KM</given-names></name><name><surname>Breiling</surname><given-names>A</given-names></name><name><surname>Lo Sardo</surname><given-names>F</given-names></name><name><surname>Saxena</surname><given-names>A</given-names></name><name><surname>Miyoshi</surname><given-names>K</given-names></name><name><surname>Siomi</surname><given-names>H</given-names></name><name><surname>Carninci</surname><given-names>P</given-names></name><name><surname>Gilmour</surname><given-names>DS</given-names></name><name><surname>Corona</surname><given-names>DF</given-names></name><name><surname>Orlando</surname><given-names>V</given-names></name></person-group><year>2011</year><article-title>Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila</article-title><source>Nature</source><volume>480</volume><fpage>391</fpage><lpage>395</lpage><pub-id pub-id-type="doi">10.1038/nature10492</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chekanova</surname><given-names>JA</given-names></name><name><surname>Gregory</surname><given-names>BD</given-names></name><name><surname>Reverdatto</surname><given-names>SV</given-names></name><name><surname>Chen</surname><given-names>H</given-names></name><name><surname>Kumar</surname><given-names>R</given-names></name><name><surname>Hooker</surname><given-names>T</given-names></name><name><surname>Yazaki</surname><given-names>J</given-names></name><name><surname>Li</surname><given-names>P</given-names></name><name><surname>Skiba</surname><given-names>N</given-names></name><name><surname>Peng</surname><given-names>Q</given-names></name><name><surname>Alonso</surname><given-names>J</given-names></name><name><surname>Brukhin</surname><given-names>V</given-names></name><name><surname>Grossniklaus</surname><given-names>U</given-names></name><name><surname>Ecker</surname><given-names>JR</given-names></name><name><surname>Belostotsky</surname><given-names>DA</given-names></name></person-group><year>2007</year><article-title>Genome-wide high-resolution mapping of exosome substrates reveals hidden features in the Arabidopsis transcriptome</article-title><source>Cell</source><volume>131</volume><fpage>1340</fpage><lpage>1353</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2007.10.056</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cheng</surname><given-names>J</given-names></name><name><surname>Kapranov</surname><given-names>P</given-names></name><name><surname>Drenkow</surname><given-names>J</given-names></name><name><surname>Dike</surname><given-names>S</given-names></name><name><surname>Brubaker</surname><given-names>S</given-names></name><name><surname>Patel</surname><given-names>S</given-names></name><name><surname>Long</surname><given-names>J</given-names></name><name><surname>Stern</surname><given-names>D</given-names></name><name><surname>Tammana</surname><given-names>H</given-names></name><name><surname>Helt</surname><given-names>G</given-names></name><name><surname>Sementchenko</surname><given-names>V</given-names></name><name><surname>Piccolboni</surname><given-names>A</given-names></name><name><surname>Bekiranov</surname><given-names>S</given-names></name><name><surname>Bailey</surname><given-names>DK</given-names></name><name><surname>Ganesh</surname><given-names>M</given-names></name><name><surname>Ghosh</surname><given-names>S</given-names></name><name><surname>Bell</surname><given-names>I</given-names></name><name><surname>Gerhard</surname><given-names>DS</given-names></name><name><surname>Gingeras</surname><given-names>TR</given-names></name></person-group><year>2005</year><article-title>Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution</article-title><source>Science</source><volume>308</volume><fpage>1149</fpage><lpage>1154</lpage><pub-id pub-id-type="doi">10.1126/science.1108625</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Clark</surname><given-names>MB</given-names></name><name><surname>Amaral</surname><given-names>PP</given-names></name><name><surname>Schlesinger</surname><given-names>FJ</given-names></name><name><surname>Dinger</surname><given-names>ME</given-names></name><name><surname>Taft</surname><given-names>RJ</given-names></name><name><surname>Rinn</surname><given-names>JL</given-names></name><name><surname>Ponting</surname><given-names>CP</given-names></name><name><surname>Stadler</surname><given-names>PF</given-names></name><name><surname>Morris</surname><given-names>KV</given-names></name><name><surname>Morillon</surname><given-names>A</given-names></name><name><surname>Rozowsky</surname><given-names>JS</given-names></name><name><surname>Gerstein</surname><given-names>MB</given-names></name><name><surname>Wahlestedt</surname><given-names>C</given-names></name><name><surname>Hayashizaki</surname><given-names>Y</given-names></name><name><surname>Carninci</surname><given-names>P</given-names></name><name><surname>Gingeras</surname><given-names>TR</given-names></name><name><surname>Mattick</surname><given-names>JS</given-names></name></person-group><year>2011</year><article-title>The reality of pervasive transcription</article-title><source>PLOS Biology</source><volume>9</volume><fpage>e1000625</fpage><pub-id pub-id-type="doi">10.1371/journal.pbio.1000625</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>David</surname><given-names>L</given-names></name><name><surname>Huber</surname><given-names>W</given-names></name><name><surname>Granovskaia</surname><given-names>M</given-names></name><name><surname>Toedling</surname><given-names>J</given-names></name><name><surname>Palm</surname><given-names>CJ</given-names></name><name><surname>Bofkin</surname><given-names>L</given-names></name><name><surname>Jones</surname><given-names>T</given-names></name><name><surname>Davis</surname><given-names>RW</given-names></name><name><surname>Steinmetz</surname><given-names>LM</given-names></name></person-group><year>2006</year><article-title>A high-resolution map of transcription in the yeast genome</article-title><source>Proceedings of the National Academy of Sciences of the United States of America</source><volume>103</volume><fpage>5320</fpage><lpage>5325</lpage><pub-id pub-id-type="doi">10.1073/pnas.0601091103</pub-id></element-citation></ref><ref id="bib90"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Derrien</surname><given-names>T</given-names></name><name><surname>Johnson</surname><given-names>R</given-names></name><name><surname>Bussotti</surname><given-names>G</given-names></name><name><surname>Tanzer</surname><given-names>A</given-names></name><name><surname>Djebali</surname><given-names>S</given-names></name><name><surname>Tilgner</surname><given-names>H</given-names></name><name><surname>Guernec</surname><given-names>G</given-names></name><name><surname>Martin</surname><given-names>D</given-names></name><name><surname>Merkel</surname><given-names>A</given-names></name><name><surname>Knowles</surname><given-names>DG</given-names></name><name><surname>Lagarde</surname><given-names>J</given-names></name><name><surname>Veeravalli</surname><given-names>L</given-names></name><name><surname>Ruan</surname><given-names>X</given-names></name><name><surname>Ruan</surname><given-names>Y</given-names></name><name><surname>Lassmann</surname><given-names>T</given-names></name><name><surname>Carninci</surname><given-names>P</given-names></name><name><surname>Brown</surname><given-names>JB</given-names></name><name><surname>Lipovich</surname><given-names>L</given-names></name><name><surname>Gonzalez</surname><given-names>JM</given-names></name><name><surname>Thomas</surname><given-names>M</given-names></name><name><surname>Davis</surname><given-names>CA</given-names></name><name><surname>Shiekhattar</surname><given-names>R</given-names></name><name><surname>Gingeras</surname><given-names>TR</given-names></name><name><surname>Hubbard</surname><given-names>TJ</given-names></name><name><surname>Notredame</surname><given-names>C</given-names></name><name><surname>Harrow</surname><given-names>J</given-names></name><name><surname>Guigò</surname><given-names>R</given-names></name></person-group><year>2012</year><article-title>The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression</article-title><source>Genome Research</source><volume>22</volume><fpage>1775</fpage><lpage>1789</lpage><pub-id pub-id-type="doi">10.1101/gr.132159.111</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ding</surname><given-names>SW</given-names></name></person-group><year>2010</year><article-title>RNA-based antiviral immunity</article-title><source>Nature Reviews Immunology</source><volume>10</volume><fpage>632</fpage><lpage>644</lpage><pub-id pub-id-type="doi">10.1038/nri2824</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Doyle</surname><given-names>M</given-names></name><name><surname>Badertscher</surname><given-names>L</given-names></name><name><surname>Jaskiewicz</surname><given-names>L</given-names></name><name><surname>Guttinger</surname><given-names>S</given-names></name><name><surname>Jurado</surname><given-names>S</given-names></name><name><surname>Hugenschmidt</surname><given-names>T</given-names></name><name><surname>Kutay</surname><given-names>U</given-names></name><name><surname>Filipowicz</surname><given-names>W</given-names></name></person-group><year>2013</year><article-title>The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal</article-title><source>RNA</source><volume>19</volume><fpage>1238</fpage><lpage>1252</lpage><pub-id pub-id-type="doi">10.1261/rna.039255.113</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Drinnenberg</surname><given-names>IA</given-names></name><name><surname>Fink</surname><given-names>GR</given-names></name><name><surname>Bartel</surname><given-names>DP</given-names></name></person-group><year>2011</year><article-title>Compatibility with killer explains the rise of RNAi-deficient fungi</article-title><source>Science</source><volume>333</volume><fpage>1592</fpage><pub-id pub-id-type="doi">10.1126/science.1209575</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Drinnenberg</surname><given-names>IA</given-names></name><name><surname>Weinberg</surname><given-names>DE</given-names></name><name><surname>Xie</surname><given-names>KT</given-names></name><name><surname>Mower</surname><given-names>JP</given-names></name><name><surname>Wolfe</surname><given-names>KH</given-names></name><name><surname>Fink</surname><given-names>GR</given-names></name><name><surname>Bartel</surname><given-names>DP</given-names></name></person-group><year>2009</year><article-title>RNAi in budding yeast</article-title><source>Science</source><volume>326</volume><fpage>544</fpage><lpage>550</lpage><pub-id pub-id-type="doi">10.1126/science.1176945</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Faghihi</surname><given-names>MA</given-names></name><name><surname>Wahlestedt</surname><given-names>C</given-names></name></person-group><year>2006</year><article-title>RNA interference is not involved in natural antisense mediated regulation of gene expression in mammals</article-title><source>Genome Biology</source><volume>7</volume><fpage>R38</fpage><pub-id pub-id-type="doi">10.1186/gb-2006-7-5-r38</pub-id></element-citation></ref><ref id="bib91"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fire</surname><given-names>A</given-names></name><name><surname>Xu</surname><given-names>S</given-names></name><name><surname>Montgomery</surname><given-names>MK</given-names></name><name><surname>Kostas</surname><given-names>SA</given-names></name><name><surname>Driver</surname><given-names>SE</given-names></name><name><surname>Mello</surname><given-names>CC</given-names></name></person-group><year>1998</year><article-title>Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans</article-title><source>Nature</source><volume>391</volume><fpage>806</fpage><lpage>811</lpage><pub-id pub-id-type="doi">10.1038/35888</pub-id></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gantier</surname><given-names>MP</given-names></name><name><surname>Williams</surname><given-names>BR</given-names></name></person-group><year>2007</year><article-title>The response of mammalian cells to double-stranded RNA</article-title><source>Cytokine & Growth Factor Reviews</source><volume>18</volume><fpage>363</fpage><lpage>371</lpage><pub-id pub-id-type="doi">10.1016/j.cytogfr.2007.06.016</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Geisler</surname><given-names>S</given-names></name><name><surname>Lojek</surname><given-names>L</given-names></name><name><surname>Khalil</surname><given-names>AM</given-names></name><name><surname>Baker</surname><given-names>KE</given-names></name><name><surname>Coller</surname><given-names>J</given-names></name></person-group><year>2012</year><article-title>Decapping of long noncoding RNAs regulates inducible genes</article-title><source>Molecular Cell</source><volume>45</volume><fpage>279</fpage><lpage>291</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2011.11.025</pub-id></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gelfand</surname><given-names>B</given-names></name><name><surname>Mead</surname><given-names>J</given-names></name><name><surname>Bruning</surname><given-names>A</given-names></name><name><surname>Apostolopoulos</surname><given-names>N</given-names></name><name><surname>Tadigotla</surname><given-names>V</given-names></name><name><surname>Nagaraj</surname><given-names>V</given-names></name><name><surname>Sengupta</surname><given-names>AM</given-names></name><name><surname>Vershon</surname><given-names>AK</given-names></name></person-group><year>2011</year><article-title>Regulated antisense transcription controls expression of cell-type specific genes in yeast</article-title><source>Molecular Cell Biology</source><volume>31</volume><fpage>1701</fpage><lpage>1709</lpage><pub-id pub-id-type="doi">10.1128/MCB.01071-10</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gerasimova</surname><given-names>TI</given-names></name><name><surname>Byrd</surname><given-names>K</given-names></name><name><surname>Corces</surname><given-names>VG</given-names></name></person-group><year>2000</year><article-title>A chromatin insulator determines the nuclear localization of DNA</article-title><source>Molecular Cell</source><volume>6</volume><fpage>1025</fpage><lpage>1035</lpage><pub-id pub-id-type="doi">10.1016/S1097-2765(00)00101-5</pub-id></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Goodman</surname><given-names>AJ</given-names></name><name><surname>Daugharthy</surname><given-names>ER</given-names></name><name><surname>Kim</surname><given-names>J</given-names></name></person-group><year>2012</year><article-title>Pervasive antisense transcription is evolutionarily conserved in budding yeast</article-title><source>Molecular Biology and Evolution</source><volume>30</volume><fpage>409</fpage><lpage>421</lpage><pub-id pub-id-type="doi">10.1093/molbev/mss240</pub-id></element-citation></ref><ref id="bib92"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gotta</surname><given-names>M</given-names></name><name><surname>Laroche</surname><given-names>T</given-names></name><name><surname>Formenton</surname><given-names>A</given-names></name><name><surname>Maillet</surname><given-names>SA</given-names></name><name><surname>Driver</surname><given-names>L</given-names></name><name><surname>Scherthan</surname><given-names>H</given-names></name><name><surname>Gasser</surname><given-names>SM</given-names></name></person-group><year>1996</year><article-title>The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae</article-title><source>Journal of Cell Biology</source><volume>134</volume><fpage>1349</fpage><lpage>1363</lpage><pub-id pub-id-type="doi">10.1083/jcb.134.6.1349</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gullerova</surname><given-names>M</given-names></name><name><surname>Moazed</surname><given-names>D</given-names></name><name><surname>Proudfoot</surname><given-names>NJ</given-names></name></person-group><year>2011</year><article-title>Autoregulation of convergent RNAi genes in fission yeast</article-title><source>Genes Development</source><volume>25</volume><fpage>556</fpage><lpage>568</lpage><pub-id pub-id-type="doi">10.1101/gad.618611</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gullerova</surname><given-names>M</given-names></name><name><surname>Proudfoot</surname><given-names>NJ</given-names></name></person-group><year>2012</year><article-title>Convergent transcription induces transcriptional gene silencing in fission yeast and mammalian cells</article-title><source>Nature Structure & Molecular Biology</source><volume>19</volume><fpage>1193</fpage><lpage>1201</lpage><pub-id pub-id-type="doi">10.1038/nsmb.2392</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hainer</surname><given-names>SJ</given-names></name><name><surname>Pruneski</surname><given-names>JA</given-names></name><name><surname>Mitchell</surname><given-names>RD</given-names></name><name><surname>Monteverde</surname><given-names>RM</given-names></name><name><surname>Martens</surname><given-names>JA</given-names></name></person-group><year>2011</year><article-title>Intergenic transcription causes repression by directing nucleosome assembly</article-title><source>Genes Development</source><volume>25</volume><fpage>29</fpage><lpage>40</lpage><pub-id pub-id-type="doi">10.1101/gad.1975011</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hamilton</surname><given-names>AJ</given-names></name><name><surname>Baulcombe</surname><given-names>DC</given-names></name></person-group><year>1999</year><article-title>A species of small antisense RNA in posttranscriptional gene silencing in plants</article-title><source>Science</source><volume>286</volume><fpage>950</fpage><lpage>952</lpage><pub-id pub-id-type="doi">10.1126/science.286.5441.950</pub-id></element-citation></ref><ref id="bib93"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hannon</surname><given-names>GJ</given-names></name></person-group><year>2002</year><article-title>RNA interference</article-title><source>Nature</source><volume>418</volume><fpage>244</fpage><lpage>251</lpage><pub-id pub-id-type="doi">10.1038/418244a</pub-id></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hirota</surname><given-names>K</given-names></name><name><surname>Miyoshi</surname><given-names>T</given-names></name><name><surname>Kugou</surname><given-names>K</given-names></name><name><surname>Hoffman</surname><given-names>CS</given-names></name><name><surname>Shibata</surname><given-names>T</given-names></name><name><surname>Ohta</surname><given-names>K</given-names></name></person-group><year>2008</year><article-title>Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs</article-title><source>Nature</source><volume>456</volume><fpage>130</fpage><lpage>134</lpage><pub-id pub-id-type="doi">10.1038/nature07348</pub-id></element-citation></ref><ref id="bib94"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hobson</surname><given-names>DJ</given-names></name><name><surname>Wei</surname><given-names>W</given-names></name><name><surname>Steinmetz</surname><given-names>LM</given-names></name><name><surname>Svejstrup</surname><given-names>JQ</given-names></name></person-group><year>2012</year><article-title>RNA polymerase II collision interrupts convergent transcription</article-title><source>Molecular Cell</source><volume>48</volume><fpage>365</fpage><lpage>374</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2012.08.027</pub-id></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hogg</surname><given-names>M</given-names></name><name><surname>Paro</surname><given-names>S</given-names></name><name><surname>Keegan</surname><given-names>LP</given-names></name><name><surname>O’Connell</surname><given-names>MA</given-names></name></person-group><year>2011</year><article-title>RNA editing by mammalian ADARs</article-title><source>Advances in Genetics</source><volume>73</volume><fpage>87</fpage><lpage>120</lpage><pub-id pub-id-type="doi">10.1016/B978-0-12-380860-8.00003-3</pub-id></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hongay</surname><given-names>CF</given-names></name><name><surname>Grisafi</surname><given-names>PL</given-names></name><name><surname>Galitski</surname><given-names>T</given-names></name><name><surname>Fink</surname><given-names>GR</given-names></name></person-group><year>2006</year><article-title>Antisense transcription controls cell fate in <italic>Saccharomyces cerevisiae</italic></article-title><source>Cell</source><volume>127</volume><fpage>735</fpage><lpage>745</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2006.09.038</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Houseley</surname><given-names>J</given-names></name></person-group><year>2012</year><article-title>Form and function of eukaryotic unstable non-coding RNAs</article-title><source>Biochemical Society Transactions</source><volume>40</volume><fpage>836</fpage><lpage>841</lpage><pub-id pub-id-type="doi">10.1042/BST20120040</pub-id></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Houseley</surname><given-names>J</given-names></name><name><surname>Kotovic</surname><given-names>K</given-names></name><name><surname>El Hage</surname><given-names>A</given-names></name><name><surname>Tollervey</surname><given-names>D</given-names></name></person-group><year>2007</year><article-title>Trf4 targets ncRNAs from telomeric and rDNA spacer regions and functions in rDNA copy number control</article-title><source>The EMBO Journal</source><volume>26</volume><fpage>4996</fpage><lpage>5006</lpage><pub-id pub-id-type="doi">10.1038/sj.emboj.7601921</pub-id></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Houseley</surname><given-names>J</given-names></name><name><surname>Rubbi</surname><given-names>L</given-names></name><name><surname>Grunstein</surname><given-names>M</given-names></name><name><surname>Tollervey</surname><given-names>D</given-names></name><name><surname>Vogelauer</surname><given-names>M</given-names></name></person-group><year>2008</year><article-title>A ncRNA modulates histone modification and mRNA induction in the yeast GAL gene cluster</article-title><source>Molecular Cell</source><volume>32</volume><fpage>685</fpage><lpage>695</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2008.09.027</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Houseley</surname><given-names>J</given-names></name><name><surname>Tollervey</surname><given-names>D</given-names></name></person-group><year>2009</year><article-title>The many pathways of RNA degradation</article-title><source>Cell</source><volume>136</volume><fpage>763</fpage><lpage>776</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2009.01.019</pub-id></element-citation></ref><ref id="bib95"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hsieh</surname><given-names>J</given-names></name><name><surname>Fire</surname><given-names>A</given-names></name></person-group><year>2000</year><article-title>Recognition and silencing of repeated DNA</article-title><source>Annual Review of Genetics</source><volume>34</volume><fpage>187</fpage><lpage>204</lpage><pub-id pub-id-type="doi">10.1146/annurev.genet.34.1.187</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ishizu</surname><given-names>H</given-names></name><name><surname>Siomi</surname><given-names>H</given-names></name><name><surname>Siomi</surname><given-names>MC</given-names></name></person-group><year>2012</year><article-title>Biology of PIWI-interacting RNAs: new insights into biogenesis and function inside and outside of germlines</article-title><source>Genes Development</source><volume>26</volume><fpage>2361</fpage><lpage>2373</lpage><pub-id pub-id-type="doi">10.1101/gad.203786.112</pub-id></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kapranov</surname><given-names>P</given-names></name><name><surname>Cheng</surname><given-names>J</given-names></name><name><surname>Dike</surname><given-names>S</given-names></name><name><surname>Nix</surname><given-names>DA</given-names></name><name><surname>Duttagupta</surname><given-names>R</given-names></name><name><surname>Willingham</surname><given-names>AT</given-names></name><name><surname>Stadler</surname><given-names>PF</given-names></name><name><surname>Hertel</surname><given-names>J</given-names></name><name><surname>Hackermuller</surname><given-names>J</given-names></name><name><surname>Hofacker</surname><given-names>IL</given-names></name><name><surname>Bell</surname><given-names>I</given-names></name><name><surname>Cheung</surname><given-names>E</given-names></name><name><surname>Drenkow</surname><given-names>J</given-names></name><name><surname>Dumais</surname><given-names>E</given-names></name><name><surname>Patel</surname><given-names>S</given-names></name><name><surname>Helt</surname><given-names>G</given-names></name><name><surname>Ganesh</surname><given-names>M</given-names></name><name><surname>Ghosh</surname><given-names>S</given-names></name><name><surname>Piccolboni</surname><given-names>A</given-names></name><name><surname>Sementchenko</surname><given-names>V</given-names></name><name><surname>Tammana</surname><given-names>H</given-names></name><name><surname>Gingeras</surname><given-names>TR</given-names></name></person-group><year>2007</year><article-title>RNA maps reveal new RNA classes and a possible function for pervasive transcription</article-title><source>Science</source><volume>316</volume><fpage>1484</fpage><lpage>1488</lpage><pub-id pub-id-type="doi">10.1126/science.1138341</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Katayama</surname><given-names>S</given-names></name><name><surname>Tomaru</surname><given-names>Y</given-names></name><name><surname>Kasukawa</surname><given-names>T</given-names></name><name><surname>Waki</surname><given-names>K</given-names></name><name><surname>Nakanishi</surname><given-names>M</given-names></name><name><surname>Nakamura</surname><given-names>M</given-names></name><name><surname>Nishida</surname><given-names>H</given-names></name><name><surname>Yap</surname><given-names>CC</given-names></name><name><surname>Suzuki</surname><given-names>M</given-names></name><name><surname>Kawai</surname><given-names>J</given-names></name><name><surname>Suzuki</surname><given-names>H</given-names></name><name><surname>Carninci</surname><given-names>P</given-names></name><name><surname>Hayashizaki</surname><given-names>Y</given-names></name><name><surname>Wells</surname><given-names>C</given-names></name><name><surname>Frith</surname><given-names>M</given-names></name><name><surname>Ravasi</surname><given-names>T</given-names></name><name><surname>Pang</surname><given-names>KC</given-names></name><name><surname>Hallinan</surname><given-names>J</given-names></name><name><surname>Mattick</surname><given-names>J</given-names></name><name><surname>Hume</surname><given-names>DA</given-names></name><name><surname>Lipovich</surname><given-names>L</given-names></name><name><surname>Batalov</surname><given-names>S</given-names></name><name><surname>Engström</surname><given-names>PG</given-names></name><name><surname>Mizuno</surname><given-names>Y</given-names></name><name><surname>Faghihi</surname><given-names>MA</given-names></name><name><surname>Sandelin</surname><given-names>A</given-names></name><name><surname>Chalk</surname><given-names>AM</given-names></name><name><surname>Mottagui-Tabar</surname><given-names>S</given-names></name><name><surname>Liang</surname><given-names>Z</given-names></name><name><surname>Lenhard</surname><given-names>B</given-names></name><name><surname>Wahlestedt</surname><given-names>C</given-names></name>, <collab>RIKEN Genome Exploration Research Group</collab>, <collab>Genome Science Group (Genome Network Project Core Group)</collab>, <collab>FANTOM Consortium</collab></person-group><year>2005</year><article-title>Antisense transcription in the mammalian transcriptome</article-title><source>Science</source><volume>309</volume><fpage>1564</fpage><lpage>1566</lpage><pub-id pub-id-type="doi">10.1126/science.1112009</pub-id></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Katiyar-Agarwal</surname><given-names>S</given-names></name><name><surname>Morgan</surname><given-names>R</given-names></name><name><surname>Dahlbeck</surname><given-names>D</given-names></name><name><surname>Borsani</surname><given-names>O</given-names></name><name><surname>Villegas</surname><given-names>A</given-names><suffix>Jnr</suffix></name><name><surname>Zhu</surname><given-names>JK</given-names></name><name><surname>Staskawicz</surname><given-names>BJ</given-names></name><name><surname>Jin</surname><given-names>H</given-names></name></person-group><year>2006</year><article-title>A pathogen-inducible endogenous siRNA in plant immunity</article-title><source>Proceedings of the National Academy of Sciences of the United States of America</source><volume>103</volume><fpage>18002</fpage><lpage>18007</lpage><pub-id pub-id-type="doi">10.1073/pnas.0608258103</pub-id></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ketting</surname><given-names>RF</given-names></name></person-group><year>2011</year><article-title>The many faces of RNAi</article-title><source>Developmental Cell</source><volume>20</volume><fpage>148</fpage><lpage>161</lpage><pub-id pub-id-type="doi">10.1016/j.devcel.2011.01.012</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kirmizis</surname><given-names>A</given-names></name><name><surname>Santos-Rosa</surname><given-names>H</given-names></name><name><surname>Penkett</surname><given-names>CJ</given-names></name><name><surname>Singer</surname><given-names>MA</given-names></name><name><surname>Vermeulen</surname><given-names>M</given-names></name><name><surname>Mann</surname><given-names>M</given-names></name><name><surname>Bahler</surname><given-names>J</given-names></name><name><surname>Green</surname><given-names>RD</given-names></name><name><surname>Kouzarides</surname><given-names>T</given-names></name></person-group><year>2007</year><article-title>Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation</article-title><source>Nature</source><volume>449</volume><fpage>928</fpage><lpage>932</lpage><pub-id pub-id-type="doi">10.1038/nature06160</pub-id></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kobayashi</surname><given-names>T</given-names></name><name><surname>Ganley</surname><given-names>AR</given-names></name></person-group><year>2005</year><article-title>Recombination regulation by transcription-induced cohesin dissociation in rDNA repeats</article-title><source>Science</source><volume>309</volume><fpage>1581</fpage><lpage>1584</lpage><pub-id pub-id-type="doi">10.1126/science.1116102</pub-id></element-citation></ref><ref id="bib96"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Langmead</surname><given-names>B</given-names></name><name><surname>Trapnell</surname><given-names>C</given-names></name><name><surname>Pop</surname><given-names>M</given-names></name><name><surname>Salzberg</surname><given-names>SL</given-names></name></person-group><year>2009</year><article-title>Ultrafast and memory-efficient alignment of short DNA sequences to the human genome</article-title><source>Genome Biology</source><volume>10</volume><fpage>R25</fpage><pub-id pub-id-type="doi">10.1186/gb-2009-10-3-r25</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Latos</surname><given-names>PA</given-names></name><name><surname>Pauler</surname><given-names>FM</given-names></name><name><surname>Koerner</surname><given-names>MV</given-names></name><name><surname>Senergin</surname><given-names>HB</given-names></name><name><surname>Hudson</surname><given-names>QJ</given-names></name><name><surname>Stocsits</surname><given-names>RR</given-names></name><name><surname>Allhoff</surname><given-names>W</given-names></name><name><surname>Stricker</surname><given-names>SH</given-names></name><name><surname>Klement</surname><given-names>RM</given-names></name><name><surname>Warczok</surname><given-names>KE</given-names></name><name><surname>Aumayr</surname><given-names>K</given-names></name><name><surname>Pasierbek</surname><given-names>P</given-names></name><name><surname>Barlow</surname><given-names>DP</given-names></name></person-group><year>2012</year><article-title>Airn transcriptional overlap, but not its lncRNA products, induces imprinted Igf2r silencing</article-title><source>Science</source><volume>338</volume><fpage>1469</fpage><lpage>1472</lpage><pub-id pub-id-type="doi">10.1126/science.1228110</pub-id></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lejeune</surname><given-names>E</given-names></name><name><surname>Bayne</surname><given-names>EH</given-names></name><name><surname>Allshire</surname><given-names>RC</given-names></name></person-group><year>2010</year><article-title>On the connection between RNAi and heterochromatin at centromeres</article-title><source>Cold Spring Harbor Symposia on Quantitative Biology</source><volume>75</volume><fpage>275</fpage><lpage>283</lpage><pub-id pub-id-type="doi">10.1101/sqb.2010.75.024</pub-id></element-citation></ref><ref id="bib51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Lu</surname><given-names>J</given-names></name><name><surname>Han</surname><given-names>Y</given-names></name><name><surname>Fan</surname><given-names>X</given-names></name><name><surname>Ding</surname><given-names>SW</given-names></name></person-group><year>2013</year><article-title>RNA interference functions as an antiviral immunity mechanism in mammals</article-title><source>Science</source><volume>342</volume><fpage>231</fpage><lpage>234</lpage><pub-id pub-id-type="doi">10.1126/science.1241911</pub-id></element-citation></ref><ref id="bib52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Maillard</surname><given-names>PV</given-names></name><name><surname>Ciaudo</surname><given-names>C</given-names></name><name><surname>Marchais</surname><given-names>A</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Jay</surname><given-names>F</given-names></name><name><surname>Ding</surname><given-names>SW</given-names></name><name><surname>Voinnet</surname><given-names>O</given-names></name></person-group><year>2013</year><article-title>Antiviral RNA interference in mammalian cells</article-title><source>Science</source><volume>342</volume><fpage>235</fpage><lpage>238</lpage><pub-id pub-id-type="doi">10.1126/science.1241930</pub-id></element-citation></ref><ref id="bib53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Martens</surname><given-names>JA</given-names></name><name><surname>Laprade</surname><given-names>L</given-names></name><name><surname>Winston</surname><given-names>F</given-names></name></person-group><year>2004</year><article-title>Intergenic transcription is required to repress the <italic>Saccharomyces cerevisiae</italic> SER3 gene</article-title><source>Nature</source><volume>429</volume><fpage>571</fpage><lpage>574</lpage><pub-id pub-id-type="doi">10.1038/nature02538</pub-id></element-citation></ref><ref id="bib54"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Martienssen</surname><given-names>RA</given-names></name><name><surname>Zaratiegui</surname><given-names>M</given-names></name><name><surname>Goto</surname><given-names>DB</given-names></name></person-group><year>2005</year><article-title>RNA interference and heterochromatin in the fission yeast Schizosaccharomyces pombe</article-title><source>Trends in Genetics</source><volume>21</volume><fpage>450</fpage><lpage>456</lpage><pub-id pub-id-type="doi">10.1016/j.tig.2005.06.005</pub-id></element-citation></ref><ref id="bib55"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nagano</surname><given-names>T</given-names></name><name><surname>Mitchell</surname><given-names>JA</given-names></name><name><surname>Sanz</surname><given-names>LA</given-names></name><name><surname>Pauler</surname><given-names>FM</given-names></name><name><surname>Ferguson-Smith</surname><given-names>AC</given-names></name><name><surname>Feil</surname><given-names>R</given-names></name><name><surname>Fraser</surname><given-names>P</given-names></name></person-group><year>2008</year><article-title>The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin</article-title><source>Science</source><volume>322</volume><fpage>1717</fpage><lpage>1720</lpage><pub-id pub-id-type="doi">10.1126/science.1163802</pub-id></element-citation></ref><ref id="bib56"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Neil</surname><given-names>H</given-names></name><name><surname>Malabat</surname><given-names>C</given-names></name><name><surname>d’Aubenton-Carafa</surname><given-names>Y</given-names></name><name><surname>Xu</surname><given-names>Z</given-names></name><name><surname>Steinmetz</surname><given-names>LM</given-names></name><name><surname>Jacquier</surname><given-names>A</given-names></name></person-group><year>2009</year><article-title>Widespread bidirectional promoters are the major source of cryptic transcripts in yeast</article-title><source>Nature</source><volume>457</volume><fpage>1038</fpage><lpage>1042</lpage><pub-id pub-id-type="doi">10.1038/nature07747</pub-id></element-citation></ref><ref id="bib57"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nejepinska</surname><given-names>J</given-names></name><name><surname>Malik</surname><given-names>R</given-names></name><name><surname>Filkowski</surname><given-names>J</given-names></name><name><surname>Flemr</surname><given-names>M</given-names></name><name><surname>Filipowicz</surname><given-names>W</given-names></name><name><surname>Svoboda</surname><given-names>P</given-names></name></person-group><year>2012a</year><article-title>dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells</article-title><source>Nucleic Acids Research</source><volume>40</volume><fpage>399</fpage><lpage>413</lpage><pub-id pub-id-type="doi">10.1093/nar/gkr702</pub-id></element-citation></ref><ref id="bib58"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nejepinska</surname><given-names>J</given-names></name><name><surname>Malik</surname><given-names>R</given-names></name><name><surname>Moravec</surname><given-names>M</given-names></name><name><surname>Svoboda</surname><given-names>P</given-names></name></person-group><year>2012b</year><article-title>Deep sequencing reveals complex spurious transcription from transiently transfected plasmids</article-title><source>PLOS ONE</source><volume>7</volume><fpage>e43283</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0043283</pub-id></element-citation></ref><ref id="bib59"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Okamura</surname><given-names>K</given-names></name><name><surname>Balla</surname><given-names>S</given-names></name><name><surname>Martin</surname><given-names>R</given-names></name><name><surname>Liu</surname><given-names>N</given-names></name><name><surname>Lai</surname><given-names>EC</given-names></name></person-group><year>2008</year><article-title>Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster</article-title><source>Nature Structure & Molecular Biology</source><volume>15</volume><fpage>581</fpage><lpage>590</lpage><pub-id pub-id-type="doi">10.1038/nsmb.1438</pub-id></element-citation></ref><ref id="bib60"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pall</surname><given-names>GS</given-names></name><name><surname>Hamilton</surname><given-names>AJ</given-names></name></person-group><year>2008</year><article-title>Improved northern blot method for enhanced detection of small RNA</article-title><source>Nature Protocols</source><volume>3</volume><fpage>1077</fpage><lpage>1084</lpage><pub-id pub-id-type="doi">10.1038/nprot.2008.67</pub-id></element-citation></ref><ref id="bib61"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pandey</surname><given-names>RR</given-names></name><name><surname>Mondal</surname><given-names>T</given-names></name><name><surname>Mohammad</surname><given-names>F</given-names></name><name><surname>Enroth</surname><given-names>S</given-names></name><name><surname>Redrup</surname><given-names>L</given-names></name><name><surname>Komorowski</surname><given-names>J</given-names></name><name><surname>Nagano</surname><given-names>T</given-names></name><name><surname>Mancini-Dinardo</surname><given-names>D</given-names></name><name><surname>Kanduri</surname><given-names>C</given-names></name></person-group><year>2008</year><article-title>Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation</article-title><source>Molecular Cell</source><volume>32</volume><fpage>232</fpage><lpage>246</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2008.08.022</pub-id></element-citation></ref><ref id="bib62"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pinskaya</surname><given-names>M</given-names></name><name><surname>Gourvennec</surname><given-names>S</given-names></name><name><surname>Morillon</surname><given-names>A</given-names></name></person-group><year>2009</year><article-title>H3 lysine 4 di- and tri-methylation deposited by cryptic transcription attenuates promoter activation</article-title><source>The EMBO Journal</source><volume>28</volume><fpage>1697</fpage><lpage>1707</lpage><pub-id pub-id-type="doi">10.1038/emboj.2009.108</pub-id></element-citation></ref><ref id="bib63"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Preker</surname><given-names>P</given-names></name><name><surname>Nielsen</surname><given-names>J</given-names></name><name><surname>Kammler</surname><given-names>S</given-names></name><name><surname>Lykke-Andersen</surname><given-names>S</given-names></name><name><surname>Christensen</surname><given-names>MS</given-names></name><name><surname>Mapendano</surname><given-names>CK</given-names></name><name><surname>Schierup</surname><given-names>MH</given-names></name><name><surname>Jensen</surname><given-names>TH</given-names></name></person-group><year>2008</year><article-title>RNA exosome depletion reveals transcription upstream of active human promoters</article-title><source>Science</source><volume>322</volume><fpage>1851</fpage><lpage>1854</lpage><pub-id pub-id-type="doi">10.1126/science.1164096</pub-id></element-citation></ref><ref id="bib65"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pryde</surname><given-names>FE</given-names></name><name><surname>Louis</surname><given-names>EJ</given-names></name></person-group><year>1999</year><article-title>Limitations of silencing at native yeast telomeres</article-title><source>The EMBO Journal</source><volume>18</volume><fpage>2538</fpage><lpage>2550</lpage><pub-id pub-id-type="doi">10.1093/emboj/18.9.2538</pub-id></element-citation></ref><ref id="bib66"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Redrup</surname><given-names>L</given-names></name><name><surname>Branco</surname><given-names>MR</given-names></name><name><surname>Perdeaux</surname><given-names>ER</given-names></name><name><surname>Krueger</surname><given-names>C</given-names></name><name><surname>Lewis</surname><given-names>A</given-names></name><name><surname>Santos</surname><given-names>F</given-names></name><name><surname>Nagano</surname><given-names>T</given-names></name><name><surname>Cobb</surname><given-names>BS</given-names></name><name><surname>Fraser</surname><given-names>P</given-names></name><name><surname>Reik</surname><given-names>W</given-names></name></person-group><year>2009</year><article-title>The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing</article-title><source>Development</source><volume>136</volume><fpage>525</fpage><lpage>530</lpage><pub-id pub-id-type="doi">10.1242/dev.031328</pub-id></element-citation></ref><ref id="bib67"><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Sambrook</surname><given-names>J</given-names></name><name><surname>Russell</surname><given-names>DW</given-names></name></person-group><year>2001</year><source>Molecular cloning: a laboratory manual</source><publisher-loc>Cold Spring Harbor, NY</publisher-loc><publisher-name>Cold Spring Harbor Laboratory Press</publisher-name><edition>3rd edition</edition></element-citation></ref><ref id="bib68"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schonborn</surname><given-names>J</given-names></name><name><surname>Oberstrass</surname><given-names>J</given-names></name><name><surname>Breyel</surname><given-names>E</given-names></name><name><surname>Tittgen</surname><given-names>J</given-names></name><name><surname>Schumacher</surname><given-names>J</given-names></name><name><surname>Lukacs</surname><given-names>N</given-names></name></person-group><year>1991</year><article-title>Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts</article-title><source>Nucleic Acids Research</source><volume>19</volume><fpage>2993</fpage><lpage>3000</lpage><pub-id pub-id-type="doi">10.1093/nar/19.11.2993</pub-id></element-citation></ref><ref id="bib70"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sinkkonen</surname><given-names>L</given-names></name><name><surname>Hugenschmidt</surname><given-names>T</given-names></name><name><surname>Filipowicz</surname><given-names>W</given-names></name><name><surname>Svoboda</surname><given-names>P</given-names></name></person-group><year>2010</year><article-title>Dicer is associated with ribosomal DNA chromatin in mammalian cells</article-title><source>PLOS ONE</source><volume>5</volume><fpage>e12175</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0012175</pub-id></element-citation></ref><ref id="bib71"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Siomi</surname><given-names>MC</given-names></name><name><surname>Sato</surname><given-names>K</given-names></name><name><surname>Pezic</surname><given-names>D</given-names></name><name><surname>Aravin</surname><given-names>AA</given-names></name></person-group><year>2011</year><article-title>PIWI-interacting small RNAs: the vanguard of genome defence</article-title><source>Nature Reviews Molecular Cell Biology</source><volume>12</volume><fpage>246</fpage><lpage>258</lpage><pub-id pub-id-type="doi">10.1038/nrm3089</pub-id></element-citation></ref><ref id="bib72"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Slotkin</surname><given-names>RK</given-names></name><name><surname>Martienssen</surname><given-names>R</given-names></name></person-group><year>2007</year><article-title>Transposable elements and the epigenetic regulation of the genome</article-title><source>Nature Reviews Genetics</source><volume>8</volume><fpage>272</fpage><lpage>285</lpage><pub-id pub-id-type="doi">10.1038/nrg2072</pub-id></element-citation></ref><ref id="bib73"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Song</surname><given-names>R</given-names></name><name><surname>Hennig</surname><given-names>GW</given-names></name><name><surname>Wu</surname><given-names>Q</given-names></name><name><surname>Jose</surname><given-names>C</given-names></name><name><surname>Zheng</surname><given-names>H</given-names></name><name><surname>Yan</surname><given-names>W</given-names></name></person-group><year>2011</year><article-title>Male germ cells express abundant endogenous siRNAs</article-title><source>Proceedings of the National Academy of Sciences of the United States of America</source><volume>108</volume><fpage>13159</fpage><lpage>13164</lpage><pub-id pub-id-type="doi">10.1073/pnas.1108567108</pub-id></element-citation></ref><ref id="bib77"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tam</surname><given-names>OH</given-names></name><name><surname>Aravin</surname><given-names>AA</given-names></name><name><surname>Stein</surname><given-names>P</given-names></name><name><surname>Girard</surname><given-names>A</given-names></name><name><surname>Murchison</surname><given-names>EP</given-names></name><name><surname>Cheloufi</surname><given-names>S</given-names></name><name><surname>Hodges</surname><given-names>E</given-names></name><name><surname>Anger</surname><given-names>M</given-names></name><name><surname>Sachidanandam</surname><given-names>R</given-names></name><name><surname>Schultz</surname><given-names>RM</given-names></name><etal/></person-group><year>2008</year><article-title>Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes</article-title><source>Nature</source><volume>453</volume><fpage>534</fpage><lpage>538</lpage><pub-id pub-id-type="doi">10.1038/nature06904</pub-id></element-citation></ref><ref id="bib97"><element-citation publication-type="journal"><person-group person-group-type="author"><collab>The ENCODE Project Constorium</collab></person-group><year>2012</year><article-title>An integrated encyclopedia of DNA elements in the human genome</article-title><source>Nature</source><volume>489</volume><fpage>57</fpage><lpage>74</lpage><pub-id pub-id-type="doi">10.1038/nature11247</pub-id></element-citation></ref><ref id="bib78"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Uhler</surname><given-names>JP</given-names></name><name><surname>Hertel</surname><given-names>C</given-names></name><name><surname>Svejstrup</surname><given-names>JQ</given-names></name></person-group><year>2007</year><article-title>A role for noncoding transcription in activation of the yeast PHO5 gene</article-title><source>Proceedings of the National Academy of Sciences of the United States of America</source><volume>104</volume><fpage>8011</fpage><lpage>8016</lpage><pub-id pub-id-type="doi">10.1073/pnas.0702431104</pub-id></element-citation></ref><ref id="bib79"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Bakel</surname><given-names>H</given-names></name><name><surname>Nislow</surname><given-names>C</given-names></name><name><surname>Blencowe</surname><given-names>BJ</given-names></name><name><surname>Hughes</surname><given-names>TR</given-names></name></person-group><year>2010</year><article-title>Most “dark matter” transcripts are associated with known genes</article-title><source>PLOS Biology</source><volume>8</volume><fpage>e1000371</fpage><pub-id pub-id-type="doi">10.1371/journal.pbio.1000371</pub-id></element-citation></ref><ref id="bib80"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Dijk</surname><given-names>EL</given-names></name><name><surname>Chen</surname><given-names>CL</given-names></name><name><surname>d’Aubenton-Carafa</surname><given-names>Y</given-names></name><name><surname>Gourvennec</surname><given-names>S</given-names></name><name><surname>Kwapisz</surname><given-names>M</given-names></name><name><surname>Roche</surname><given-names>V</given-names></name><name><surname>Bertrand</surname><given-names>C</given-names></name><name><surname>Silvain</surname><given-names>M</given-names></name><name><surname>Legoix-Ne</surname><given-names>P</given-names></name><name><surname>Loeillet</surname><given-names>S</given-names></name><name><surname>Nicolas</surname><given-names>A</given-names></name><name><surname>Thermes</surname><given-names>C</given-names></name><name><surname>Morillon</surname><given-names>A</given-names></name></person-group><year>2011</year><article-title>XUTs are a class of Xrn1-sensitive antisense regulatory non-coding RNA in yeast</article-title><source>Nature</source><volume>475</volume><fpage>114</fpage><lpage>117</lpage><pub-id pub-id-type="doi">10.1038/nature10118</pub-id></element-citation></ref><ref id="bib82"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Werven</surname><given-names>FJ</given-names></name><name><surname>Neuert</surname><given-names>G</given-names></name><name><surname>Hendrick</surname><given-names>N</given-names></name><name><surname>Lardenois</surname><given-names>A</given-names></name><name><surname>Buratowski</surname><given-names>S</given-names></name><name><surname>van Oudenaarden</surname><given-names>A</given-names></name><name><surname>Primig</surname><given-names>M</given-names></name><name><surname>Amon</surname><given-names>A</given-names></name></person-group><year>2012</year><article-title>Transcription of two long noncoding RNAs mediates mating-type control of gametogenesis in budding yeast</article-title><source>Cell</source><volume>150</volume><fpage>1170</fpage><lpage>1181</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2012.06.049</pub-id></element-citation></ref><ref id="bib98"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Velmurugan</surname><given-names>S</given-names></name><name><surname>Yang</surname><given-names>XM</given-names></name><name><surname>Chan</surname><given-names>CSM</given-names></name><name><surname>Dobson</surname><given-names>M</given-names></name><name><surname>Jayaram</surname><given-names>M</given-names></name></person-group><year>2000</year><article-title>Partitioning of the 2-microm circle plasmid of Saccharomyces cerevisiae. Functional coordination with chromosome segregation and plasmid-encoded rep protein distribution</article-title><source>Journal of Cell Biology</source><volume>149</volume><fpage>553</fpage><lpage>566</lpage><pub-id pub-id-type="doi">10.1083/jcb.149.3.553</pub-id></element-citation></ref><ref id="bib83"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Watanabe</surname><given-names>T</given-names></name><name><surname>Takeda</surname><given-names>A</given-names></name><name><surname>Tsukiyama</surname><given-names>T</given-names></name><name><surname>Mise</surname><given-names>K</given-names></name><name><surname>Okuno</surname><given-names>T</given-names></name><name><surname>Sasaki</surname><given-names>H</given-names></name><name><surname>Minami</surname><given-names>N</given-names></name><name><surname>Imai</surname><given-names>H</given-names></name></person-group><year>2006</year><article-title>Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes</article-title><source>Genes Development</source><volume>20</volume><fpage>1732</fpage><lpage>1743</lpage><pub-id pub-id-type="doi">10.1101/gad.1425706</pub-id></element-citation></ref><ref id="bib84"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Watanabe</surname><given-names>T</given-names></name><name><surname>Totoki</surname><given-names>Y</given-names></name><name><surname>Toyoda</surname><given-names>A</given-names></name><name><surname>Kaneda</surname><given-names>M</given-names></name><name><surname>Kuramochi-Miyagawa</surname><given-names>S</given-names></name><name><surname>Obata</surname><given-names>Y</given-names></name><name><surname>Chiba</surname><given-names>H</given-names></name><name><surname>Kohara</surname><given-names>Y</given-names></name><name><surname>Kono</surname><given-names>T</given-names></name><name><surname>Nakano</surname><given-names>T</given-names></name><name><surname>Surani</surname><given-names>MA</given-names></name><name><surname>Sakaki</surname><given-names>Y</given-names></name><name><surname>Sasaki</surname><given-names>H</given-names></name></person-group><year>2008</year><article-title>Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes</article-title><source>Nature</source><volume>453</volume><fpage>539</fpage><lpage>543</lpage><pub-id pub-id-type="doi">10.1038/nature06908</pub-id></element-citation></ref><ref id="bib99"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wong</surname><given-names>MC</given-names></name><name><surname>Scott-Drew</surname><given-names>SR</given-names></name><name><surname>Hayes</surname><given-names>MJ</given-names></name><name><surname>Howard</surname><given-names>PJ</given-names></name><name><surname>Murray</surname><given-names>JA</given-names></name></person-group><year>2002</year><article-title>RSC2, encoding a component of the RSC nucleosome remodeling complex, is essential for 2 microm plasmid maintenance in Saccharomyces cerevisiae</article-title><source>Molecular and Cellular Biology</source><volume>22</volume><fpage>4218</fpage><lpage>4229</lpage><pub-id pub-id-type="doi">10.1128/MCB.22.12.4218-4229.2002</pub-id></element-citation></ref><ref id="bib85"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Xu</surname><given-names>Z</given-names></name><name><surname>Wei</surname><given-names>W</given-names></name><name><surname>Gagneur</surname><given-names>J</given-names></name><name><surname>Perocchi</surname><given-names>F</given-names></name><name><surname>Clauder-Munster</surname><given-names>S</given-names></name><name><surname>Camblong</surname><given-names>J</given-names></name><name><surname>Guffanti</surname><given-names>E</given-names></name><name><surname>Stutz</surname><given-names>F</given-names></name><name><surname>Huber</surname><given-names>W</given-names></name><name><surname>Steinmetz</surname><given-names>LM</given-names></name></person-group><year>2009</year><article-title>Bidirectional promoters generate pervasive transcription in yeast</article-title><source>Nature</source><volume>457</volume><fpage>1033</fpage><lpage>1037</lpage><pub-id pub-id-type="doi">10.1038/nature07728</pub-id></element-citation></ref><ref id="bib86"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yamada</surname><given-names>M</given-names></name><name><surname>Hayatsu</surname><given-names>N</given-names></name><name><surname>Matsuura</surname><given-names>A</given-names></name><name><surname>Ishikawa</surname><given-names>F</given-names></name></person-group><year>1998</year><article-title>Y’-Help1, a DNA helicase encoded by the yeast subtelomeric Y’ element, is induced in survivors defective for telomerase</article-title><source>The Journal of Biological Chemistry</source><volume>273</volume><fpage>33360</fpage><lpage>33366</lpage><pub-id pub-id-type="doi">10.1074/jbc.273.50.33360</pub-id></element-citation></ref><ref id="bib87"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yamanaka</surname><given-names>S</given-names></name><name><surname>Mehta</surname><given-names>S</given-names></name><name><surname>Reyes-Turcu</surname><given-names>FE</given-names></name><name><surname>Zhuang</surname><given-names>F</given-names></name><name><surname>Fuchs</surname><given-names>RT</given-names></name><name><surname>Rong</surname><given-names>Y</given-names></name><name><surname>Robb</surname><given-names>GB</given-names></name><name><surname>Grewal</surname><given-names>SI</given-names></name></person-group><year>2012</year><article-title>RNAi triggered by specialized machinery silences developmental genes and retrotransposons</article-title><source>Nature</source><volume>493</volume><fpage>557</fpage><lpage>560</lpage><pub-id pub-id-type="doi">10.1038/nature11716</pub-id></element-citation></ref><ref id="bib88"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname><given-names>N</given-names></name><name><surname>Kazazian</surname><given-names>HH</given-names><suffix>Jnr</suffix></name></person-group><year>2006</year><article-title>L1 retrotransposition is suppressed by endogenously encoded small interfering RNAs in human cultured cells</article-title><source>Nature Structure & Molecular Biology</source><volume>13</volume><fpage>763</fpage><lpage>771</lpage><pub-id pub-id-type="doi">10.1038/nsmb1141</pub-id></element-citation></ref><ref id="bib89"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhang</surname><given-names>H</given-names></name><name><surname>Zhu</surname><given-names>JK</given-names></name></person-group><year>2011</year><article-title>RNA-directed DNA methylation</article-title><source>Current Opinion in Plant Biology</source><volume>14</volume><fpage>142</fpage><lpage>147</lpage><pub-id pub-id-type="doi">10.1016/j.pbi.2011.02.003</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01581.027</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Proudfoot</surname><given-names>Nick J</given-names></name><role>Reviewing editor</role><aff><institution>University of Oxford</institution>, <country>United Kingdom</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Endogenous RNA interference is driven by copy number” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>This study employs the so-called RNAi+ <italic>S. cerevisiae</italic> strain generated in the Bartel lab (<xref ref-type="bibr" rid="bib23">Drinnenberg et al 2009</xref>) that is transformed with <italic>S. Castellii Dicer</italic> and <italic>Ago</italic> genes. This original study suggested that this RNAi+ yeast would be a good model system to explore RNAi gene silencing mechanisms. It is clear that this is the case as this new study makes excellent use of this strain. What is shown here is that endogenous convergent transcription (especially mRNA sense and ncRNA antisense arrangement) generates siRNAs in RNAi+ cells but this does not correlate with reduced mRNA abundance (<xref ref-type="fig" rid="fig1">Figure 1</xref>). Also repetitive sequences (Y′ RNA and rRNA) generate abundant siRNAs and show significant RNAi dependent RNA degradation. Some of these data are however predicted from the original Drinnenberg et al study. New results are presented in <xref ref-type="fig" rid="fig3">Figure 3</xref> showing in two test gene cases (<italic>MAL32</italic> and <italic>GAL4</italic>) that forced sense antisense expression of these genes on multicopy transformed plasmids generate both siRNAs and also show clear gene silencing. These results lead to the key observation that it isn't the overall level of expression (sense antisense) that is critical but rather it is the fact that expression comes from multicopy genes (rather than one highly expressed gene). This point is initially made from bioinformatic analysis of the RNAi+ transcriptiome (Drinneneberg et al) where multicopy genes generate more siRNA than single copy rather than simply correlating with totals RNA levels (<xref ref-type="fig" rid="fig4">Figure 4</xref>). <xref ref-type="fig" rid="fig5 fig6">Figures 5 and 6</xref> then validate the bioinformatics very clearly. The integrated single copy <italic>MAL32</italic> expressing high level sense and antisense transcription due to the use of strong convergent arranged promoters does not generate as much siRNA as multicopy low expressed <italic>MAL32</italic>. Also sense antisense ncRNAs expressed on multicopy plasmids generate significant amounts of siRNA. Finally some evidence was provided that siRNAs from high-copy <italic>GAL10</italic> constructs (making sense and antisense RNAs) can generate siRNA that silence another <italic>GAL10</italic> gene in trans.</p><p>Overall the three reviewers of this paper while aware of the somewhat artificial nature of the RNAi+ <italic>S. cerevisiae</italic> strain consider that the experimental benefit of this system has been put to excellent use and has allowed significant new insight in RNAi mechanism to be obtained. The key additional experiments that we feel are necessary to make a revised manuscript acceptable for publication by <italic>eLife</italic> are as follows.</p><p>1) The actual copy number of the 2µ plasmids transformed into the RNAi+ strain should be directly measured by Southern blotting</p><p>2) The <italic>Castellii</italic> Dcr1 gene should be knocked out to show loss of the high-copy number silencing observed for <italic>MAL32</italic> and <italic>GAL4</italic>.</p><p>3) The location of dsRNA and <italic>Dicer</italic> in the transformed RNAi+ cells should be directly visualised (J2 antibody for dsRNA and Tagged <italic>Dcr1</italic>/anti <italic>Dcr1</italic>) to show either chromatin or cytolasmic association. See Schönborn, J. et al. NAR 19, 2993-3000, 1991; Gullerova and Proudfoot NSMB 19, 1193-1201 2013 for J2 antibody.</p><p>4) Better quantitation of RNAi effect on mRNA levels and level of dsRNA is required. This can be achieved by RTqPCR and for dsRNA level of J2 dsRNA immunoprecipitation.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01581.028</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) The actual copy number of the 2µ plasmids transformed into the RNAi+ strain should be directly measured by</italic> <italic>Southern blotting</italic></p><p>We have included a southern for 2µ copy number as <xref ref-type="fig" rid="fig3s5">Figure 3–figure supplement 5</xref>. We have also shown a northern blot for the <italic>MAL32</italic> high-copy plasmid in which the plasmid signals are clearly visible. Both analyses show that the copy number does not decrease in the RNAi+ strain – it actually increases, an observation we discuss in the text.</p><p><italic>2) The</italic> Castellii <italic>Dcr1 gene should be knocked out to show loss of the high-copy number silencing observed for</italic> MAL32 <italic>and</italic> GAL4<italic>.</italic></p><p>We decided to combine this with changing the genetic background of the RNAi+ strain. We cloned Dcr1 and Ago1 onto single copy plasmids and introduced these into BY4741 cells (the original background is W303), along with the high-copy plasmids. The result for <italic>MAL32</italic> is very clear, showing that both genes are required for knockdown (<xref ref-type="fig" rid="fig3s3">Figure 3–figure supplement 3</xref>). However, in this genetic background <italic>GAL4</italic> is only expressed very weakly from the high-copy plasmid; the RNA forms dsRNA efficiently as expected and this is removed by Dicer, although this does not lead to a clear knockdown. The <italic>GAL4</italic> knockdown was the weakest we observed in the original experiments, and RNAi knockdowns in this new strain are not as strong as in the original strain (60% in new strain vs. 80% in the original strain for the <italic>MAL32</italic> mRNA). Combined with the confounding effect of the increased plasmid copy number in RNAi+ cells, the <italic>GAL4</italic> knockdown becomes too weak to test the importance of Dicer. We do not think that this compromises the conclusions of the paper.</p><p><italic>3) The location of dsRNA and</italic> Dicer <italic>in the transformed RNAi+ cells should be directly visualised (J2 antibody for dsRNA and Tagged</italic> Dcr1<italic>/anti</italic> Dcr1<italic>) to show either chromatin or cytolasmic association. See Schönborn, J. et al. NAR 19, 2993-3000, 1991; Gullerova and Proudfoot NSMB 19, 1193-1201 2013 for J2 antibody</italic>.</p><p>We have included this data as <xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1</xref>. We could not convincingly detect Dicer in nuclei, and the dsRNA signal is dominated by strong cytoplasmic signals, most likely from Killer virus RNAs. Although RNAi should technically clear Killer virus, the analysis performed by the Bartel lab shows that this clearance is far from complete. We have used a different approach to improve confidence in nuclear formation of dsRNA; if dsRNA forms in the nucleus then the juxtaposition of transcribing copies should be important, and we demonstrate that this is true for the <italic>TRP1</italic> gene in the new <xref ref-type="fig" rid="fig6">Figure 6</xref>. We have referred to both of these experiments in the Discussion.</p><p><italic>4) Better quantitation of RNAi effect on mRNA levels and level of dsRNA is required. This can be achieved by RTqPCR and for dsRNA level of J2 dsRNA immunoprecipitation</italic>.</p><p>We have added a number of additional results here. Firstly, we have added quantification for the effect of RNAi on endogenous <italic>MAL32</italic> and <italic>GAL4</italic> mRNA along with full length northern blot images that show degradation patterns (<xref ref-type="fig" rid="fig3s2">Figure 3–figure supplement 2</xref>). We have also performed the J2 immunoprecipitations (<xref ref-type="fig" rid="fig3s6">Figure 3–figure supplement 6</xref>) – these show a clear loss dsRNA in the RNAi strain, and confirm the Northern blot results for mRNA knockdown.</p></body></sub-article></article> |