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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">01849</article-id><article-id pub-id-type="doi">10.7554/eLife.01849</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Genes and chromosomes</subject></subj-group><subj-group subj-group-type="heading"><subject>Neuroscience</subject></subj-group></article-categories><title-group><article-title>Natural antisense transcripts regulate the neuronal stress response and excitability</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-8999"><name><surname>Zheng</surname><given-names>Xingguo</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-9000"><name><surname>Valakh</surname><given-names>Vera</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-9001"><name><surname>DiAntonio</surname><given-names>Aaron</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-2516"><name><surname>Ben-Shahar</surname><given-names>Yehuda</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="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Biology</institution>, <institution>Washington University in St. Louis</institution>, <addr-line><named-content content-type="city">St. Louis</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Developmental Biology</institution>, <institution>Washington University School of Medicine</institution>, <addr-line><named-content content-type="city">St. Louis</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Hope Center for Neurological Disorders</institution>, <institution>Washington University School of Medicine</institution>, <addr-line><named-content content-type="city">St. Louis</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Griffith</surname><given-names>Leslie C</given-names></name><role>Reviewing editor</role><aff><institution>Brandeis University</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>benshahary@wustl.edu</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>18</day><month>03</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e01849</elocation-id><history><date date-type="received"><day>08</day><month>11</month><year>2013</year></date><date date-type="accepted"><day>18</day><month>02</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Zheng et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Zheng et al</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="elife01849.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.01849.001</object-id><p>Neurons regulate ionic fluxes across their plasma membrane to maintain their excitable properties under varying environmental conditions. However, the mechanisms that regulate ion channels abundance remain poorly understood. Here we show that <italic>pickpocket 29</italic> (<italic>ppk29</italic>), a gene that encodes a <italic>Drosophila</italic> degenerin/epithelial sodium channel (DEG/ENaC), regulates neuronal excitability via a protein-independent mechanism. We demonstrate that the mRNA 3′UTR of <italic>ppk29</italic> affects neuronal firing rates and associated heat-induced seizures by acting as a natural antisense transcript (NAT) that regulates the neuronal mRNA levels of <italic>seizure</italic> (<italic>sei</italic>), the <italic>Drosophila</italic> homolog of the human <italic>Ether-à-go-go</italic> Related Gene (hERG) potassium channel. We find that the regulatory impact of <italic>ppk29</italic> mRNA on <italic>sei</italic> is independent of the sodium channel it encodes. Thus, our studies reveal a novel mRNA dependent mechanism for the regulation of neuronal excitability that is independent of protein-coding capacity.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.001">http://dx.doi.org/10.7554/eLife.01849.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.01849.002</object-id><title>eLife digest</title><p>Neurons communicate with one another via electrical signals known as action potentials. These signals are generated when a stimulus causes sodium and potassium ion channels in the cell membrane to open, leading to an influx of sodium ions, followed by an efflux of potassium ions. Changes in temperature affect the rate at which ion channels open and close, and thus affect how easy it is for a stimulus to trigger an action potential. In response to a sudden rise in temperature, neurons must adjust the number of ion channels in their membranes to ensure that they do not become hyperexcitable, which could result in epilepsy.</p><p>Now, Zheng et al. have revealed one possible mechanism for how neurons do this. In the fruit fly, <italic>Drosophila</italic>, a gene for a potassium channel is found on the same chromosomal location as a gene for a sodium channel, and some of the genetic elements that regulate the expression of these two genes even overlap. However, the genes are on opposite strands of the DNA double helix. This means that when the genes are transcribed to produce molecules of messenger RNA (mRNA), which is usually single stranded, some of the mRNA molecules will pair up to form double-stranded mRNA molecules. This is significant because such RNA ‘duplexes’ have been shown to inhibit the translation of conventional single-stranded mRNA molecules into proteins, or to lead to their complete degradation.</p><p>Zheng et al. found that flies with mutations in the potassium channel gene display seizures in response to sudden changes in temperature. However, insects with mutations in the sodium channel gene are not affected because, surprisingly, they have a higher than expected number of potassium channels. It turns out that the mutant sodium channel mRNA molecules are unable to form RNA duplexes with potassium channel mRNA molecules: these duplexes would normally limit the number of potassium channels so, in their absence, the number of potassium channels increases, and this protects the flies from seizures.</p><p>Zheng et al. also uncovered a novel mechanism by which mRNA molecules can regulate gene expression independent of their role as templates for proteins. Further work is required to determine whether this mechanism is also present in other organisms, including humans.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.002">http://dx.doi.org/10.7554/eLife.01849.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>Degenerin</kwd><kwd>Epithelial sodium channel</kwd><kwd>DEG/ENaC</kwd><kwd><italic>Drosophila</italic></kwd><kwd>fruit fly</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>D. melanogaster</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>DC010244</award-id><principal-award-recipient><name><surname>Ben-Shahar</surname><given-names>Yehuda</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>National Science Foundation</institution></institution-wrap></funding-source><award-id>1322783</award-id><principal-award-recipient><name><surname>Ben-Shahar</surname><given-names>Yehuda</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>The Esther A & Joseph Klingenstein Fund</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Ben-Shahar</surname><given-names>Yehuda</given-names></name></principal-award-recipient></award-group><funding-statement>The funder 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 mRNA that encodes a <italic>Drosophila</italic> sodium channel enables neurons to adapt to acute temperature changes, via a mechanism independent of its protein-coding role.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>The neuronal action potential is sensitive to abrupt changes in environmental temperatures (<xref ref-type="bibr" rid="bib28">Peng et al., 2007</xref>; <xref ref-type="bibr" rid="bib7">Buzatu, 2009</xref>). Thus, the failure of neurons to adjust their physiological properties in response to a fast rise in temperature can lead to neurological disorders such as febrile seizures (<xref ref-type="bibr" rid="bib3">Bassan et al., 2013</xref>). Previous theoretical and experimental studies suggested that one of the main mechanisms for maintaining normal neuronal excitability, circuit integrity, and behavioral robustness under varying environmental temperatures depends on changes in the abundance and membrane half-life of various voltage-dependent ion channels (<xref ref-type="bibr" rid="bib23">Marder and Prinz, 2003</xref>; <xref ref-type="bibr" rid="bib25">O’Leary et al., 2013</xref>; <xref ref-type="bibr" rid="bib32">Rinberg et al., 2013</xref>; <xref ref-type="bibr" rid="bib34">Rosati and McKinnon, 2004</xref>; <xref ref-type="bibr" rid="bib36">Tang et al., 2010</xref>, <xref ref-type="bibr" rid="bib37">2012</xref>). However, the actual molecular mechanisms that mediate these processes are largely unknown.</p><p>Several whole genome transcriptomics studies revealed that natural antisense non-coding transcripts (NATs) are prevalent in eukaryotes (<xref ref-type="bibr" rid="bib18">Lapidot and Pilpel, 2006</xref>; <xref ref-type="bibr" rid="bib26">Okamura et al., 2008</xref>). Although the function of the majority of NATs is still unknown, evidence suggests that at least some <italic>cis</italic>-NATs are likely to act as regulatory RNAs of protein-coding transcripts (<xref ref-type="bibr" rid="bib6">Borsani et al., 2005</xref>; <xref ref-type="bibr" rid="bib26">Okamura et al., 2008</xref>; <xref ref-type="bibr" rid="bib43">Watanabe et al., 2008</xref>), including a recent report about a non-coding NAT that regulates a neuronal potassium channel (<xref ref-type="bibr" rid="bib46">Zhao et al., 2013</xref>). Furthermore, some NATs have been shown to play a role in the physiological response to various stresses in plants (<xref ref-type="bibr" rid="bib6">Borsani et al., 2005</xref>; <xref ref-type="bibr" rid="bib17">Katiyar-Agarwal et al., 2006</xref>). Whether NATs play a role in the post-transcriptional regulation of ion channels and neuronal excitability was unknown.</p></sec><sec id="s2" sec-type="results|discussion"><title>Results and discussion</title><sec id="s2-1"><title><italic>ppk29</italic> and <italic>sei</italic> are convergently transcribed ion channels</title><p>The response of neurons to acute heat stress is likely to require rapid changes in ion channel functions. We hypothesized that NATs play a role in the posttranscriptional regulation of ion channel function in response to stress. Therefore, we screened the well-annotated genome of the fruit fly <italic>Drosophila melanogaster</italic> to identify known excitability-related ion channels that might be regulated by endogenous NATs.</p><p>Using this approach, we found that the gene <italic>seizure</italic> (<italic>sei</italic>), which encodes the sole fly homolog of the human <italic>Ether-à-go-go</italic> Related Gene (hERG) inward rectifying K<sup>+</sup> channel (<xref ref-type="bibr" rid="bib40">Titus et al., 1997</xref>; <xref ref-type="bibr" rid="bib42">Wang et al., 1997</xref>), is located downstream of the degenerin/epithelial sodium channel (DEG/ENaC) <italic>ppk29</italic> (<xref ref-type="bibr" rid="bib19">Liu et al., 2012</xref>; <xref ref-type="bibr" rid="bib38">Thistle et al., 2012</xref>; <xref ref-type="bibr" rid="bib45">Zelle et al., 2013</xref>). The two genes are convergently transcribed on opposite DNA strands, and have complementary 3′UTRs that overlap by 88 nucleotides, which we confirmed by fully sequenced cDNAs deposited in NCBI and 3’RACE analysis (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). The two opposing physiological functions of <italic>sei</italic> and <italic>ppk29</italic> (K<sup>+</sup> and Na<sup>+</sup> channels respectively), and the realization that their transcripts could form natural sense/antisense RNA duplexes (<xref ref-type="bibr" rid="bib16">Katayama et al., 2005</xref>; <xref ref-type="bibr" rid="bib10">Czech et al., 2008</xref>) led us to hypothesize that the mRNAs of <italic>sei</italic> and <italic>ppk29</italic> may regulate each other via the formation of natural endogenous dsRNAs. Since mRNA-dependent interaction between <italic>sei</italic> and <italic>ppk29</italic> requires that the two genes will be co-transcribed we first analyzed expression data from the modENcode (<xref ref-type="bibr" rid="bib8">Cherbas et al., 2011</xref>) and the FlyExpress (<xref ref-type="bibr" rid="bib33">Robinson et al., 2013</xref>) projects. Although previous studies suggested that <italic>ppk29</italic> function might be a sensory-specific (<xref ref-type="bibr" rid="bib19">Liu et al., 2012</xref>; <xref ref-type="bibr" rid="bib38">Thistle et al., 2012</xref>), our analysis revealed that <italic>sei</italic> and <italic>ppk29</italic> are co-expressed in neuronal cell lines (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>) and are both enriched in the fly central nervous system (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>). <italic>In situ</italic> hybridization in the fly brain also demonstrated neuronal co-expression (<xref ref-type="fig" rid="fig1">Figure 1B–D</xref>). Furthermore, we used cell-specific mRNA enrichment (<xref ref-type="bibr" rid="bib39">Thomas et al., 2012</xref>) to demonstrate that both genes are co-expressed in motor neurons <italic>in vivo</italic> (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). Together, these data support spatial co-expression of <italic>sei</italic> and <italic>ppk29</italic>.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01849.003</object-id><label>Figure 1.</label><caption><title><italic>sei</italic> and <italic>ppk29</italic> are co-expressed in the nervous system.</title><p>(<bold>A</bold>) The chromosomal architecture of <italic>sei</italic> and <italic>ppk29</italic> (2R:19,934,934- 19,944,660). Coding exons are in black. 3′ and 5′ untranslated regions (UTRs) are in gray. AY058350, fully sequenced <italic>sei</italic> cDNA; BT029266, fully sequenced <italic>ppk29</italic> cDNA. Black triangles represent transposons insertion sites. Arrows represent direction of transcription. Yellow boxes, <italic>sei</italic> 3′RACE product. Green boxes, <italic>ppk29</italic> 3′RACE product. (<bold>B</bold>) <italic>In situ</italic> hybridization shows <italic>sei</italic> and <italic>ppk29</italic> are co-expressed in neuronal tissues. Antisense riboprobes. Scale bar, 100 μm. (<bold>C</bold>) Higher magnification of white box in <bold>B</bold>. White arrowheads, optic lobe neurons. Red, <italic>ppk29</italic> signal; Green, <italic>sei</italic> signal; Blue, DAPI nuclear stain. Scale bar, 10 μm. (<bold>D</bold>) Sense riboprobe controls. Scale bar, 100 μm. (<bold>E</bold>) Translating Ribosome Affinity Purification (TRAP) of mRNAs from larval motor neurons shows that <italic>sei</italic> and <italic>ppk29</italic> are co-enriched in these cells relative to total body RNA. mRNA levels for each gene were measured with Real-Time qRT-PCR. N = 4 per gene. **p<0.01.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.003">http://dx.doi.org/10.7554/eLife.01849.003</ext-link></p></caption><graphic xlink:href="elife01849f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01849.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title><italic>ppk29</italic> and <italic>sei</italic> are co-expressed in Drosophila neuronal tissues.</title><p>(<bold>A</bold>) <italic>ppk29</italic> and <italic>sei</italic> are co-expressed in neuronal cell lines. Data are from the modEncode database. Expression levels represent average strand-specific unique RNA-seq reads. BG1 and BG2 are neuronal cell lines. Schneider 2 (S2) is an undefined embryonic cell line. (<bold>B</bold>) Expression of <italic>ppk29</italic> and <italic>sei</italic> in different tissues. Orange bars highlight neuronal tissues. Average data are presented as mean ± SEM (n = 4 arrays per tissue).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.004">http://dx.doi.org/10.7554/eLife.01849.004</ext-link></p></caption><graphic xlink:href="elife01849fs001"/></fig></fig-group></p><p>Previous studies suggested that transcriptional changes in ion channel transcript abundance could play a role in the adaptation of neurons to changes in environmental temperatures (<xref ref-type="bibr" rid="bib22">Marder, 2011</xref>). Thus, as a first test of our hypothesis that these two ion channels might interact antagonistically to regulate the neuronal response to heat we measured the relative expression levels of both genes in wild type animals that were adapted to variable environmental temperatures. In agreement with our hypothesis, we found that when animals adapted to high temperature (37°C) the transcripts levels of <italic>sei</italic> went up and <italic>ppk29</italic> went down relative to their levels at 25°C. In contrast, adaptation to colder temperature (13°C) led to an opposite effect on the expression of both genes (<xref ref-type="fig" rid="fig2">Figure 2</xref>). We conclude that both <italic>sei</italic> and <italic>ppk29</italic> are likely to play opposite roles in the regulation of neuronal activity in response to changes in ambient temperature, and that the possible interaction between these two genes is physiologically relevant.<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.01849.005</object-id><label>Figure 2.</label><caption><title><italic>sei</italic> and <italic>ppk29</italic> transcripts are inversely regulated in response to changes in ambient temperature.</title><p>(<bold>A</bold>) Temperature adaptation protocol. Total time from 25–37°C or 25–13°C is 7 hr. (<bold>B</bold>) Real-time qRT-PCR data. Different letters above bars represent statistically significant post hoc analyses (Tukey’s, p<0.05, N = 4 per group).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.005">http://dx.doi.org/10.7554/eLife.01849.005</ext-link></p></caption><graphic xlink:href="elife01849f002"/></fig></p></sec><sec id="s2-2"><title>Mutations in <italic>ppk29</italic> and <italic>sei</italic> have opposing effects on the behavioral and neuronal responses to heat stress</title><p>The data presented in <xref ref-type="fig" rid="fig2">Figure 2</xref>, and previous reports that indicated that mutations in <italic>sei</italic> are highly sensitive to heat stress (<xref ref-type="bibr" rid="bib40">Titus et al., 1997</xref>; <xref ref-type="bibr" rid="bib42">Wang et al., 1997</xref>), led us to hypothesize that mutations in <italic>ppk29</italic> might lead to a protection from heat stress. Based on our model presented in Figure 6, such a protective effects for <italic>ppk29</italic> mutations may arise from the loss of sodium currents or alternatively due to the upregulation of SEI-dependent potassium currents. As was previously reported, we found that multiple independent mutations in <italic>sei</italic> lead to rapid seizures and paralysis in response to acute heat stress (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). In contrast, flies carrying independent insertional alleles of <italic>ppk29</italic> demonstrated protection from the effects of heat stress relative to wild type and <italic>sei</italic> mutant animals (<xref ref-type="fig" rid="fig3">Figure 3A</xref>, <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1A,B</xref>; <italic>ppk29</italic><sup>P1</sup> and <italic>ppk29</italic><sup>P2</sup> are described in <xref ref-type="fig" rid="fig1">Figure 1A</xref>). These data confirmed our hypothesis that <italic>sei</italic> and <italic>ppk29</italic> play opposing roles in the neuronal response to heat stress, and are likely playing an important adaptive role in environmentally induced neuronal plasticity. We also observed contrasting behavioral responses to heat stress in animals that carry single copy insertional alleles of <italic>sei</italic> or <italic>ppk29 in trans</italic> with a chromosomal deficiency that covers both loci (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1C,D</xref>). These data indicate that the effects of either mutation on behavior are specific and not due to other background mutations.<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.01849.006</object-id><label>Figure 3.</label><caption><title>RNAi-dependent knockdowns of <italic>ppk29</italic> and <italic>sei</italic> expression lead to opposing effects on heat-induced paralysis.</title><p>(<bold>A</bold>) The behavioral response to heat stress in <italic>sei</italic> and <italic>ppk29</italic> mutants. Left panel, cumulative paralyzed flies over time. Right panel, same data as in left panel presented as time to total paralysis (n = 16, p<0.001, one-way ANOVA). Different letters above bars represent significantly different groups (Tukey <italic>post hoc</italic> analysis, p<0.05). (<bold>B</bold>) Representative extracellular recordings from motor neurons from each genotype at 25°C and 38°C. (<bold>C</bold>) Summary neurophysiological data (n = 8-10 per genotype, **p<0.01, ***p<0.001, one-way ANOVA with a Tukey <italic>post-hoc</italic> test). (<bold>D</bold>) Neuronal downregulation of <italic>sei</italic> or <italic>ppk29</italic> with gene-specific RNAi constructs. Data presented as in A (n = 16, p<0.001, one-way ANOVA). (<bold>E</bold>) <italic>sei</italic> and <italic>ppk29</italic> mRNA levels in <italic>sei</italic> and <italic>ppk29</italic> mutant lines. Analyses were by relative real-time quantitative RT-PCR analyses. Left panel, <italic>sei</italic> mRNA. Right panel, <italic>ppk29</italic> mRNA (n = 4 per genotype, p<0.05, one-way ANOVA). (<bold>F</bold>) <italic>sei</italic> and <italic>ppk29</italic> mRNA levels in <italic>sei</italic> and <italic>ppk29</italic> RNAi-knockdown lines. Analyses as in E (n = 4 per genotype, p<0.05, one-way ANOVA). Data are presented as mean ± SEM. Different letters above bars represent significantly different groups (Tukey <italic>post hoc</italic> analysis, p<0.05).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.006">http://dx.doi.org/10.7554/eLife.01849.006</ext-link></p></caption><graphic xlink:href="elife01849f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01849.007</object-id><label>Figure 3—figure supplement 1.</label><caption><title><italic>ppk29</italic> mutations confer protection from heat-induced paralysis.</title><p>(<bold>A</bold>) Two independent <italic>ppk29</italic> transposon-insertional alleles do not complement each other. Data presented as cumulative paralyzed flies over time. (<bold>B</bold>) Same data as in <bold>A</bold> presented as total time to paralysis. Different letters above bars represent significantly different genotypes (one-way ANOVA Tukey's <italic>post hoc</italic> test; n = 16, p<0.001). (<bold>C</bold> and <bold>D</bold>) <italic>sei</italic> or <italic>ppk29</italic> transposon-insertional alleles in trans across a deficiency chromosome (<italic>Df</italic><sup><italic>BSC136</italic></sup>) that covers both loci. Control <italic>Df</italic><sup><italic>BSC652</italic></sup> has the same genetic background as <italic>Df</italic><sup><italic>BSC136</italic></sup> but does not cover the <italic>sei/ppk29</italic> loci. Analyses and data presentations are as in panels <bold>A</bold> and <bold>B</bold>. (<bold>E</bold> and <bold>F</bold>) Gene knockdowns of <italic>sei</italic> or <italic>ppk29</italic> by mutations or neuronal RNAi in larvae lead to heat sensitivity or protection phenotypes respectively that are analogous to the adult phenotypes (<bold>G</bold>) Acute RNAi-dependent targeting of <italic>ppk29</italic> or <italic>sei</italic> in the adult nervous system with the GeneSwitch (GS) version of the pan-neuronal promoter <italic>elav</italic> was sufficient to phenocopy the mutant phenotypes. Mutant phenotypes were apparent only in the RU486 feeding group (RU486+) (n = 16, ***p<0.001; two-way ANOVA with a Tukey's <italic>post-hoc</italic> test). The interaction term between genotype and drug was also significant (<italic>p</italic>=<0.001). Data are presented as mean ± SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.007">http://dx.doi.org/10.7554/eLife.01849.007</ext-link></p></caption><graphic xlink:href="elife01849fs002"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01849.008</object-id><label>Figure 3—figure supplement 2.</label><caption><title>Mutations in <italic>sei</italic> and <italic>ppk29</italic> do not affect gross locomotion at room temperature.</title><p>(NS; n = 10 per genotype, one-way ANOVA with Tukey's <italic>post-hoc</italic> test). Data are presented as mean ± SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.008">http://dx.doi.org/10.7554/eLife.01849.008</ext-link></p></caption><graphic xlink:href="elife01849fs003"/></fig></fig-group></p><p>Previous studies indicated that the temperature-sensitive phenotype of <italic>sei</italic> mutants is associated with heat-induced neuronal hyperexcitability (<xref ref-type="bibr" rid="bib15">Kasbekar et al., 1987</xref>). Therefore, we hypothesized that mutations in <italic>ppk29</italic> will lead to a hypoexcitable neuronal phenotype under heat stress. We found that the spontaneous neuronal activity of larval motor neurons is not different between <italic>ppk29</italic>, <italic>sei</italic> and wild type animals at 25°C. In contrast, at 38°C wild type neurons show a small but significant increase in neuronal activity, while <italic>sei</italic> mutant neurons become hyperexcitable. In contrast to <italic>sei</italic> null and wild type animals, <italic>ppk29</italic> mutants are unable to increase neuronal firing rates in response to heat stress, which is consistent with a hypoexcitability phenotype (<xref ref-type="fig" rid="fig3">Figure 3B,C</xref>). We also confirmed that the larval excitability phenotypes of <italic>sei</italic> and <italic>ppk29</italic> mutants are correlated with behavior. As in our neurophysiological studies, we found that at 25°C all genotypes show normal larval locomotion (<xref ref-type="other" rid="video1 video2 video3">Videos 1–3</xref>). However, exposure to 38°C lead to an abnormal seizure-like locomotion in wild type animals (twitching and rolling). This phenotype is significantly higher in <italic>sei</italic> mutant and RNAi knockdown larvae but completely absent in <italic>ppk29</italic> mutant larvae (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1E,F</xref>; <xref ref-type="other" rid="video4 video5 video6">Videos 4–6</xref>). We conclude that <italic>sei</italic> and <italic>ppk29</italic> affect the behavioral sensitivity to heat stress via the contrasting regulation of neuronal excitability in both larval and adult stages.<media content-type="glencoe play-in-place height-250 width-310" id="video1" mime-subtype="mov" mimetype="video" xlink:href="elife01849v001.mov"><object-id pub-id-type="doi">10.7554/eLife.01849.009</object-id><label>Video 1.</label><caption><p>Wild type larva at 25<bold>°</bold>C.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.009">http://dx.doi.org/10.7554/eLife.01849.009</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video2" mime-subtype="mov" mimetype="video" xlink:href="elife01849v002.mov"><object-id pub-id-type="doi">10.7554/eLife.01849.010</object-id><label>Video 2.</label><caption><p><italic>sei</italic><sup>P</sup> larva at 25<bold>°</bold>C.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.010">http://dx.doi.org/10.7554/eLife.01849.010</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video3" mime-subtype="mov" mimetype="video" xlink:href="elife01849v003.mov"><object-id pub-id-type="doi">10.7554/eLife.01849.011</object-id><label>Video 3.</label><caption><p><italic>ppk29</italic><sup>P1</sup> larva at 25<bold>°</bold>C.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.011">http://dx.doi.org/10.7554/eLife.01849.011</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video4" mime-subtype="mov" mimetype="video" xlink:href="elife01849v004.mov"><object-id pub-id-type="doi">10.7554/eLife.01849.012</object-id><label>Video 4.</label><caption><p>Wild type larva at 38<bold>°</bold>C.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.012">http://dx.doi.org/10.7554/eLife.01849.012</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video5" mime-subtype="mov" mimetype="video" xlink:href="elife01849v005.mov"><object-id pub-id-type="doi">10.7554/eLife.01849.013</object-id><label>Video 5.</label><caption><p><italic>sei</italic><sup>P</sup> larva at 38<bold>°</bold>C.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.013">http://dx.doi.org/10.7554/eLife.01849.013</ext-link></p></caption></media><media content-type="glencoe play-in-place height-250 width-310" id="video6" mime-subtype="mov" mimetype="video" xlink:href="elife01849v006.mov"><object-id pub-id-type="doi">10.7554/eLife.01849.014</object-id><label>Video 6.</label><caption><p><italic>ppk29</italic><sup>P1</sup> larva at 38<bold>°</bold>C.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.014">http://dx.doi.org/10.7554/eLife.01849.014</ext-link></p></caption></media></p><p>Similarly to the mutant adult phenotypes, neuronal RNAi-dependent knockdown of <italic>sei</italic> and <italic>ppk29</italic> mRNAs with the neuronal <italic>elav</italic>-GAL4 driver lead to contrasting phenotypes that are identical to the phenotypes observed in mutants (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). These data demonstrate that the observed phenotypes are neuronal-specific and suggest that quantitative changes in neuronal mRNA levels of either <italic>sei</italic> or <italic>ppk29</italic> are sufficient to induce high-sensitivity or protective phenotypes respectively. Analyses of mRNA levels in mutants and RNAi-expressing animals support the hypothesis that downregulation of <italic>ppk29</italic> mRNA is associated with increased <italic>sei</italic> mRNA levels, but the converse effect is not evident (<xref ref-type="fig" rid="fig3">Figure 3E,F</xref>). Together, these data demonstrate that the regulatory interaction between the mRNAs of <italic>sei</italic> and <italic>ppk29</italic> is not symmetric; changes in <italic>ppk29</italic> mRNA level downregulate <italic>sei</italic> mRNA, but not the other way around. We also observed contrasting phenotypes when we expressed the same gene-specific RNAi constructs in adult neurons only by using the hormonally-induced GeneSwitch version of the <italic>elav</italic>-GAL4 (<xref ref-type="bibr" rid="bib27">Osterwalder et al., 2001</xref>; <xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1G</xref>). These data show that the contrasting effects of <italic>sei</italic> and <italic>ppk29</italic> mRNA dowregulation on the neuronal response to heat stress are physiological rather than developmental. We did not observe any general locomotion defects in <italic>sei</italic> or <italic>ppk29</italic> mutants at 25°C (<xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2</xref>).</p><p>Together, data presented in <xref ref-type="fig" rid="fig2 fig3">Figures 2 and 3</xref> suggest that the protective effect of mutations in <italic>ppk29</italic> are symptomatic in the sense that they lead to a pre-stress increase in <italic>sei</italic> transcript levels, which leads to a higher ability of the nervous system to deal with the acute heat stress even without prior adaptation to slow temperature increase.</p></sec><sec id="s2-3"><title>The Protective effect of <italic>ppk29</italic> mutations are mediated by <italic>sei</italic> channel activity</title><p>Although our data suggest that the contrasting heat-induced phenotypes of <italic>sei</italic> and <italic>ppk29</italic> mutants are possibly mediated via mRNA-dependent interactions, they do not exclude the possibility that the two channels also interact at the protein level. Therefore, we investigated whether the protection from heat stress in <italic>ppk29</italic> mutants is mediated by the loss of PPK29 channel activity or the up-regulation of <italic>sei</italic> mRNAs (As shown in <xref ref-type="fig" rid="fig3">Figure 3E,F</xref>). To test this we first blocked SEI channel activity in wild type and <italic>ppk29</italic> mutant animals by using two different hERG channel blockers (<xref ref-type="bibr" rid="bib2">Afrasiabi et al., 2010</xref>). These studies reveal that blocking SEI activity in wild type animals phenocopies the heat sensitivity phenotype of the <italic>sei</italic> mutation, which indicate that the drugs are successfully blocking SEI channels in the fly. Similar to wild type animals, blocking SEI activity in <italic>ppk29</italic> mutants reduce their resistance to heat stress to a level comparable to wild type animals in a dose-dependent manner (<xref ref-type="fig" rid="fig4">Figure 4A</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>). These data are in agreement with the expression data (<xref ref-type="fig" rid="fig3">Figure 3E,F</xref>), and strongly indicate that the <italic>ppk29</italic>-mediated protection from heat stress is due, at least in part, to increased SEI K<sup>+</sup> channel activity rather then the loss of <italic>ppk29</italic>-dependent Na<sup>+</sup> currents.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.01849.015</object-id><label>Figure 4.</label><caption><title>The Protective Effect of <italic>ppk29</italic> Mutations is Mediated by SEI Channel Activity.</title><p>(<bold>A</bold>) Blocking SEI channel activity in <italic>ppk29</italic> mutants with the hERG channel blocker Cisapride eliminate the protective effect in a dose dependent manner (n = 8 per genotype, p<0.01, two-way ANOVA; genotype, dose, and genotype by dose showed significant effects, <italic>p</italic>=<0.001). (<bold>B</bold>) Schematic representation of transgenic constructs. (<bold>C</bold>) Neuronal expression of <italic>ppk29</italic>-3′UTR is sufficient to rescue the majority of the protective effect of the <italic>ppk29</italic> mutation (n = 12, p<0.01, one-way ANOVA). Data are presented as mean ± SEM. Different letters above bars represent significantly different groups (Tukey <italic>post hoc</italic> analysis, p<0.05). (<bold>D</bold>) Neuronal expression of <italic>sei</italic> cDNA with or without its endogenous 3′UTR, but not the 3′UTR alone<italic>,</italic> is sufficient to rescue the <italic>sei</italic> mutation (n = 12, p<0.001, one-way ANOVA).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.015">http://dx.doi.org/10.7554/eLife.01849.015</ext-link></p></caption><graphic xlink:href="elife01849f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.01849.016</object-id><label>Figure 4—figure supplement 1.</label><caption><title>The protective effect of <italic>ppk29</italic> mutations depends on SEI K<sup>+</sup> channel activity.</title><p>Treating <italic>ppk29</italic> mutants flies with hERG inhibitors cisapride (<bold>A</bold>) and E−4301 (<bold>B</bold>) lead to a significantly faster heat-induced seizures and paralyses in all tested genotypes (n = 8 for each genotype, two-way ANOVA with a Tukey's <italic>post-hoc</italic> test; the interaction between genotype and concentration is significant for both drugs, <italic>p</italic>=<0.001). Average data are presented as mean ± SEM. Different letters above bars represent significantly different groups.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.016">http://dx.doi.org/10.7554/eLife.01849.016</ext-link></p></caption><graphic xlink:href="elife01849fs004"/></fig></fig-group></p></sec><sec id="s2-4"><title>The <italic>ppk29</italic> mRNA affects <italic>sei</italic> function by serving as a natural antisense regulatory RNA</title><p>Our hypothesis predicts that the 3’UTR of <italic>ppk29</italic> can regulate <italic>sei</italic> function by acting as a natural antisense RNA. To test directly this hypothesis we generated transgenic fly lines that can express the cDNAs of either <italic>sei</italic> or <italic>ppk29</italic> with or without their endogenous 3′UTR, or their 3′UTRs alone (<xref ref-type="fig" rid="fig4">Figure 4B</xref>) by using the UAS-GAL4 system. Remarkably, we found that the expression of the <italic>ppk29</italic> endogenous 3′UTR alone or the cDNA with the 3′UTR is sufficient to rescue the <italic>ppk29</italic> mutation. In contrast, expression of <italic>ppk29</italic> cDNA alone is not sufficient to completely rescue the phenotype of the <italic>ppk29</italic> mutation (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). In agreement with the pharmacological studies, these data demonstrate that the main protective effect of <italic>ppk29</italic> mutations is mediated via 3′UTR-dependent regulation of SEI, independent of PPK29 channel functions. Nevertheless, we also found that a complete rescue of the <italic>ppk29</italic> mutation phenotype require the expression of the <italic>ppk29</italic> cDNA with its endogenous 3′UTR. Therefore, PPK29 channel activity may also contribute neuronal excitability independent of <italic>sei</italic> regulation. In addition, since the observed effects of <italic>ppk29</italic> transgenes on <italic>sei</italic> function are <italic>in trans,</italic> these data show that the two genes can interact at the transcript level independent of their chromosomal proximity. Unlike for <italic>ppk29</italic>, the neuronal expression of <italic>sei</italic> cDNA with or without its endogenous 3′UTR, but not the 3′UTR alone, is sufficient to rescue the <italic>sei</italic> mutation (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). These data further show that <italic>sei</italic> is the focal physiological element in the neuronal response to heat stress, and that the mRNA 3′UTR-dependent interaction between <italic>sei</italic> and <italic>ppk29</italic> is not symmetric.</p><p>We next investigated the role of <italic>ppk29</italic> 3′UTR in regulating <italic>sei</italic> mRNA expression and heat-induced seizures and paralysis. Consistent with our model, neuronal overexpression of <italic>sei</italic> cDNA (with or without its endogenous 3′UTR but not the 3′UTR alone) is sufficient to protect animals from heat stress as in <italic>ppk29</italic> mutants (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). We also found that neuronal overexpression of a <italic>ppk29</italic> cDNA with its endogenous 3′UTR or <italic>ppk29</italic> 3′UTR alone, but not the cDNA alone, is sufficient to induce heat sensitivity as in <italic>sei</italic> mutants (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). In agreement with the behavioral data, overexpression of the <italic>ppk29</italic>-3′UTR is sufficient to reduce endogenous <italic>sei</italic> mRNA levels but overexpression of <italic>sei</italic>-3′UTR alone does not have a similar effect on <italic>ppk29</italic> (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). These data demonstrate that elevated levels of <italic>ppk29</italic>-3′UTR alone in <italic>trans</italic> are sufficient to affect neuronal physiology by downregulating <italic>sei</italic> mRNA levels. Expression of the <italic>ppk29</italic> related constructs specifically in the adult nervous system by using the GeneSwitch <italic>elav</italic>-GAL4 driver demonstrate that the observed effects of <italic>ppk29</italic>-3′UTR overexpression on <italic>sei</italic> function and behavior are physiological and not developmental (<xref ref-type="fig" rid="fig5">Figure 5D</xref>). Thus, our data prove that downregulation of <italic>sei</italic> expression leads to neuronal heat sensitivity while increased <italic>sei</italic> expression leads to a protection, and that the relative abundance of <italic>sei</italic> transcripts in neurons is affected by the expression levels of <italic>ppk29</italic>.<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.01849.017</object-id><label>Figure 5.</label><caption><title><italic>ppk29</italic>-dependent regulation of <italic>sei</italic> depends on the canonical RISC pathway.</title><p>(<bold>A</bold>) Neuronal overexpression of <italic>sei</italic> cDNA with or without its endogenous 3′UTR in wild type animals leads to a protection from heat-induced paralysis (n = 12, p<0.001, one-way ANOVA). (<bold>B</bold>) Neuronal overexpression of the <italic>ppk29</italic> cDNA with its endogenous 3′UTR or the 3′UTR alone, but not the <italic>ppk29</italic> cDNA lone, is sufficient to induce <italic>sei</italic> mutant-like heat sensitivity phenotype (n = 12, p<0.001, one-way ANOVA). (<bold>C</bold>) Real-time qRT-PCR analyses of <italic>sei</italic> and <italic>ppk29</italic> mRNA level. Overexpression of <italic>ppk29</italic> cDNA with its 3′UTR or the 3′UTR alone<italic>,</italic> but not the cDNA alone, is sufficient to downregulate endogenous <italic>sei</italic> mRNA levels (left panel) but not conversely (right panel) (n = 4, p<0.05, one-way ANOVA). (<bold>D</bold>) Adult-specific neuronal overexpression of <italic>ppk29</italic>-3′UTR with the hormone inducible GeneSwitch <italic>elav</italic>-GAL4 is sufficient to induce <italic>sei</italic> mutant-like phenotype (n = 12, ***p<0.001; two-way ANOVA, genotype, RU486, and their interaction are significant, <italic>p</italic>=<0.001). (<bold>E</bold> and <bold>F</bold>) The effect of <italic>ppk29</italic> 3′UTR overexpression on heat sensitivity and sei mRNA downregulation is abolished in the <italic>Dcr-2</italic> mutant background (n = 12, one-way ANOVA). (<bold>F</bold>) Real-time qRT-PCR (n = 4, NS, one-way ANOVA). Data are presented as mean ± SEM. Different letters above bars represent significantly different groups (Tukey post hoc analysis, p<0.05).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.017">http://dx.doi.org/10.7554/eLife.01849.017</ext-link></p></caption><graphic xlink:href="elife01849f005"/></fig></p></sec><sec id="s2-5"><title>The mRNA-dependent interaction between <italic>ppk29</italic> and <italic>sei</italic> depends on the canonical endogenous siRNA pathway</title><p>The above data show that <italic>ppk29</italic> mRNA can serve as a regulatory antisense RNA in addition to its capacity to encode for a DEG/ENaC subunit. The processing of endo-siRNAs depends on <italic>Dicer2</italic> (<italic>Dcr-2</italic>) in flies (<xref ref-type="bibr" rid="bib10">Czech et al., 2008</xref>). Thus, we investigated whether the regulatory impact of <italic>ppk29</italic>-3′UTR on <italic>sei</italic> mRNA levels and behavior depends on the RNAi machinery. We find that in the background of the <italic>Dcr-2</italic> mutation neuronal overexpression of the <italic>ppk29</italic>-3′UTR has no effect on the behavioral response to heat stress (<xref ref-type="fig" rid="fig5">Figure 5E</xref>) or on the expression levels of <italic>sei</italic> (<xref ref-type="fig" rid="fig5">Figure 5F</xref>). These data demonstrate that the regulatory function of <italic>ppk29</italic>-3′UTR depends on the endogenous siRNA pathway.</p><p>The finding that <italic>ppk29</italic> can regulate <italic>sei</italic> mRNA levels via the canonical siRNA pathway may also explain why the mRNA 3′UTR-dependent interaction between sei and <italic>ppk29</italic> is not symmetric. Recent studies of the molecular mechanism that underly the specificity of the RNAi machinery indicate that the protein complex that mediate the recognition of the target RNA by the short dsRNA are not symmetrical. Thus, via mechanisms that are not fully understood, RISC treats only one of the strands as a guide (<xref ref-type="bibr" rid="bib41">Tomari et al., 2004</xref>; <xref ref-type="bibr" rid="bib29">Rand et al., 2005</xref>; <xref ref-type="bibr" rid="bib5">Betancur and Tomari, 2012</xref>; <xref ref-type="bibr" rid="bib24">Noland and Doudna, 2013</xref>). It is likely that a similar mechanism is at play here. Our data indicate that when forming siRNA duplexes, <italic>ppk29</italic> 3′UTR is the preferred guide strand during RISC loading and the subsequent mRNA target identification.</p></sec><sec id="s2-6"><title>Concluding remarks</title><p>Here we describe a novel mechanism for the regulation of ion channel functions and neuronal excitability via a natural antisense mRNA (<xref ref-type="fig" rid="fig6">Figure 6</xref>). While this is a novel mechanism, it is by no means the only known RNA-dependent mechanism for the regulation of ion channel functions. For example, the double-stranded RNA helicase <italic>maleless</italic> (<italic>mle</italic>) regulates the <italic>Drosophila</italic> voltage-gated sodium channel <italic>paralytic</italic> (<italic>para</italic>) via A-to-I RNA editing. Mutations in <italic>mle</italic> lead to aberrant editing of <italic>para</italic>, splicing errors, and subsequent low channel activity (<xref ref-type="bibr" rid="bib31">Reenan et al., 2000</xref>). Other examples include the putative transcription factor <italic>down and out</italic> (<italic>dao</italic>), which seem to affect <italic>sei</italic> transcription levels (<xref ref-type="bibr" rid="bib11">Fergestad et al., 2010</xref>), the potassium-independent effects of the <italic>sei</italic>-related mammalian EAG potassium channel on cellular signaling (<xref ref-type="bibr" rid="bib13">Hegle et al., 2006</xref>), and other diverse mechanisms for the co-regulation of various ion channels (<xref ref-type="bibr" rid="bib21">MacLean et al., 2005</xref>; <xref ref-type="bibr" rid="bib30">Ransdell et al., 2013</xref>).<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.01849.018</object-id><label>Figure 6.</label><caption><title>Cartoon depicting a model for the molecular interaction between <italic>sei</italic> and <italic>ppk29</italic>.</title><p>The chromosomal organization of these two genes suggest they could generate endogenous siRNA by convergent transcription. (I) The complementary 3′UTRs of <italic>sei</italic> and <italic>ppk29</italic> mRNAs form a dsRNA. (II) <italic>Dicer-2</italic> cleaves dsRNAs into siRNAs. (III) The loaded RISC complex targets <italic>sei</italic> transcripts for degradation via the canonical siRNA pathway.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.018">http://dx.doi.org/10.7554/eLife.01849.018</ext-link></p></caption><graphic xlink:href="elife01849f006"/></fig></p><p>It is unlikely that the type of interaction we have identified between the mRNAs of <italic>sei</italic> and <italic>ppk29</italic> is unique. Bioinformatic analyses of genome sequences show that at least two of the three fly and three out of the eight human <italic>eag</italic>-like (KCNH-type) channels are organized in a chromosomal architecture that is similar to that of <italic>sei</italic> and <italic>ppk29</italic> (<xref ref-type="table" rid="tbl1">Table 1</xref>). The functional diversity of the converging genes in each of the pairs we uncovered suggest that, like in the case of <italic>sei/ppk29</italic>, the actual protein identity is secondary to the mRNA level interactions. However, to conclusively test this hypothesis will require additional experimental molecular and biochemical analyses of these loci in the fly and mammalian systems.<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.01849.019</object-id><label>Table 1.</label><caption><p>Fly and human <italic>eag</italic>-like channels that are possibly regulated via convergent transcription with an unrelated mRNA</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.01849.019">http://dx.doi.org/10.7554/eLife.01849.019</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Species</th><th>eag-like gene</th><th>Converging gene</th></tr></thead><tbody><tr><td rowspan="2"><italic>Drosophila</italic></td><td><italic>sei</italic></td><td><italic>ppk29</italic></td></tr><tr><td><italic>eag</italic></td><td><italic>hiw</italic></td></tr><tr><td rowspan="3">Human</td><td>KCNH1</td><td>HHAT</td></tr><tr><td>KCNH3</td><td>MCRS1</td></tr><tr><td>KCNH7</td><td>GCA</td></tr></tbody></table><table-wrap-foot><fn><p>Please note that the converging genes are functionally diverse, which suggest that their protein identities might not play a role in their regulatory functions.</p></fn></table-wrap-foot></table-wrap></p><p>Our findings also indicate that the regulatory interaction between <italic>sei</italic> and <italic>ppk29</italic> may play a role in the homeostatic response to slow changes in environmental temperature (<xref ref-type="fig" rid="fig2">Figure 2</xref>). However, our current genetic and transgenic tools make it impossible for us to completely disentangle the direct effects of temperature changes on <italic>sei</italic> and <italic>ppk29</italic> transcription and the indirect effects via the interactions of their mRNAs. Thus, more studies will be needed to further establish <italic>ppk29</italic> mRNA as a homeostatic factor, beyond its effects on the acute heat response.</p><p>In contrast to the linear simplicity of the ‘central dogma of molecular biology’ (<xref ref-type="bibr" rid="bib9">Crick, 1970</xref>), we now know that the true molecular landscape of cells is complex and far from linear. In this regard, our studies provide an additional layer of regulatory complexity, and support the idea that mRNAs, which are typically thought to solely act as the template for protein translation, can also serve as regulatory RNAs, independent of their protein-coding capacity. Thus, the abundance of convergent transcription of protein-coding genes in eukaryotic genomes suggests that many other mRNAs might serve dual functions that are not necessary associated with the same cellular or physiological processes. Furthermore, although the phenomenon of mRNA-dependent interaction between the two genes we describe here occurs in <italic>cis</italic> (<xref ref-type="fig" rid="fig6">Figure 6</xref>), we currently have no reason to assume that similar interactions between RNAs cannot occur in <italic>trans</italic> as well. Consequently, it is likely that some of the evolutionary changes observed in mRNAs, including those that are considered ‘neutral’, should be re-evaluated in light of the possible regulatory function that some mRNAs might exert independently of the proteins they encode.</p></sec></sec><sec id="s3" sec-type="materials|methods"><title>Materials and methods</title><sec id="s3-1"><title>Fly strains</title><p>Flies (<italic>Drosophila melanogaster</italic>) were raised on standard cornmeal-agar food at 25°C and 60% relative humidity with a 12 hr light: dark cycle. Unless stated differently, the <italic>w</italic><sup><italic>1118</italic></sup> strain was used a ‘wild type’. In our hands, the heat-induced behavior and physiology of these flies were not different from the <italic>Canton</italic>-S strain. The original stocks for <italic>ppk29</italic><sup>P1</sup> and <italic>sei</italic><sup>P</sup> were obtained from the Bloomington Stock Center (Stock No. 19016, and 21935). The <italic>ppk29</italic><sup>P2</sup> stock (f04205) was from the Exelixis collection at Harvard Medical School. All insertional alleles used in our studies were backcrossed into the <italic>w</italic><sup>1118</sup> background for six generation. The <italic>sei</italic><sup>ts1</sup> EMS-allele was from the Ganetzki lab (U of Wisconsin). The deficiency lines <italic>Df(2R)BSC136</italic> and <italic>Df(2R)BSC652</italic> (9424 and 25742), <italic>elav</italic>-GAL4; <italic>UAS-Dicer2</italic> (25750), <italic>UAS-ppk29</italic><sup>RNAi</sup> (27241), <italic>elav-Gal4</italic> (33805) <italic>,elav</italic>-<italic>GeneSwitch</italic>-GAL4 (43642) and <italic>Dicer-2</italic> mutant (32064) were from the Bloomington Stock Center (stock no.). <italic>UAS-sei</italic><sup>RNAi</sup> was from VDRC (v3606GD).</p></sec><sec id="s3-2"><title>Transgenic constructs</title><p>The transgene <italic>sei</italic>Δ3′UTR was generated by amplifying <italic>sei</italic> coding sequence (variant RA, NP_476713) with primers 5′-AAAAGCGGCCGCATGTCCCACAAATCTTGCGT-3′ and 5′-AAAATCTAGACTAATTATTATTATCGAACAAGTCAAGGTG-3′ from cDNA clone GH12235. The transgene <italic>sei</italic>-3′UTR was generated by amplifying the same <italic>sei</italic> coding sequence plus its 3′UTR (<italic>sei</italic>-RA, length = 95) with primers 5′-AAAAGCGGCCGCATGT-CCCACAAATCTTGCGT-3′ and 5′-AAAATCTAGATTTTCGGTTAGGACCTTTATTGC-3′. The transgene <italic>ppk29</italic>Δ3′UTR was generated by amplifying <italic>ppk29</italic> coding sequence (variant PD, NP_001097442) with 5′-AAAAGCGGCCGCATGTGGCGGAAGTCAGTA-ATG-3′ and 5′-AAAATCTAGACTAACCGAAAATCATGGTCTTGA-3′ from cDNA clone IP06558. The transgene <italic>ppk29</italic>-3′UTR was generated by amplifying the same <italic>ppk29</italic> coding sequence plus its 3′UTR (ppk29-RD, length = 112) with primers 5′-AAAAGCGGC-CGCATGTGGCGGAAGTCAGTAATG-3′ and 5′-AAATCTAGATTGACTTGTTCGATAAT-AATAATTAGGGC-3′. The transgene GFP-3′UTR<sub>sei</sub> was generated by amplifying the EGFP ORF from the pEGFP-N3 vector with primers 5′-AAAAGCGGCCGCATGGTGA-GCAAGGGCGA-3′ and 5′-CTTGTGCACAAATAAATAAGATTCACTTGTACAGCTCGT-CCATG-3′ and the <italic>sei</italic>-3′UTR from cDNA clone GH12235 with primers 5′-CATGGACG-AGCTGTACAAGTGAGGCTCACTTATGCTCGCTCAATCCGAATTATCTTATTTATTTGTGCACAAGCTGTTGCGAGGCTAAAGAG-3′ and 5′-AAAATCTAGATTTTCGGTTAGGA-CCTTTATTGCTTTTCGCTCTTTAGCCTCGCAACAGCTTGTGCACAAATAAATAAGAT-3′ followed by PCR fusion of the two DNA fragments. The transgene mCherry-3′UTR<sub>ppk29</sub> was generated by amplifying the mCherry ORF from the pCAMBIA-1300 vector with primers 5′-AAAAGCGGCCGCATGGTGAGCAAGGGCGA-3′ and 5′-TAAAGAGCGAA-AAGCAATAAAGGTCTTACTTGTACAGCTCGTCCATGC-3′ and <italic>ppk29</italic>-3′UTR from cDNA clone IP06558 with primers 5′-GCATGGACGAGCTGTACAAGTAAGACCTTTATTGCTTTTCGCTCTTTAGCCTCGCAACAGCTTGTGCACAAATAAATAAGATAATTCGGATTG-3′ and 5′-AAAATCTAGATTGACTTGTTCGATAATAATAATTAGGGCTCACT-TATGCTCGCTCAATCCGAATTATCTTATTTATTTGT-3′ followed by PCR fusion of the two DNA fragments. All transgenes were verified by sequencing and subsequently subcloned into the <italic>pUASTattB</italic> plasmid. Each of the six individual transgenes was transformed by <italic>Phi</italic>C31 integrase-based transgenesis into two different landing chromosomal landing sites (2L:1476459 and 3L:11837236) (<xref ref-type="bibr" rid="bib4">Bateman et al., 2006</xref>).</p></sec><sec id="s3-3"><title>Adult heat-induced paralysis assay</title><p>20–30 flies (2–3 days post eclosion) were anesthetized by CO<sub>2</sub> and transferred to standard <italic>Drosophila</italic> vials containing fresh food for 24 hr. On test day, 10 flies (1:1 male/female ratio) were transferred to an empty polystyrene vial (Genesee Scientific, San Diego, CA) without anesthesia. Flies were allowed to recover for 10 min before vials were immersed in a 41 ± 1°C water bath (ISOTEMP105; Fisher Scientific, Pittsburgh, PA). The number of cumulative paralyzed flies was counted every 15 s until all flies were paralyzed at the bottom of the vial. The proportion of paralyzed flies and the time it takes to reach total paralysis for all 10 flies were used to generate heat-induced paralysis scores.</p></sec><sec id="s3-4"><title>Negative geotaxis assay</title><p>We used the negative geotaxis response as an assay for general locomotion as we previously described (<xref ref-type="bibr" rid="bib20">Lu et al., 2012</xref>). In short, groups of ten flies were introduced into an empty vial without anesthesia. Additional empty vial was taped on top. To assay locomotion, bottom vial was tapped down lightly and the number of flies that climbed above a marked 15 cm line in 15 s was recorded.</p></sec><sec id="s3-5"><title>Larval locomotion</title><p>Feeding stage 3<sup>rd</sup>-instar larvae were used. Each larva was briefly washed in distilled water to remove all food debris and then transferred to a 3% agar plate that was equilibrated to 25 ± 1°C or 38 ± 1°C. Recording of behavior started 3 min post introduction by videotaping animals for 2 min. The total numbers of larval side-twitching events were used to quantify larval ‘seizure’ like behavior (<xref ref-type="other" rid="video1 video2 video3 video4 video5 video6">Videos 1–6</xref>).</p></sec><sec id="s3-6"><title>Pharmacological treatments</title><p>Stock solutions of hERG blockers were kept as 10 mM Cisapride (Sigma-Aldrich, St. Louis, MO, USA) in DMSO and 100 mM E 4301 (Alomone labs, Jerusalem, Israel) in distilled water. Working solution were made by diluting the stock solutions in in 2% (wt/vol) sucrose solution. Flies were treated in groups of 20 adults (1–2 days post eclosion, 1:1 mixed sex) in a vial containing a Kimwipe tissue paper soaked with 1 ml of the drug. Flies were allowed to feed on the drug for three days at 25°C and 60% humidity. Prior to the heat stress test, treated flies were transferred to a new vial containing standard fly food without drugs for two hours. Heat-induced paralysis was assayed as above.</p></sec><sec id="s3-7"><title>RU-486 activation of the <italic>elav</italic>-GeneSwitch GAL4 line</title><p>A 10 mM stock solution of RU486 (mifepristone, Sigma-Aldrich, St. Louis, MO, USA) was prepared in 80% ethanol. Then, the RU486 working solution was diluted to the final concentration (500 μM) in 2% sucrose. The drug was delivered to flies as described above. Flies were treated with RU-486 or 2% sucrose for 7 days at 25°C and 60% humidity. During the feeding period 200 μl RU486 working solution or a 2% sucrose solution control were added to each vial every 2 days. Prior to behavioral tests, flies were transferred into vials containing fresh standard fly food without drugs for two hours. Heat-induced paralysis was assayed as above.</p></sec><sec id="s3-8"><title>Extracellular electrophysiological recording</title><p>Extracellular recordings of larval segmental nerves were as previously reported (<xref ref-type="bibr" rid="bib35">Simon et al., 2009</xref>). Although these neuronal bundles include both motor and sensory fibers, previous studies demonstrated that the majority of the burst firing activity patterns observed in this preparation are generated by motor neurons alone (<xref ref-type="bibr" rid="bib12">Fox et al., 2006</xref>). Feeding stage 3<sup>rd</sup>-instar larvae were dissected in HL-3 solution containing 2.0 mM CaCl<sub>2</sub>, 70 mM NaCl, 5 mM KCl, 4 mM MgCl<sub>2</sub>, 10 mM NaHCO<sub>3</sub>, 5 mM trehalose, 115 mM sucrose, 5 mM HEPES, pH 7.2. Segmental nerves connecting to the ventral nerve cord were left intact. We preferentially recorded from segmental nerves that innervate the anterior segments with a polished glass electrode to suck up the nerves. Neuronal signals were filtered by a high-pass filter set at 100 Hz and a low-pass filter set at 10 kHz (Clampex software package). The extracellular temperature was manipulated in the recording chamber by using a temperature-control perfusion system (Multi Channel Systems MCS, Baden-Württemberg, Germany) using the following protocol: (1) Recording of neuronal activity started once the perfusion system was stable at 25°C for at least 1 min. Neuronal spikes were recorded at baseline for 3 min. (2) To acutely raise the temperature, perfusion was turned off until it reached 38°C stabley for at least 1 min. (3) Recording at 38°C was initiated 1 min after perfusion was turned on again for 3 min.</p></sec><sec id="s3-9"><title>Real-time qRT-PCR</title><p>Total RNA from adult fly heads or whole flies was extracted with the TRIzol reagent (Applied Biosystems, Grand Island, NY). First strand cDNA pool was made from total RNA (1 μg) with random hexamere oligos <italic>SuperScript</italic> II reverse transcriptase (Invitrogen, Grand Island, NY) in 20 μl reacting volume. cDNA pool was diluted (1:5) in distilled water. Gene specific assays were used to quantify genes with the SybrGreen method using the PowerSYBR Green Super PCR Mix (ABI Inc., Grand Island, NY) on an ABI7500 machine (Applied Biosystems) using default parameters. Gene specific assays were designed with the PrimeTime qPCR Assay design tool (Integrated DNA Technologies). The housekeeping gene <italic>rp49</italic> was used as an RNA loading control as previously described (<xref ref-type="bibr" rid="bib20">Lu et al., 2012</xref>). Data were transformed and analyzed according to the ΔΔCt method and are represented as relative fold differences (<xref ref-type="bibr" rid="bib20">Lu et al., 2012</xref>). Primer sequences used are: <italic>sei-</italic>forward: 5′-TTATTCAAAGGCTGTACTCGGG-3′; <italic>sei-</italic>reverse: 5′-GATGCCATTCGTATAGGTCCAG-3′; <italic>ppk29-</italic>forward: 5′-CCTCTCAGGTATTCTTCGTTGG-3′; <italic>ppk29-</italic>reverse: 5′-TCGGTG-GAGATGGTATAGGTC-3′; <italic>rp49-</italic>forward: 5′-CACCAAGCACTTCATCCG-3′; <italic>rp49-</italic>reverse: 5′-TCGATCCGTAACCGATGT-3′.</p></sec><sec id="s3-10"><title>Double fluorescence <italic>in situ</italic> hybridization</title><p>The double fluorescence in situ hybridization in fresh brain sections was performed following published protocols (<xref ref-type="bibr" rid="bib14">Jones et al., 2007</xref>). Briefly, templates for the anti-sense (AS) and sense (S) control riboprobes targeting either <italic>ppk29</italic> or <italic>sei</italic> transcripts were synthesized by PCR reactions from pUAST-<italic>ppk29</italic> or pUAST-<italic>sei</italic> plasmids with the following primers: <italic>sei</italic>-AS left: 5′-TAATACGACTCA-CTATAGGGCATCGATTTGATTGTGGACG-3′;<italic>sei</italic>-AS right: 5′-CAGTATTCGGTGC-CACATTG-3′; <italic>sei</italic>-S left: 5′-CATCGATTTGATTGTGGACG-3′<italic>; sei</italic>-S right: 5′-TAATAC-GACTCACTATAGGGCAGTATTCGGTGCCACATTG-3′<italic>; ppk29-AS</italic> left: 5′-TAATACG-ACTCACTATAGGGAATACGAAATGTGGCGGAAG-3′<italic>; ppk29</italic>-AS right: 5′-GCATTTCTTCGATGCTGTCA-3′<italic>; ppk29</italic>-S left: 5′-AATACGAAATGTGGCGGAAG-3′; <italic>ppk29</italic>-S right: 5′-TAATACGACTCACTATAGGGGCATTTCTTCGATGCTGTCA-3′. The <italic>sei</italic> riboprobes were labeled by DIG (DIG RNA Labeling Kit, Roche), and the <italic>ppk29</italic> riboprobes were labeled by fluorescein (Fluorescein RNA Labeling Kit, Roche). Freshly dissected female brains (4–5 days old) were embedded in cryo-embedding medium (Tisse-Tek OCT, Fisher Scientific, Pittsburgh, PA). Frozen tissue were cryo-sectioned at 15 μm and fixed in 4% paraformaldehyde for 5 min. Probes were used at 2 ng/μl standard ISH hybridization buffer, 65°C overnight. Post-hybridization, tissues were blocked with TNB for 30 min followed by an incubation with a peroxidase-conjugated anti-DIG antibody in TNB buffer (1:500; Anti-Digoxigenin-POD, Fab fragments, Roche) for 2 hr to detect <italic>sei</italic>-specific signal. To increase signal-to-noise ratio, the Tyramide Signal Amplification system (TSA) with Horseradish Peroxidase (HRP) was used. Samples were treated for 1 hr (1:50, TSA Plus Cy3, PerkinElmer, Waltham, MA). Then, samples were transferred to 0.3% hydrogen peroxide in TNT buffer to quench HRP activity for 20 min. Subsequently, the <italic>ppk29</italic> antisense riboprobe was detected with a peroxidase-conjugated anti-Fluorescein antibody in TNB buffer (1:500; Anti-Fluorescein-POD, Fab fragments, Roche) for 2 hr. To amplify <italic>ppk29</italic> signal, samples were treated with the primary antibody in TSA signal amplification buffer (1:50, TSA Plus Fluorescein, PerkinElmer) for 1 hr. Tissue sections were mounted with Vectashield mounting medium with DAPI and imaged with a confocal microscope.</p></sec><sec id="s3-11"><title>3′RACE</title><p>The FirstChoice RLM-RACE Kit (Life Technologies, Grand Island, NY) was used to characterize the 3′UTRs of <italic>sei</italic> and <italic>ppk29</italic> by following manufacturer′s instructions. Total RNA was isolated from mixed adults and 5 μg total RNA was used for first strand cDNA synthesis. Gene specific primers for PCRs were: <italic>ppk29</italic>: 5′-ACTTGCGACTGCTCTCTATTC-3′; <italic>sei</italic>: 5′-AAACTGCACAGGGACGATTT-3′; 3′RACEOuterPrimer: 5′-GCGAGCACAGAATTAATACGACT-3′. Positive PCR products were sequenced from both ends with the PCR primers.</p></sec><sec id="s3-12"><title>Motor neuron mRNA profiling</title><p>Translating Ribosome Affinity Purification (TRAP) was used to isolate mRNAs specifically from larval motor neurons according to a recently published protocol (<xref ref-type="bibr" rid="bib39">Thomas et al., 2012</xref>). In short, a GFP tagged version of the ribosomal protein <italic>RpL10A</italic> was specifically expressed in larval motor neurons with the motor-neuron specific driver OK6-GAL4 (<xref ref-type="bibr" rid="bib1">Aberle et al., 2002</xref>; <xref ref-type="bibr" rid="bib44">Xiong et al., 2010</xref>). Total RNA was extracted using the TRizol reagent (Life Sciences, Grand Island, NY) from 35 3<sup>rd</sup> instar larvae. Enrichment for <italic>sei</italic> and <italic>ppk29</italic> transcripts in motor neurons was measured with Real-Time qRT-PCR in TRAPped mRNAs by comparing enriched vs total RNA from the <italic>OK6-Gal4>UAS-GFP::RpL10A</italic> genotype. Real-time qRT-PCR was performed as described above.</p></sec><sec id="s3-13"><title>Statistical analyses</title><p>All quantitative behavioral, molecular, and neurophysiological data were analyzed using the most recent version of the SAS package (SAS Inc.). One-way and Two-way ANOVAs were used to analyze parametric data followed by a Tukey <italic>post hoc</italic> analyses (p<0.05) when comparisons between individual groups were required. Data distributions are presented as error bars that denote Standard Error of the Mean.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank members of the Ben-Shahar and DiAntonio labs for useful comments on the manuscript, the Bloomington Stock Center for fly strains, and Paula Kiefel for assistance in generating transgenic flies.</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>XZ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>VV, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con3"><p>ADA, Conception and design, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>YB-S, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aberle</surname><given-names>H</given-names></name><name><surname>Haghighi</surname><given-names>AP</given-names></name><name><surname>Fetter</surname><given-names>RD</given-names></name><name><surname>McCabe</surname><given-names>BD</given-names></name><name><surname>Magalhaes</surname><given-names>TR</given-names></name><name><surname>Goodman</surname><given-names>CS</given-names></name></person-group><year>2002</year><article-title>wishful thinking encodes a BMP type II receptor that regulates synaptic growth in 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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://elife.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>[Editors’ note: although it is not typical of the review process at <italic>eLife</italic>, in this case the editors decided to include the reviews in their entirety for the authors’ consideration as they prepared their revised submission.]</p><p>Thank you for sending your work entitled “Natural Antisense Transcripts Regulate the Neuronal Stress Response and Excitability” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor and 2 reviewers, one of whom, Leslie Griffith, is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other reviewer 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>The full text of the original reviews is attached for your consideration. It would be good to address as many of the comments as you can, but the 3 key issues are enumerated below.</p><p>The consensus of the reviewers was that the paper was very novel but there were several things that should be addressed to make the argument for the proposed mechanism stronger. In addition, perhaps because of the novelty of this finding, it was felt that some of the conclusions should be tempered by acknowledgement that there might be other potential mechanisms to explain the findings. Specifically:</p><p>1) Since the identification of the antisense region was based on the (sometimes imperfectly) annotated genome, the authors should isolate the mRNAs and sequence them to verify that the UTRs from the annotation are present in the actual transcripts.</p><p>2) The data on colocalization of the <italic>ppk</italic> and <italic>sei</italic> transcripts is very weak and the figure hard to interpret. A better demonstration of coexpression would substantially strengthen the argument for example, cell specific expression profiling, high quality in situ, etc.</p><p>3) Authors should include the other interpretations and caveats of the results. Specifically, the UAS-FP-3'UTR-<italic>ppk29</italic> thermoprotective phenotype and <italic>sei</italic> transcript reduction suggests the antisense mechanism, but does not prove it conclusively. The 3'UTR might have additional targets, and the evidence for this mechanism in the endogenous rather than over-expression case is still weak.</p><p><italic>Reviewer</italic> <italic>#1:</italic></p><p>This is a very novel study demonstrating temperature/stress-dependent regulation of seizure via an antisense transcript that is part of a neighboring gene. The importance of this mechanism for regulation of CNS function is made clear by the behavioral outcomes.</p><p>The data seem very clear and consistent. There is one thing lacking, however, and that is information on where and how temperature acts transcriptionally. The model would suggest that transcription of <italic>ppk</italic> is regulated in a temp-dependent manner, but <italic>sei</italic> is not (its mRNA levels are controlled exclusively posttranscriptionally). Some measure of temperature effects on transcription rate or nascent transcripts would add substantially to the story and make it completely solid that the NAT is the major regulator and not some transcriptional effect of temperature on <italic>sei</italic>.</p><p><italic>Reviewer</italic> <italic>#2:</italic></p><p>The authors show that the sodium channel ppk29 and the potassium channel sei are transcribed on opposite strands from mRNA with overlapping 3'UTRs. Reduced expression of <italic>sei</italic> makes flies more vulnerable to heat-induced paralysis, while reduction of ppk29 makes them more resistant. The authors propose that the 3'UTR of <italic>ppk29</italic> targets the <italic>sei</italic> transcript for destruction via the endogenous RNA interference pathway (siRNA and RISC complex).</p><p>The straightforward hypothesis is that the amount of ion exchange through neural membranes could be achieved by co-regulating the protein expression levels of sodium and potassium channels. The surprise here is that the 3'UTR of one channel (ppk29) seems to affect the mRNA levels of the other, so the regulation might be at the transcriptional stage.</p><p>This is a very interesting concept and, if supportable, makes us think about how the balance of ion channels is achieved in new ways. Because it is such a surprising and potentially impactful finding, it needs to be extremely well supported, and I think it still falls short.</p><p>1) The overlapping transcripts of <italic>ppk29</italic> and <italic>sei</italic> are suggested by computational annotation of the <italic>Drosophila</italic> genome. Is there any independent verification that the identification of the 3'UTRs is correct? RT-PCR, with polyA primers and <italic>sei</italic> or <italic>ppk29</italic>-specific 5' primers, should amplify the actual mRNA transcripts and confirm that the 3' UTRs exist as predicted. Do full-length cDNAs exist for these genes already, and do they support the transcript annotation?</p><p>2) For the proposed mechanism to work, <italic>sei</italic> and <italic>ppk29</italic> do have to be precisely co-expressed, and I do not find any of the data presented convincing on this point. The cell lines are next to useless, whole CNS or whole tissue profiling is at best suggestive, and the in situ figure, which would be the most helpful is too low resolution and dim to be interpretable. Adult brain in situs in flies are notoriously hard, so I applaud the authors for trying, but <xref ref-type="fig" rid="fig1">Figure 1C–E</xref> does not persuade me that these two transcripts are co-expressed. In addition, my reading of the Methods using the Tyramide amplification system and the HRP quenching makes me nervous that there may be artifactual cross-talk as well.</p><p>3) A lot of these data rest on the “thermo-protective” phenotype of <italic>ppk29</italic>, and this is a new result, so I would like to see a bit more about how it is measured and how variable it is.</p><p>4) The temperature-sensitive paralysis occurs very quickly (a few minutes), while the affect of temperature rearing on ion channel transcript levels shown if <xref ref-type="fig" rid="fig2">Figure 2</xref> is very slow (7 hr). At what speed do the authors think the natural antisense mechanism normally is acting? To set the basal levels of sei K<sup>+</sup> channel expression appropriate for current environmental conditions? This should be clarified.</p><p>5) The authors use transgenes with a fluorescent protein fused to the <italic>ppk29</italic> or <italic>sei</italic> 3'UTR to assess whether the UTRs alone are sufficient. The levels of endogenous fluorescence would be a quantifiable measure of how much the expression levels are changes and this could provide an alternative measure to the qRT-PCR data. Not essential, but should be considered.</p><p>6) The result that the <italic>ppk29</italic> 3'UTR alone has a thermo-protective effect is certainly interesting. Is it certain that all of its effect is due to increased levels of sei? Does the UAS-sei without UTR rescue better than the version with the UTR? Presumably it would bypass the <italic>ppk29</italic> regulation and produce more sei. Given the chromosomal proximity, it would be hard to make a double mutant of <italic>sei</italic> and <italic>ppk29</italic>, but perhaps RNAi could be used to confirm that reducing sei modifies the <italic>ppk29</italic> mutant phenotype? The sei drug blockers (<xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1</xref>) address this a bit, but RNAi would corroborate.</p><p>The authors should connect their findings to other regulatory mechanisms that have been shown to act on channel transcripts, specifically the RNA helicase NAP thought to regulate the para sodium transcript levels and RNA editing (Reenan and Ganetzky, Neuron 2000), the Dao protein proposed to regulate potassium and sodium channel levels (Fergestad and Ganetzky, PNAS 2010), the conductance-independent affects of the eag potassium channel on intracellular signaling (Hegle and Wilson PNAS, 2006), and the co-regulation of various ion channels as described in McLean and Harris-Warrick, J. Neurophys (2005), for example. The current proposal of RNA degradation of sei by ppk29 is an interesting idea, but adding some historical and intellectual context seems appropriate for the Discussion.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.01849.021</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>The consensus of the reviewers was that the paper was very novel but there were several things that should be addressed to make the argument for the proposed mechanism stronger. In addition, perhaps because of the novelty of this finding, it was felt that some of the conclusions should be tempered by acknowledgement that there might be other potential mechanisms to explain the findings</italic>.</p><p>We appreciate the consensus view that our work is novel. As described later, we now acknowledge that some of our data cannot exclude other possible explanations for our findings (see specific responses below).</p><p><italic>1) Since the identification of the antisense region was based on the (sometimes imperfectly) annotated genome, the authors should isolate the mRNAs and sequence them to verify that the UTRs from the annotation are present in the actual transcripts</italic>.</p><p>We agree with the reviewer that the prediction of 3’UTRs is often imperfect. However, in the case of the two genes in question, both sei and ppk29 gene boundaries are supported by fully sequenced cDNAs as well as RNA-seq data from the <italic>Drosophila</italic> modEncode database. Nevertheless, as suggested by the reviewer, we performed 3’RACE for both genes, which confirmed the data obtained from fully sequenced cDNAs available at NCBI (revised <xref ref-type="fig" rid="fig1">Figure 1A</xref>).</p><p><italic>2) The data on colocalization of the</italic> ppk <italic>and</italic> sei <italic>transcripts is very weak and the figure hard to interpret. A better demonstration of coexpression would substantially strengthen the argument for example cell specific expression profiling, high quality in situ, etc</italic>.</p><p>We now further support the co-localization of <italic>sei</italic> and <italic>ppk29</italic> by demonstrating that both genes are enriched in motor neurons by using in vivo Translating Ribosome Affinity Purification (TRAP) (new <xref ref-type="fig" rid="fig1">Figure 1E</xref> and new paragraph in the Results section).</p><p><italic>3) Authors should include the other interpretations and caveats of the results. Specifically, the UAS-FP-3'UTR-</italic>ppk29 <italic>thermoprotective phenotype and</italic> sei <italic>transcript reduction suggests the antisense mechanism, but does not prove it conclusively. The 3'UTR might have additional targets, and the evidence for this mechanism in the endogenous rather than over-expression case is still weak</italic>.</p><p>We toned down our interpretation of the data throughout the revised manuscript.</p><p>Reviewer #1:</p><p><italic>The data seem very clear and consistent. There is one thing lacking, however, and that is information on where and how temperature acts transcriptionally. The model would suggest that transcription of</italic> ppk <italic>is regulated in a temp-dependent manner, but</italic> sei <italic>is not (its mRNA levels are controlled exclusively posttranscriptionally). Some measure of temperature effects on transcription rate or nascent transcripts would add substantially to the story and make it completely solid that the NAT is the major regulator and not some transcriptional effect of temperature on</italic> sei.</p><p>The reviewer is absolutely correct that it’s very hard to disentangle the effects of direct impact of temperature on <italic>sei</italic> expression from the effect of the interaction with <italic>ppk29</italic>. We are currently following up on this line of investigation as part of larger study on the role of NATs in regulating neuronal homeostasis. We’re generating new sets of transgenic reporters that will enable us to measure activity levels of both promoters independent of transcript levels in vivo. Although these studies are beyond the scope of this manuscript, we now address this caveat in the interpretation of our data: “Our findings also indicate that the regulatory interaction between sei and ppk29 may play a role in the homeostatic response to slow changes in environmental temperature (<xref ref-type="fig" rid="fig2">Figure 2</xref>). However, our current genetic and transgenic tools make it impossible for us to completely disentangle the direct effects of temperature changes on <italic>sei</italic> and <italic>ppk29</italic> transcription and the indirect effects via the interactions of their mRNAs. Thus, more studies will be needed to further establish <italic>ppk29</italic> mRNA as a homeostatic factor, beyond its effects on the acute heat response.”</p><p>Reviewer #2:</p><p><italic>1) The overlapping transcripts of</italic> ppk29 <italic>and</italic> sei <italic>are suggested by computational annotation of the Drosophila genome. Is there any independent verification that the identification of the 3'UTRs is correct? RT-PCR, with polyA primers and</italic> sei <italic>or</italic> ppk29<italic>-specific 5' primers, should amplify the actual mRNA transcripts and confirm that the 3' UTRs exist as predicted. Do full-length cDNAs exist for these genes already, and do they support the transcript annotation</italic>?</p><p>See our response to Comment 1<bold>.</bold></p><p><italic>2) For the proposed mechanism to work,</italic> sei <italic>and</italic> ppk29 <italic>do have to be precisely co-expressed, and I do not find any of the data presented convincing on this point. The cell lines are next to useless, whole CNS or whole tissue profiling is at best suggestive, and the in situ figure, which would be the most helpful is too low resolution and dim to be interpretable. Adult brain in situs in flies are notoriously hard, so I applaud the authors for trying, but</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1C–E</italic></xref> <italic>does not persuade me that these two transcripts are co-expressed. In addition, my reading of the Methods using the Tyramide amplification system and the HRP quenching makes me nervous that there may be artifactual cross-talk as well</italic>.</p><p>We respectfully disagree with the reviewer that the cell line data are useless. These are clonal cell lines and thus represent a single population. These data are consistent with our hypothesis. As described in our response to Comment 2, we added new data in further support of the co-localization of <italic>sei</italic> and <italic>ppk29</italic> by demonstrating that both genes are enriched in motor neurons (new <xref ref-type="fig" rid="fig1">Figure 1E</xref> and new paragraph in the Results section).</p><p><italic>3) A lot of these data rest on the “thermo-protective” phenotype of</italic> ppk29<italic>, and this is a new result, so I would like to see a bit more about how it is measured and how variable it is</italic>.</p><p>We show in <xref ref-type="fig" rid="fig3">Figure 3</xref>, and <xref ref-type="fig" rid="fig3s1">Figure 3–figure supplement 1</xref> data for paralysis as a function of time. We use standard error of the mean to represent variability. To ensure clarity, we revised and expanded our description of the method used.</p><p><italic>4) The temperature-sensitive paralysis occurs very quickly (a few minutes), while the affect of temperature rearing on ion channel transcript levels shown if</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2</italic></xref> <italic>is very slow (7 hr). At what speed do the authors think the natural antisense mechanism normally is acting? To set the basal levels of sei K</italic><sup><italic>+</italic></sup> <italic>channel expression appropriate for current environmental conditions? This should be clarified</italic>.</p><p>With our current tools it is impossible for us to accurately compare the kinetics of NAT activity versus transcription. Based on general principles of eukaryotic transcription, the acute effects of heat on paralysis are not likely to involve transcriptional changes. We interpret our data to indicate that when <italic>seizure</italic> mRNA levels are high due to a mutation in <italic>ppk29</italic> it leads to an increase in baseline resistance to heat. Thus, we predict that the slow adaptation to heat as seen in <xref ref-type="fig" rid="fig2">Figure 2</xref> is the natural process in which flies slowly become desensitized to the effects of slowly increasing temperatures by increasing <italic>seizure</italic> expression and lowering <italic>ppk29</italic> expression. We now make this distinction clearer in our Discussion.</p><p><italic>5) The authors use transgenes with a fluorescent protein fused to the</italic> ppk29 <italic>or</italic> se<italic>i 3'UTR to assess whether the UTRs alone are sufficient. The levels of endogenous fluorescence would be a quantifiable measure of how much the expression levels are changes and this could provide an alternative measure to the qRT-PCR data. Not essential, but should be considered</italic>.</p><p>We thank the reviewer for this excellent suggestion. We did try to use these in vivo reporters quantitatively. However, because GFP and mCherry are highly stable proteins in this system, the fluorescent signal does not faithfully report transcript levels. Although beyond the scope of the current study, we are currently developing new transgenic reporters that will hopefully enable us to address this point directly.</p><p>6) <italic>The result that the</italic> ppk29 <italic>3'UTR alone has a thermo-protective effect is certainly interesting. Is it certain that all of its effect is due to increased levels of sei? Does the UAS-sei without UTR rescue better than the version with the UTR? Presumably it would bypass the</italic> ppk29 <italic>regulation and produce more sei. Given the chromosomal proximity, it would be hard to make a double mutant of</italic> sei <italic>and</italic> ppk29, <italic>but perhaps RNAi could be used to confirm that reducing sei modifies the</italic> ppk29 <italic>mutant phenotype? The sei drug blockers (</italic><xref ref-type="fig" rid="fig4s1"><italic>Figure 4–figure supplement 1</italic></xref><italic>) address this a bit, but RNAi would corroborate</italic>.</p><p>As the reviewer suggested, we tried and failed to produce a double <italic>sei/ppk29</italic> mutant line. We agree with the reviewer that testing this hypothesis with RNAi would help. However, generating these lines is not trivial since it requires us to combine in a single genetic background elav-Gal4, UAS-<italic>Dcr2</italic>, UAS-sei<sup>RNAi</sup> and the <italic>ppk29</italic> mutation. The realization that this would be a very difficult line to generate with matching congenic controls led us to use pharmacology instead.</p><p><italic>The authors should connect their findings to other regulatory mechanisms that have been shown to act on channel transcripts, specifically the RNA helicase NAP thought to regulate the para sodium transcript levels and RNA editing (Reenan and Ganetzky, Neuron 2000), the Dao protein proposed to regulate potassium and sodium channel levels (Fergestad and Ganetzky, PNAS 2010), the conductance-independent affects of the eag potassium channel on intracellular signaling (Hegle and Wilson PNAS, 2006), and the co-regulation of various ion channels as described in McLean and Harris-Warrick, J. Neurophys (2005), for example. The current proposal of RNA degradation of sei by ppk29 is an interesting idea, but adding some historical and intellectual context seems appropriate for the Discussion</italic>.</p><p>As suggested by the reviewer, we now expand our Discussion to include these important studies in the context of our findings.</p></body></sub-article></article> |