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• W2117613120 abstract "Decapping represents a critical control point in regulating expression of protein coding genes. Here, we demonstrate that decapping also modulates expression of long noncoding RNAs (lncRNAs). Specifically, levels of >100 lncRNAs in yeast are controlled by decapping and are degraded by a pathway that occurs independent of decapping regulators. We find many lncRNAs degraded by DCP2 are expressed proximal to inducible genes. Of these, we show several genes required for galactose utilization are associated with lncRNAs that have expression patterns inversely correlated with their mRNA counterpart. Moreover, decapping of these lncRNAs is critical for rapid and robust induction of GAL gene expression. Failure to destabilize a lncRNA known to exert repressive histone modifications results in perpetuation of a repressive chromatin state that contributes to reduced plasticity of gene activation. We propose that decapping and lncRNA degradation serve a vital role in transcriptional regulation specifically at inducible genes. Decapping represents a critical control point in regulating expression of protein coding genes. Here, we demonstrate that decapping also modulates expression of long noncoding RNAs (lncRNAs). Specifically, levels of >100 lncRNAs in yeast are controlled by decapping and are degraded by a pathway that occurs independent of decapping regulators. We find many lncRNAs degraded by DCP2 are expressed proximal to inducible genes. Of these, we show several genes required for galactose utilization are associated with lncRNAs that have expression patterns inversely correlated with their mRNA counterpart. Moreover, decapping of these lncRNAs is critical for rapid and robust induction of GAL gene expression. Failure to destabilize a lncRNA known to exert repressive histone modifications results in perpetuation of a repressive chromatin state that contributes to reduced plasticity of gene activation. We propose that decapping and lncRNA degradation serve a vital role in transcriptional regulation specifically at inducible genes. lncRNAs are degraded by a DCP2-dependent decapping pathway Many lncRNAs targeted to decapping are associated with inducible gene loci Decapping of lncRNAs is required for robust activation of GAL gene expression lncRNA degradation contributes plasticity to gene regulatory mechanisms Gene transcription in the nucleus and degradation in the cytoplasm together dictate the level of messenger RNA (mRNA) that is available as a template for protein synthesis. mRNA turnover, therefore, represents a critical control point in regulating gene expression (Franks and Lykke-Andersen, 2008Franks T.M. Lykke-Andersen J. The control of mRNA decapping and P-body formation.Mol. Cell. 2008; 32: 605-615Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). In eukaryotes, mRNA decay is initiated by removal of the 3′ poly(A) tail (deadenylation) and is typically followed by cleavage of the 5′ end 7-methyl-guanosine (m7G) cap and rapid 5′→3′ exonucleolytic degradation of the transcript body. Cleavage of the mRNA 5′ cap is catalyzed by a holoenzyme composed of the decapping proteins, DCP1 and DCP2, with DCP2 harboring a conserved NUDIX domain required for catalysis (Dunckley and Parker, 1999Dunckley T. Parker R. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif.EMBO J. 1999; 18: 5411-5422Crossref PubMed Scopus (270) Google Scholar). mRNA decapping is regulated by a suite of activators, including DHH1, PAT1, and the LSM1-7 complex (Franks and Lykke-Andersen, 2008Franks T.M. Lykke-Andersen J. The control of mRNA decapping and P-body formation.Mol. Cell. 2008; 32: 605-615Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). While the role of decapping in controlling mRNA levels is well documented, the contribution of decapping in modulating the levels and function of other RNAs has been largely unexplored. Eukaryotic genomes express a complex repertoire of RNA molecules that are not protein coding—thousands of which are classified as small noncoding RNAs (i.e., microRNA, small interfering RNA, Piwi-interacting RNA) or large noncoding RNAs (lncRNAs; i.e., intergenic, antisense, and intronic) (Wilusz et al., 2009Wilusz J.E. Sunwoo H. Spector D.L. Long noncoding RNAs: functional surprises from the RNA world.Genes Dev. 2009; 23: 1494-1504Crossref PubMed Scopus (1867) Google Scholar, Djuranovic et al., 2011Djuranovic S. Nahvi A. Green R. A parsimonious model for gene regulation by miRNAs.Science. 2011; 331: 550-553Crossref PubMed Scopus (413) Google Scholar). While some lncRNA transcripts may represent transcriptional “noise,” several lncRNAs have now been shown to have biological function as bona fide regulators of gene expression both transcriptionally and posttranscriptionally (Wilusz et al., 2009Wilusz J.E. Sunwoo H. Spector D.L. Long noncoding RNAs: functional surprises from the RNA world.Genes Dev. 2009; 23: 1494-1504Crossref PubMed Scopus (1867) Google Scholar, Nagano and Fraser, 2011Nagano T. Fraser P. No-nonsense functions for long noncoding RNAs.Cell. 2011; 145: 178-181Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). Notwithstanding, our understanding of the mechanisms and biological importance of lncRNAs is comparatively scant to that of small noncoding RNAs, which have been the recent focus of intense research (Djuranovic et al., 2011Djuranovic S. Nahvi A. Green R. A parsimonious model for gene regulation by miRNAs.Science. 2011; 331: 550-553Crossref PubMed Scopus (413) Google Scholar). lncRNAs have been implicated in regulating a large array of processes in eukaryotic cells, including gene imprinting, dosage compensation, cell-cycle regulation, innate immunity, pluripotency, retrotransposon silencing, meiotic entry, and telomere length (Wilusz et al., 2009Wilusz J.E. Sunwoo H. Spector D.L. Long noncoding RNAs: functional surprises from the RNA world.Genes Dev. 2009; 23: 1494-1504Crossref PubMed Scopus (1867) Google Scholar, Nagano and Fraser, 2011Nagano T. Fraser P. No-nonsense functions for long noncoding RNAs.Cell. 2011; 145: 178-181Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). Moreover, altered expression of lncRNAs has been linked to disease states such as cancer and neurological disorders (Qureshi et al., 2010Qureshi I.A. Mattick J.S. Mehler M.F. Long non-coding RNAs in nervous system function and disease.Brain Res. 2010; 1338: 20-35Crossref PubMed Scopus (377) Google Scholar, Tsai et al., 2011Tsai M.C. Spitale R.C. Chang H.Y. Long intergenic noncoding RNAs: new links in cancer progression.Cancer Res. 2011; 71: 3-7Crossref PubMed Scopus (584) Google Scholar). Regulation of gene expression by lncRNAs can be mediated at the level of transcription by interference with mRNA expression, competition at genomic loci for transcription factors, or chromatin remodeling (Berretta and Morillon, 2009Berretta J. Morillon A. Pervasive transcription constitutes a new level of eukaryotic genome regulation.EMBO Rep. 2009; 10: 973-982Crossref PubMed Scopus (183) Google Scholar, Wilusz et al., 2009Wilusz J.E. Sunwoo H. Spector D.L. Long noncoding RNAs: functional surprises from the RNA world.Genes Dev. 2009; 23: 1494-1504Crossref PubMed Scopus (1867) Google Scholar). Posttranscriptionally, lncRNAs influence pre-mRNA splicing, nuclear trafficking, and mRNA degradation (Wilusz et al., 2009Wilusz J.E. Sunwoo H. Spector D.L. Long noncoding RNAs: functional surprises from the RNA world.Genes Dev. 2009; 23: 1494-1504Crossref PubMed Scopus (1867) Google Scholar, Nagano and Fraser, 2011Nagano T. Fraser P. No-nonsense functions for long noncoding RNAs.Cell. 2011; 145: 178-181Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, Gong and Maquat, 2011Gong C.G. Maquat L.E. lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3′ UTRs via Alu elements.Nature. 2011; 470: 284-288Crossref PubMed Scopus (949) Google Scholar). Based on the emerging emphasis of lncRNAs on regulating gene expression, the metabolism of the lncRNA itself will likely be a vital aspect of its function. Similar to most mRNAs transcribed by RNA polymerase II, lncRNAs are both capped and polyadenylated (Berretta and Morillon, 2009Berretta J. Morillon A. Pervasive transcription constitutes a new level of eukaryotic genome regulation.EMBO Rep. 2009; 10: 973-982Crossref PubMed Scopus (183) Google Scholar, Khalil et al., 2009Khalil A.M. Guttman M. Huarte M. Garber M. Raj A. Rivea Morales D. Thomas K. Presser A. Bernstein B.E. van Oudenaarden A. et al.Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.Proc. Natl. Acad. Sci. USA. 2009; 106: 11667-11672Crossref PubMed Scopus (2359) Google Scholar, Guttman et al., 2009Guttman M. Amit I. Garber M. French C. Lin M.F. Feldser D. Huarte M. Zuk O. Carey B.W. Cassady J.P. et al.Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals.Nature. 2009; 458: 223-227Crossref PubMed Scopus (3277) Google Scholar). We therefore set out to evaluate whether the decapping enzyme, DCP2, and its associated factors play a role in lncRNA metabolism and whether lncRNA turnover impinges on the ability of lncRNAs to regulate gene expression. Using RNA sequencing to profile transcriptome-wide expression patterns, we determined that over 100 lncRNAs are elevated in cells lacking RNA decapping activity. Importantly, decapping of lncRNA occurs independently of all known regulators of the decapping holoenzyme and thus represents a unique pathway for RNA turnover. Our study reveals that lncRNAs are often found proximal to inducible genes, and degradation of a lncRNA is required for proper induction of genes involved in galactose metabolism. We propose that lncRNAs are used as a means to tightly maintain repression at inducible genes and that efficient clearance of the lncRNA by DCP2-dependent decapping is vital for robust gene activation. RNA polymerase II transcribes a large number of lncRNAs that are predicted to receive a 5′ m7G cap structure cotranscriptionally (Berretta and Morillon, 2009Berretta J. Morillon A. Pervasive transcription constitutes a new level of eukaryotic genome regulation.EMBO Rep. 2009; 10: 973-982Crossref PubMed Scopus (183) Google Scholar, Bentley, 2005Bentley D.L. Rules of engagement: co-transcriptional recruitment of pre-mRNA processing factors.Curr. Opin. Cell Biol. 2005; 17: 251-256Crossref PubMed Scopus (391) Google Scholar). We anticipated that decapping might, therefore, play an important role in modulating abundance and perhaps biological activity of lncRNAs. We monitored the contribution of decapping to global lncRNA levels in budding yeast by high-throughput RNA sequencing (RNA-seq). Total RNA was isolated from wild-type (WT) cells and a strain lacking the catalytic subunit of the decapping enzyme (i.e., dcp2Δ), and complementary DNA (cDNA) libraries were prepared for analysis by the Illumina sequencing platform (see the Experimental Procedures). Notably, RNA was not subjected to poly(A)+ selection since, without decapping, mRNA (and perhaps lncRNA) accumulate as a deadenylated [i.e., poly(A)−] species (Dunckley and Parker, 1999Dunckley T. Parker R. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif.EMBO J. 1999; 18: 5411-5422Crossref PubMed Scopus (270) Google Scholar). In addition, cDNA libraries were constructed from RNA with strand-specific information retained (see the Experimental Procedures). RNA-seq analysis resulted in 84.4 million and 61.2 million mappable reads from WT and dcp2Δ libraries, respectively. Of these, 5.2 million WT and 5.5 million dcp2Δ reads mapped to nonribosomal loci. Consistent with our prediction that lncRNAs would be substrates for decapping, we observed a dramatic elevation in the level of several previously characterized lncRNAs in decapping-deficient cells compared to the WT (Figures 1A and 1B and Table S1 available online). Moreover, our analysis identified approximately 100 putative and previously unannotated lncRNAs based on their accumulation in dcp2Δ cells (Figures 1C–1F and Table S2). Notably, several of the putative lncRNAs we identified were predicted in previous studies interrogating the yeast transcriptome, but remain uncharacterized (Nagalakshmi et al., 2008Nagalakshmi U. Wang Z. Waern K. Shou C. Raha D. Gerstein M. Snyder M. The transcriptional landscape of the yeast genome defined by RNA sequencing.Science. 2008; 320: 1344-1349Crossref PubMed Scopus (1808) Google Scholar, Xu et al., 2009Xu Z. Wei W. Gagneur J. Perocchi F. Clauder-Münster S. Camblong J. Guffanti E. Stutz F. Huber W. Steinmetz L.M. Bidirectional promoters generate pervasive transcription in yeast.Nature. 2009; 457: 1033-1037Crossref PubMed Scopus (688) Google Scholar, Yassour et al., 2010Yassour M. Pfiffner J. Levin J.Z. Adiconis X. Gnirke A. Nusbaum C. Thompson D.A. Friedman N. Regev A. Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species.Genome Biol. 2010; 11: R87Crossref PubMed Scopus (107) Google Scholar, van Dijk et al., 2011van Dijk E.L. Chen C.L. d'Aubenton-Carafa Y. Gourvennec S. Kwapisz M. Roche V. Bertrand C. Silvain M. Legoix-Né P. Loeillet S. et al.XUTs are a class of Xrn1-sensitive antisense regulatory non-coding RNA in yeast.Nature. 2011; 475: 114-117Crossref PubMed Scopus (268) Google Scholar). In general, lncRNAs that accumulated in dcp2Δ cells mapped to three types of genomic loci: (1) intergenic regions between previously annotated protein-coding genes, (2) locations proximal to telomeres, and (3) antisense to either the 5′ end or the entire length of known protein-coding genes (Table S2). Unexpectedly, the majority of DCP2-sensitive lncRNAs map proximal to genes that could be grouped into specific biological pathways. These pathways include, but are not limited to, iron sensing (i.e., FRE1, FRE5, and FRE7), glucose usage (i.e., HXT5, HXT8, HXT10, and RGS2), maltose metabolism (i.e., MAL11, MAL12, MAL13, and MAL32), flocculation (i.e., FLO5, FLO9, FLO10, and FLO11), inorganic phosphate uptake and utilization (i.e., PHO5 and PHO84), and galactose utilization (i.e., GAL1, GAL10, GAL2, and GAL4). Importantly, most genes within this subset were repressed, and therefore not transcriptionally active, under the conditions assayed in our RNA-seq analysis (Table S3). Their expression is, however, induced by specific environmental cues, which suggests that a large proportion of these lncRNAs map to highly regulated genes. We analyzed RNA by Northern blot to confirm that the steady-state levels of seven of our identified lncRNAs were indeed elevated in dcp2Δ cells compared to the WT. Specifically, lncRNAs that map antisense to GAL10, GAL1, GAL2, GAL4, PHO84, and FRE5 genes were dramatically increased in decapping-deficient cells (Figure 1G, WT versus dcp2Δ). Similarly, a lncRNA mapping intergenic to the OCR2 and TRM7 genes was poorly expressed in WT cells but accumulated in dcp2Δ mutants (Figure 1G). Our analyses in dcp2Δ cells confirm decapping modulates levels of lncRNAs. Recently, lncRNAs termed XUTs were identified based on their sensitivity to XRN1, a cytoplasmic 5′→3′ exonuclease implicated in degrading decapped mRNA (van Dijk et al., 2011van Dijk E.L. Chen C.L. d'Aubenton-Carafa Y. Gourvennec S. Kwapisz M. Roche V. Bertrand C. Silvain M. Legoix-Né P. Loeillet S. et al.XUTs are a class of Xrn1-sensitive antisense regulatory non-coding RNA in yeast.Nature. 2011; 475: 114-117Crossref PubMed Scopus (268) Google Scholar). Consistent with the requirement for removal of the 5′ m7Gpp cap before RNA degradation by XRN1 (Stevens and Poole, 1995Stevens A. Poole T.L. 5′-exonuclease-2 of Saccharomyces cerevisiae. Purification and features of ribonuclease activity with comparison to 5′-exonuclease-1.J. Biol. Chem. 1995; 270: 16063-16069Crossref PubMed Scopus (96) Google Scholar), 70% of the lncRNAs upregulated in dcp2Δ cells were also classified as XUTs (van Dijk et al., 2011van Dijk E.L. Chen C.L. d'Aubenton-Carafa Y. Gourvennec S. Kwapisz M. Roche V. Bertrand C. Silvain M. Legoix-Né P. Loeillet S. et al.XUTs are a class of Xrn1-sensitive antisense regulatory non-coding RNA in yeast.Nature. 2011; 475: 114-117Crossref PubMed Scopus (268) Google Scholar). Interestingly, 30% of lncRNAs we identified were not identified as XUTs (Table S2). It is unclear whether this discrepancy represents differences in annotation of RNA-seq data or whether some lncRNAs are degraded by an alternative pathway. Indeed, in eukaryotes there are two 5′→3′ exonucleases (i.e., XRN1 and RAT1), both of which act downstream of RNA decapping to degrade RNA. Our observation that lncRNAs are sensitive to decapping prompted us to evaluate whether they are also modulated by additional proteins implicated in mRNA turnover. Degradation of cytoplasmic mRNA is initiated by removal of the 3′ poly(A) tail by the CCR4-NOT deadenylase and is followed by decapping catalyzed by the DCP1/DCP2 holoenzyme and 5′→3′ exonucleolytic degradation by XRN1. Additional factors, including DHH1, PAT1, and LSM1-7, play an important role in mRNA stability as activators of decapping (Franks and Lykke-Andersen, 2008Franks T.M. Lykke-Andersen J. The control of mRNA decapping and P-body formation.Mol. Cell. 2008; 32: 605-615Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar) (Figure S1). We performed northern blot analysis on RNA isolated from WT or cells lacking an activity important for mRNA turnover. As shown in Figures 2A and 2B , RNA levels for seven lncRNAs elevated in DCP2-deficient cells (Figure 1G) were also increased in cells lacking XRN1 (5- to 47-fold). Consistent with our northern analysis, qRT-PCR of the GAL10 lncRNA confirmed its accumulation in dcp2Δ and xrn1Δ cells compared to the WT (4.8- and 6-fold, respectively; Figure 2E, top). Surprisingly, lncRNA levels were unchanged in cells lacking either deadenylase activity (i.e., ccr4Δ) or regulators of mRNA decapping (i.e., dhh1Δ and lsm1Δ; Figures 2A and 2B). Additionally, inactivation of either nuclear or cytoplasmic 3′→5′ exosome activity (i.e., rrp6Δ, trf4Δ, and ski2Δ) failed to result in elevated levels of these lncRNAs (Figures 2A and 2B). The abundance, therefore, of lncRNAs we identified as decapping substrates, unlike mRNAs, is unaffected by deadenylation, 3′→5′ degradation, and, most unexpectedly, proteins required for activating mRNA decapping. Eukaryotic cells possess two enzymes catalyzing 5′→3′ exonucleolytic degradation, XRN1 and RAT1, which are predominantly present in the cytoplasm and nucleus, respectively (Johnson, 1997Johnson A.W. Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively.Mol. Cell. Biol. 1997; 17: 6122-6130Crossref PubMed Scopus (160) Google Scholar). To determine whether RAT1 plays a role in modulating lncRNA levels, we used a temperature-sensitive allele of the essential RAT1 gene, rat1-1, that abrogates RAT1 function at restrictive temperature of 37°C (Amberg et al., 1992Amberg D.C. Goldstein A.L. Cole C.N. Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA.Genes Dev. 1992; 6: 1173-1189Crossref PubMed Scopus (302) Google Scholar). WT and rat1-1 cells were grown at permissive temperature and shifted to 37°C for 2 hr before harvesting and isolation of RNA. Northern analysis determined several but not all lncRNAs elevated in dcp2Δ cells also accumulated when RAT1 was inactivated (Figures 2C and 2D). qRT-PCR analysis of the GAL10 lncRNA confirmed these findings (Figure 2E, bottom). These observations suggest that nuclear 5′→3′ exonucleolytic digestion by RAT1 contributes to lncRNA decay. Steady-state accumulation of lncRNAs in the absence of DCP2, XRN1, or RAT1 activity strongly implies a role in their degradation. To demonstrate direct involvement of these proteins in mediating lncRNA turnover, we performed kinetic analysis of RNA degradation. We specifically focused on the GAL10 lncRNA because its transcription is regulated by the sugar in the growth media (Houseley et al., 2008Houseley J. Rubbi L. Grunstein M. Tollervey D. Vogelauer M. A ncRNA modulates histone modification and mRNA induction in the yeast GAL gene cluster.Mol. Cell. 2008; 32: 685-695Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Cells grown in raffinose, where the GAL10 lncRNA is transcriptionally active, were shifted to galactose-containing media, which represses GAL10 lncRNA transcription. RNA isolated from cells at various times after galactose addition was analyzed by northern blot to evaluate changes in GAL10 lncRNA levels over time and determine lncRNA half-life. As seen in Figure 2F, the half-life of GAL10 lncRNA in WT cells was 17 min. In contrast, GAL10 lncRNA stability increased to 49 min in dcp2Δ cells, demonstrating that DCP2 is directly involved in the turnover of this lncRNA. Stabilization of GAL10 lncRNA is dependent on DCP2's pyrophosphatase activity (Figures S2B and S2C). In cells lacking the noncatalytic subunit of the decapping enzyme, DCP1 (dcp1Δ), GAL10 lncRNA was also stabilized with a half-life of 39 min. This result is consistent with observations that DCP1 is required for decapping activity both in vivo and in vitro and that the two proteins constitute a holoenzyme (Steiger et al., 2003Steiger M. Carr-Schmid A. Schwartz D.C. Kiledjian M. Parker R. Analysis of recombinant yeast decapping enzyme.RNA. 2003; 9: 231-238Crossref PubMed Scopus (144) Google Scholar). Moreover, as expected from steady-state analysis, GAL10 lncRNA was stabilized in cells lacking XRN1 (xrn1Δ; half-life of 36 min), but unaffected by inactivation of mRNA decapping activators (lsm1Δ, lsm6Δ, dhh1Δ), the deadenylase complex (ccr4Δ), nonsense-mediated mRNA decay (upf1Δ), the nuclear exosome (rrp6Δ), and the TRAMP complex (trf4Δ) (Figure 2F). We also evaluated the role of the nuclear 5′→3′ exonuclease RAT1 in GAL10 lncRNA stability. Transcriptional shut-off analysis of GAL10 lncRNA in rat1-1 cells at the permissive temperature, where RAT1 function is impaired but not completely abrogated, demonstrated that RAT1 plays an important role in its stability (half-life of 42 min; Figure 2F). In contrast, RAI1, a known cofactor of RAT1 that itself has pyrophosphatase activity (Jiao et al., 2010Jiao X. Xiang S. Oh C. Martin C.E. Tong L. Kiledjian M. Identification of a quality-control mechanism for mRNA 5′-end capping.Nature. 2010; 467: 608-611Crossref PubMed Scopus (124) Google Scholar), did not impact either the steady-state level or stability of GAL10 lncRNA (Figures 2A and 2F). Taken together, our data demonstrate that GAL10 lncRNA turnover is mediated by DCP2, DCP1, XRN1, and RAT1, but not other proteins implicated in decay of mRNA, and therefore its metabolism involves a distinct decay pathway. Given the steady-state data for a number of lncRNAs (Figure 2A), we predict that this decay pathway is used to degrade many, if not most, lncRNAs in the cell. Moreover, these observations support the existence of two separate, yet partially redundant pathways for decay of lncRNAs—a cytoplasmic, XRN1-dependent pathway and a nuclear, RAT1-dependent pathway. Considering that decapping is required to generate a substrate for both 5′→3′ exonuclease enzymes (i.e., XRN1 or RAT1), DCP2 constitutes a critical regulator in lncRNA metabolism that is likely to function in both the cytoplasm and the nucleus based on observations that DCP2 can shuttle (Grousl et al., 2009Grousl T. Ivanov P. Frýdlová I. Vasicová P. Janda F. Vojtová J. Malínská K. Malcová I. Nováková L. Janosková D. et al.Robust heat shock induces eIF2alpha-phosphorylation-independent assembly of stress granules containing eIF3 and 40S ribosomal subunits in budding yeast, Saccharomyces cerevisiae.J. Cell Sci. 2009; 122: 2078-2088Crossref PubMed Scopus (174) Google Scholar). GAL-inducible gene regulation represents a classic genetic switch regulating sugar metabolism in eukaryotic cells (Lohr et al., 1995Lohr D. Venkov P. Zlatanova J. Transcriptional regulation in the yeast GAL gene family: a complex genetic network.FASEB J. 1995; 9: 777-787Crossref PubMed Scopus (336) Google Scholar). The GAL system has been extensively characterized in yeast and consists of four structural genes—GAL1, GAL10, GAL7, and GAL2—that are coordinately regulated at the level of transcription by GAL4, GAL80, and GAL3. In repressed or noninduced conditions (glucose and raffinose sugar sources, respectively), GAL80 inhibits the ability of GAL4 to recruit the transcription machinery to drive expression of the structural genes. In the presence of galactose, GAL3 sequesters GAL80 in the cytoplasm, thus allowing robust transcriptional activation of GAL1, GAL10, GAL7, and GAL2 by GAL4. We were interested in determining whether GAL lncRNAs, like their mRNA counterparts, exhibit expression patterns in response to sugar availability. To this end, we grew WT and dcp2Δ cells under conditions where GAL gene transcription is either repressed (glucose), noninduced (raffinose), or induced (galactose) with respect to the mRNA at these loci (Figure 3A ) and analyzed lncRNA levels by northern blot. In WT cells grown in glucose, GAL2, GAL1, and GAL10 lncRNAs are present at very low or undetectable levels (Figure 3B, lane 1), consistent with our RNA-seq data and previous reports characterizing GAL10 lncRNA expression (Figures 1A, 1C, 2A, and 2B) (Houseley et al., 2008Houseley J. Rubbi L. Grunstein M. Tollervey D. Vogelauer M. A ncRNA modulates histone modification and mRNA induction in the yeast GAL gene cluster.Mol. Cell. 2008; 32: 685-695Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Similarly, low levels of these lncRNA were observed for WT cells grown in raffinose or galactose (Figure 3B, lanes 3 and 5). GAL lncRNAs from cells lacking DCP2 were significantly elevated in glucose-grown cells (Figure 3B, lane 2), in agreement with their detection by RNA-seq. These lncRNA levels were also elevated in raffinose-grown cells (Figure 3B, lane 4), but not in cells grown in galactose (Figure 3B, lane 6). Critically, GAL lncRNAs fail to accumulate in cells where GAL gene expression is induced, despite the absence of destabilizing DCP2 activity, indicating a reciprocal pattern of expression and suggesting a role for these lncRNAs in regulating their cognate protein-coding genes (see below). Our RNA-Seq data indicated a lncRNA expressed antisense to the GAL4 locus (Figure 1D), and we were interested in determining whether the GAL4 lncRNA also displayed reciprocal expression patterns with regards to its cognate mRNA. Importantly, GAL4, the transcriptional activator of GAL-inducible genes, while subject to glucose repression, is itself not induced by galactose (Lohr et al., 1995Lohr D. Venkov P. Zlatanova J. Transcriptional regulation in the yeast GAL gene family: a complex genetic network.FASEB J. 1995; 9: 777-787Crossref PubMed Scopus (336) Google Scholar). We determined that, similar to GAL2, GAL1, and GAL10 lncRNAs, GAL4 lncRNA levels were low in WT cells grown under all conditions tested (Figure 3C, lanes 1, 3, and 5). Abrogation of decapping activity resulted in robust accumulation of GAL4 lncRNA in glucose-grown cells as expected (Figure 3C, lane 2) but also led to elevated levels in raffinose and galactose-grown cells, where GAL4 mRNA is also expressed (Figure 3C, lanes 4 and 6). GAL4 mRNA and lncRNA do not, therefore, show an inverse expression pattern. GAL4 mRNA levels were, however, reduced in decapping-deficient cells in which GAL4 lncRNA accumulated (dcp2Δ; Figure 3C, lanes 4 and 6), suggesting that GAL4 lncRNA may impair GAL4 mRNA expression. To evaluate whether GAL4 lncRNA impinges upon GAL4 mRNA levels, we attenuated GAL4 lncRNA expression by introducing mutations within the region of its promoter (see the Experimental Procedures). WT and dcp2Δ cells in which GAL4 lncRNA was either present or absent (gal4 lncRNAmut) were grown under conditions in which GAL4 mRNA is expressed (i.e., raffinose or galactose-containing media) to determine the influence of GAL4 lncRNA on GAL4 mRNA expression. Importantly, GAL4 lncRNA levels were reduced greater than 90% in dcp2Δ cells harboring the gal4 lncRNAmut mutation (Figures 3D and 3E). Critically, in the absence of GAL4 lncRNA, GAL4 mRNA levels increased 2- to 3-fold in dcp2Δ cells grown in either condition (Figures 3D and 3F, lanes 2 versus 4; lanes 6 versus 8). Our data indicate that GAL4 lncRNA levels regulate expression of GAL4 mRNA. We observed that in dcp2Δ cells, several GAL lncRNAs accumulate in cells grown in glucose or raffinose but fail to accumulate under conditions where GAL mRNAs are expressed (i.e., in the presence of galactose; Figures 3A and 3B). We hypothesized that GAL lncRNAs are absent in cells grown in galactose because their presence would impinge upon the transcriptional induction of GAL structural genes. GAL lncRNAs would, therefore, need to be rapidly removed from the cell upon an environmental shift from transcriptionally inactive to active conditions. We evaluated whether the GAL10 lncRNA, which spans both GAL10 and GAL1 gene loci, influenced transcriptional activation of GAL1 mRNA upon induction by galactose. Prior to induction, cells were grown in raffinose, noninducing conditions for GAL1 mRNA, and samples were removed over time after galactose addition. In WT cells, GAL10 lncRNA was present at low levels and decayed quickly upon addition of galactose (half-life = 17 min; Figure 4B ). Consistent with reciprocal expression patterns, GAL1 mRNA levels accumulated quickly in these cells and attained maximum" @default.
• W2117613120 created "2016-06-24" @default.
• W2117613120 creator A5006981419 @default.
• W2117613120 creator A5039458149 @default.
• W2117613120 creator A5052749945 @default.
• W2117613120 creator A5056030932 @default.
• W2117613120 creator A5074950343 @default.
• W2117613120 date "2012-02-01" @default.
• W2117613120 modified "2023-10-12" @default.
• W2117613120 title "Decapping of Long Noncoding RNAs Regulates Inducible Genes" @default.
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