Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription
2019; Springer Nature; Volume: 38; Issue: 16 Linguagem: Inglês
10.15252/embj.2019101955
ISSN1460-2075
AutoresJulieta Rivosecchi, Marc Larochelle, Camille Teste, Frédéric Grenier, Amélie Malapert, Emiliano P. Ricci, Pascal Bernard, François Bachand, Vincent Vanoosthuyse,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle11 July 2019free access Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription Julieta Rivosecchi Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author Marc Larochelle Département de Biochimie, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Camille Teste Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author Frédéric Grenier Département de Biochimie, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Amélie Malapert Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author Emiliano P Ricci Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author Pascal Bernard orcid.org/0000-0003-2732-9685 Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author François Bachand Département de Biochimie, Université de Sherbrooke, Sherbrooke, QC, Canada Centre de Recherche du CHUS, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Vincent Vanoosthuyse Corresponding Author [email protected] orcid.org/0000-0002-4416-1557 Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author Julieta Rivosecchi Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author Marc Larochelle Département de Biochimie, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Camille Teste Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author Frédéric Grenier Département de Biochimie, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Amélie Malapert Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author Emiliano P Ricci Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author Pascal Bernard orcid.org/0000-0003-2732-9685 Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author François Bachand Département de Biochimie, Université de Sherbrooke, Sherbrooke, QC, Canada Centre de Recherche du CHUS, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Vincent Vanoosthuyse Corresponding Author [email protected] orcid.org/0000-0002-4416-1557 Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France Search for more papers by this author Author Information Julieta Rivosecchi1, Marc Larochelle2, Camille Teste1, Frédéric Grenier2, Amélie Malapert1, Emiliano P Ricci1, Pascal Bernard1,‡, François Bachand2,3,‡ and Vincent Vanoosthuyse *,1 1Laboratoire de Biologie et Modélisation de la Cellule, Université de Lyon, CNRS, UMR 5239, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Lyon, France 2Département de Biochimie, Université de Sherbrooke, Sherbrooke, QC, Canada 3Centre de Recherche du CHUS, Université de Sherbrooke, Sherbrooke, QC, Canada ‡These authors contributed equally to this work *Corresponding author. Tel: +33 4727 28197; E-mail: [email protected] EMBO J (2019)38:e101955https://doi.org/10.15252/embj.2019101955 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract R-loop disassembly by the human helicase Senataxin contributes to genome integrity and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin also contributes to transcription termination at other classes of genes has remained unclear. Here, we show that Sen1, one of two fission yeast homologues of Senataxin, promotes efficient termination of RNA polymerase III (RNAP3) transcription in vivo. In the absence of Sen1, RNAP3 accumulates downstream of RNAP3-transcribed genes and produces long exosome-sensitive 3′-extended transcripts. Importantly, neither of these defects was affected by the removal of R-loops. The finding that Sen1 acts as an ancillary factor for RNAP3 transcription termination in vivo challenges the pre-existing view that RNAP3 terminates transcription autonomously. We propose that Sen1 is a cofactor for transcription termination that has been co-opted by different RNA polymerases in the course of evolution. Synopsis Fission yeast expresses two Senataxin helicase homologs, Sen1 and Dbl8. Sen1 is now shown to be required for efficient RNA polymerase III transcription termination of a subset of transcripts, challenging models of RNAP3 terminating transcription autonomously. RNAP3 interacts with Sen1 but not Dbl8 in fission yeast. Sen1 is required for normal RNAP3 transcription. Sen1 prevents the accumulation of exosome-sensitive read-through transcripts. Sen1 acts independently of R-loops at RNAP3-transcribed genes. Introduction Senataxin is a conserved DNA/RNA helicase whose deficiency has been implicated in the neurological disorders amyotrophic lateral sclerosis type 4 (ALS4) and ataxia–ocular apraxia type 2 (AOA2; Chen et al, 2004; Moreira et al, 2004). How different Senataxin mutations contribute to the development of diseases with distinct pathologies remains unclear (Groh et al, 2017). Senataxin is the homologue of the yeast Sen1 helicase, and concordant observations have established that both human Senataxin and budding yeast Sen1 are important for transcription termination of at least a subset of RNAP2-transcribed genes (Ursic et al, 1997; Steinmetz et al, 2006; Skourti-Stathaki et al, 2011; Porrua & Libri, 2013). However, the mechanisms involved probably differ in both species as budding yeast Sen1 contributes to RNAP2 transcription termination as part of the Nrd1-Nab3-Sen1 (NNS) complex, which is not conserved in human cells. In addition, both budding yeast Sen1 and human Senataxin have been implicated in the repair of DNA damage (Li et al, 2016; Andrews et al, 2018; Cohen et al, 2018) and in the resolution of transcription–replication conflicts (Alzu et al, 2012; Richard et al, 2013; Yüce & West, 2013). Both budding and fission yeast Sen1 can translocate in a 5′–3′ direction on either single-stranded DNA or RNA in vitro (Kim et al, 1999; Martin-Tumasz & Brow, 2015; Han et al, 2017), and it is believed that long, co-transcriptional RNA-DNA hybrids (also known as R-loops) represent a critical substrate of budding yeast Sen1 and human Senataxin in vivo (Mischo et al, 2011; Skourti-Stathaki et al, 2011 and reviewed in Groh et al, 2017). Current models propose that the stabilization of R-loops upon Senataxin inactivation underlies the associated transcription termination and DNA repair defects. This proposal however has been somewhat challenged by the observation that budding yeast Sen1 could directly dissociate pre-assembled RNAP2 transcription elongation complexes in vitro by translocating on the nascent RNA, even in the absence of RNA-DNA hybrids (Porrua & Libri, 2013; Han et al, 2017), suggesting that Sen1 has potentially R-loop-independent functions in the control of transcription. In addition, it has been suggested that the very low processivity of Sen1′s helicase activity might actually prevent it from unwinding long R-loops in vivo (Han et al, 2017). The fission yeast Schizosaccharomyces pombe expresses two non-essential homologues of Senataxin, Sen1 and Dbl8. Surprisingly, transcription termination at RNAP2-transcribed genes is largely unaffected by lack of either or both homologues (Lemay et al, 2016; Larochelle et al, 2018) and to date, the roles of the fission yeast Senataxin enzymes have remained largely unknown. Dbl8 was shown to localize at sites of double-strand breaks (Yu et al, 2013), and we reported previously that fission yeast Sen1 physically associates with RNAP3 and is recruited to specific tRNA genes (Legros et al, 2014). The function of Sen1 at RNAP3-transcribed genes has remained unclear, however. RNAP3 is predominantly implicated in the transcription of the short and abundant tRNA and 5S rRNA species. Internal promoter sequences called A- and B-box recruit the TFIIIC complex, which helps to position TFIIIB upstream of the transcription start site (TSS). TFIIIB in turn recruits the 17 subunits of RNAP3 complex at the TSS to initiate transcription (reviewed in Schramm and Hernandez 2002). In fission yeast, an upstream TATA box assists TFIIIC in recruiting TFIIIB and is essential for the proper recruitment of RNAP3 (Hamada et al, 2001). In vitro transcription assays have indicated that once loaded, RNAP3 can terminate transcription autonomously upon reaching a transcription termination signal, which is constituted by a simple stretch of five thymine residues on the non-template strand (Mishra & Maraia, 2019), although this number may vary for different genes and organisms (reviewed in Arimbasseri et al 2013). The C37/53/11 subcomplex of RNAP3 is particularly important for this intrinsic transcription termination mechanism (Arimbasseri et al, 2013). Interestingly, however, low levels of read-through transcription are observed at many tRNA genes in vivo in budding yeast, especially at tRNA genes with weaker terminator sequences (Turowski et al, 2016). The resulting 3′-extended transcripts are degraded by specific mechanisms involving the RNA exosome and the poly(A)-binding protein Nab2 (Turowski et al, 2016). In addition, human RNAP3 was also found in the 3′ regions of many tRNA genes (Orioli et al, 2011), suggesting that in distantly related systems, RNAP3 frequently overrides primary termination signals. These observations suggest that efficient RNAP3 transcription termination might be more challenging in vivo than suggested by in vitro studies. Yet, to what extent robust RNAP3 transcription termination requires the support of ancillary factors in vivo remains elusive. We previously showed that unstable R-loops form at tRNA genes in fission yeast (Legros et al, 2014). Similar observations have been made in budding yeast (El Hage et al, 2014), humans (Chen et al, 2017) and the plant Arabidopsis thaliana (Xu et al, 2017), suggesting that R-loop formation is a conserved feature of RNAP3 transcription. The genome-wide stabilization of R-loops by the deletion of RNase H had a mild impact on pre-tRNA processing in budding yeast (El Hage et al, 2014). However, whether these mild perturbations were a direct consequence of R-loop stabilization in cis at tRNA genes was not addressed. Thus, the contribution of R-loops to RNAP3 transcription still remains largely unknown. Our previous work revealed that fission yeast tRNA genes form R-loops and recruit Sen1. As both R-loops and Senataxin were previously proposed to facilitate transcription termination of RNAP2 in humans (Skourti-Stathaki et al, 2011), we investigated the possibility that Sen1 together with R-loops might participate in transcription termination of RNAP3 in fission yeast. We find that Sen1 primarily associates with RNAP3 transcription units at the genome-wide level and does indeed promote proper RNAP3 transcription termination, albeit in a manner that is insensitive to the presence of R-loops. Thus, in contrast to conclusions drawn from in vitro studies, our work reveals the need for a protein cofactor to ensure robust RNAP3 transcription termination in vivo. Results Sen1 but not Dbl8 associates primarily with RNAP3-transcribed genes There are two homologues of Senataxin in fission yeast, Sen1 and Dbl8. Our previous mass spectrometry (MS) analysis of the protein partners of GFP-tagged Sen1 indicated that Sen1 associates physically with RNAP3 but not with RNAP1 or RNAP2 (Legros et al, 2014). Using affinity purification coupled to MS with Flag-tagged proteins expressed from their endogenous chromosomal loci, we show here that Sen1 and Dbl8 associate with different sets of proteins (Appendix Table S1). Sen1 primarily associated with RNAP3 (Fig EV1A), whereas Dbl8 associated with RNAP1 subunits but not with RNAP3 (Fig EV1A and B). The association between Sen1 and RNAP3 was resistant to a benzonase treatment, indicating that it is mediated by direct protein contacts rather than by DNA or RNA (Appendix Fig S1). Taken together, these data suggested that Sen1 but not Dbl8 could play a direct role at RNAP3-transcribed genes. Accordingly, the current study will focus on the role of Sen1 in RNAP3 transcription. Click here to expand this figure. Figure EV1. Sen1 and Dbl8 associate with different RNA polymerases Flag-tagged Sen1 and Dbl8 were affinity-purified and their associated proteins identified using mass spectrometry analysis. The table lists the RNA polymerase components that were recovered with either Sen1 or Dbl8. The full list of proteins identified is shown in Appendix Table S1. Co-immunoprecipitation between Flag-tagged Dbl8 and the GFP-tagged RNAP1 subunit Rpa43. * Aspecific protein recognized by the anti-Flag antibody; ** IgG. Download figure Download PowerPoint Consistent with the idea that Sen1 acts at RNAP3-transcribed genes, we reported previously that Sen1 associates with specific tRNA genes in an R-loop-independent manner (Legros et al, 2014). However, the extent of Sen1 recruitment to all RNAP3-transcribed genes (tRNA, 5S rRNA, srp7 and U6 snRNA) had remained unclear. We therefore used chromatin immunoprecipitation (ChIP) assays coupled to high-throughput sequencing (ChIP-seq) to analyse the genome-wide distribution of Sen1 relative to RNAP2 and RNAP3 occupancy. Analysis of ChIP-seq data confirmed that Sen1 is primarily enriched at tRNA and 5S rRNA genes, which also showed specific and robust RNAP3 binding (Fig 1A). In contrast, RNAP2-transcribed genes, as exemplified by the strongly expressed tef3 gene in Fig 1A, showed only background Sen1 signal. Sen1 was also enriched at the RNAP3-transcribed U6 snRNA snu6 (Fig 1B) and srp7 (Fig 1C) loci. A breakdown of all aligned ChIP-seq reads from two independent experiments revealed that more than 85% of Sen1-associated regions correspond to RNAP3-transcribed genes (Fig 1D, tRNA and 5S rRNA). This contrasts to Seb1 (Lemay et al, 2016), the fission yeast homologue of the NNS component Nrd1, that primarily associates with RNAP2-transcribed genes (Fig 1D). This is consistent with previous observations that Sen1 and Seb1 do not form a functional complex in fission yeast (Legros et al, 2014; Lemay et al, 2016; Larochelle et al, 2018). Overall, we observed a positive genome-wide correlation between ChIP-seq signals of Sen1 and two independent subunits of RNAP3 but not with RNAP2 at all loci (Fig 1E and Appendix Fig S2). We confirmed the enrichment of Sen1 at RNAP3-transcribed sites using ChIP-qPCR (Fig 1F). Consistent with the observation that Dbl8 does not form a complex with RNAP3, Dbl8 was not enriched at RNAP3-transcribed genes (Fig 1F) and lack of Dbl8 did not affect the recruitment of Sen1 at RNAP3-transcribed genes (Appendix Fig S3). Interestingly, chromosome-organizing clamp (COC) sites, which recruit TFIIIC but not RNAP3 (Noma et al, 2006), were not enriched for Sen1 (Fig 1F). This is consistent with our observation that Sen1 physically associates with RNAP3 but not with TFIIIC (Legros et al, 2014) and suggests that the RNAP3 transcription complex might recruit Sen1 to target genes. To test this possibility, we mutated the upstream TATA box of a model tRNA gene, SPCTRNAARG.10 (tRNAARGUCG), in order to interfere with the recruitment of RNAP3 specifically at this locus. The mutated TATA-less SPCTRNAARG.10 locus showed reduced levels of both RNAP3 and Sen1 (Fig 1G), indicating that the TATA box-dependent recruitment of RNAP3 is important for Sen1 occupancy at RNAP3-transcribed genes. Collectively, these data indicate that fission yeast Sen1 is primarily enriched at RNAP3-transcribed genes, where it is likely to perform a function that is not shared with Dbl8. Figure 1. Sen1 associates predominantly with RNAP3-transcribed genes A–C. Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from Larochelle et al 2018) across (A) a 25-kb region of chromosome 3 containing several tRNA and 5S rRNA genes, (B) the U6 snRNA snu6 and (C) the srp7 loci. D. Comparison of the distribution of ChIP-seq reads across the indicated categories of genes for Sen1 (this study) and Seb1 (data from Lemay et al 2016). E. Genome-wide pairwise Pearson's correlation coefficient matrix at a resolution of 10 bp. F. ChIP-qPCR analysis of Flag-tagged Sen1 at the indicated loci in a population of cycling cells (mean ± SD from 4 biological replicates). G. ChIP-qPCR analysis of the Flag-tagged RNAP3 subunit Rpc37 and Flag-tagged Sen1 across the SPCTRNAARG.10 tRNA locus, whose TATA box was either mutated (TATA−) or not (TATA+). The mutations introduced to disrupt the putative TATA boxes are indicated in red above the graphs. The enrichment values were normalized to SPCTRNATHR.10 (mean ± SD from four biological replicates; P-value obtained using the Wilcoxon–Mann–Whitney statistical test). Download figure Download PowerPoint Sen1 is required for normal RNAP3 transcription Next, we investigated whether lack of Sen1 affects RNAP3 transcription. Using ChIP-qPCR, we detected increased amount of RNAP3 at all sites tested in the absence of Sen1 (Fig 2A). Strikingly, this accumulation of RNAP3 over its target genes was not associated with an increased amount of RNAP3 transcripts in the cell (Fig 2B, compare lanes 1–2). The steady state level of 5S RNAs remained unchanged, and the overall levels of tRNA detected in the absence of Sen1 were even slightly reduced (Fig 2B), as confirmed using gene-specific RT–qPCR (Fig 2C). Interestingly, this reduction was even more apparent when Dis3-mediated tRNA degradation (Gudipati et al, 2012; Schneider et al, 2012) was impaired (Fig 2B, compare lanes 3–4). Since the steady state levels of tRNAs are controlled by the equilibrium between synthesis and Dis3-mediated degradation (Gudipati et al, 2012; Schneider et al, 2012), the reduction in nuclear exosome activity associated with the depletion of Dis3 is expected to uncover the direct impact that lack of Sen1 has on RNAP3 transcription. Taken together, these results therefore indicate that lack of Sen1 impairs RNAP3 transcription. Figure 2. Sen1 is required for normal RNAP3 transcription (left) ChIP-qPCR analysis of Rpc37 in the presence or absence of Sen1 at the indicated loci in a population of cycling cells (mean ± SD from four biological replicates). (right) Western blot analysis of Rpc37 protein levels in the presence or absence of Sen1. Tubulin was used as a loading control. (left) Total RNA from the indicated strains was separated on a 2.8% agarose gel. (right) Quantification of overall tRNA levels (mean ± SD from three biological replicates). Strand-specific RT–qPCR was used to quantify the indicated RNAP3 transcripts. Transcript levels were normalized to act1 (mean ± SD from three biological replicates). Download figure Download PowerPoint The accumulation of RNAP3 on its target genes in the absence of Sen1 is independent of R-loops Fission yeast Sen1 is able to unwind RNA-DNA hybrids in vitro (Kim et al, 1999), and the budding yeast and human homologues of Sen1 are believed to antagonize R-loop formation in vivo (Mischo et al, 2011; Skourti-Stathaki et al, 2011). Interestingly, R-loops were shown to interfere with transcription elongation, at least when they form close to the TSS (Belotserkovskii et al, 2017), and we have shown previously that RNase H-sensitive R-loops form at tRNA genes in fission yeast (Legros et al, 2014; Hartono et al, 2018). To test whether the stabilization of R-loops at tRNA genes could underlie the accumulation of RNAP3 in the absence of Sen1, we expressed RNase H1 from Escherichia coli (RnhA) under the control of the strong nmt1 promoter in fission yeast cells. We showed previously that this strategy was sufficient to remove R-loops at tRNA genes (Legros et al, 2014; Hartono et al, 2018). Using R-ChIP to monitor R-loop formation at tRNA genes (Legros et al, 2014), we confirmed that RnhA expression was sufficient to completely remove R-loops in the absence of Sen1 (Fig 3A). However, this treatment did not alter the accumulation of RNAP3 (Fig 3B), indicating that R-loops do not contribute to the accumulation of RNAP3 in the sen1∆ mutant. Conversely, stabilization of R-loops at tRNA genes by the deletion of both endogenous RNase H1 and RNase H2 (rnh1∆rnh201∆) (Legros et al, 2014) did not result in the accumulation of RNAP3 (Fig 3B). These results therefore establish that the stabilization of R-loops does not account for the accumulation of RNAP3 in Sen1-deficient cells. Figure 3. Sen1 regulates RNAP3 recruitment in an R-loop-independent mannerCells were grown in minimal medium during 18 hours to induce the strong expression of RnhA R-ChIP using a catalytically inactive RNase H1 (Rnh1-D129N) was used to quantify R-loop formation at RNAP3-transcribed genes (mean ± SD from four biological replicates). (left) ChIP-qPCR of the 13myc-tagged RNAP3 subunit Rpc25 in the indicated strains at the indicated loci (mean ± SD of four biological replicates). (right) Western blot analysis of Myc-tagged Rpc25 and Flag-tagged RnhA protein levels in the indicated strains. Tubulin was used as a loading control. Download figure Download PowerPoint Sen1 is required for effective RNAP3 transcription termination We next analysed the effect of a Sen1 deficiency on the genome-wide distribution of RNAP3 by comparing ChIP-seq profiles of Rpc1 and Rpc2 in sen1+ and sen1Δ strains. Strikingly, in the absence of Sen1, the distribution of both Rpc1 and Rpc2 displayed increased density downstream of most tRNA and 5S rRNA genes (as exemplified on Fig 4A and B), as well as at srp7 (Fig 4C), consistent with read-through transcription by RNAP3. Evidence of delayed transcription termination in the sen1Δ mutant was also noted at the snu6 gene, albeit at more modest levels (Appendix Fig S4). Importantly, averaging Rpc1 and Rpc2 ChIP-seq signals over all isolated tRNA and 5S rRNA genes confirmed that the distribution pattern of RNAP3 is globally extended at the 3′ end in the absence of Sen1 (Fig 4D). ChIP followed by qPCR analysis at several candidate loci confirmed that the domain occupied by RNAP3 was wider in the absence of Sen1 and that RNAP3 accumulated downstream of its natural transcription termination sites (Fig 4E). Notably, this accumulation downstream of the transcription termination site was not altered upon RnhA expression, confirming that it did not result from the stabilization of R-loops (Fig EV2). Together, these results reveal that Sen1 is required for RNAP3 termination at the genome-wide level in an R-loop-independent manner. Figure 4. Sen1 is required for efficient RNAP3 transcription termination A–C. Snapshots of ChIP-seq signals of the RNAP3 subunits Rpc1 and Rpc2 in the presence or absence of Sen1 across a representative (A) tRNA gene, (B) 5S rRNA gene and (C) srp7. Boxed regions highlight the increased density of reads in the downstream region of genes in the absence of Sen1. D. Average ChIP-seq profile of Rpc1 and Rpc2 across all isolated tRNA and 5S rRNA genes in the presence and absence of Sen1. E. Scanning of Rpc37-3flag occupancy at three different tDNA loci in the absence of Sen1 by ChIP-qPCR (mean ± SD from six biological replicates). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Loss of R-loops upon expression of RnhA does not impact the distribution of RNAP3 in the absence of Sen1ChIP-qPCR analysis of the RNAP3 subunit Rpc25 in the indicated genotypes and at the indicated loci in a population of cycling cells (mean ± SD from two biological replicates). Download figure Download PowerPoint Read-through tRNA transcripts accumulate in the absence of Sen1 We used several independent assays to demonstrate that the transcription termination defects associated with lack of Sen1 resulted in the production of 3′-extended transcripts. First, we used a genetic assay that translates a transcription termination defect at the synthetic tRNA DRT5T construct into a change of colour of yeast colonies from red to white (Iben et al, 2011; Fig 5A, see scheme of the construct at the top). Briefly, a transcription termination defect allows the synthesis of a suppressor tRNA that suppresses the accumulation of a red pigment caused by the ade6-704 mutation, resulting in white colonies in limiting adenine conditions. As a positive control for this assay, we mutated the valine residue at position 189 in the Rpc37 subunit of RNAP3 into an aspartate residue (rpc37-V189D), as overexpression of this mutant was shown to interfere with transcription termination in a dominant-negative manner (Rijal & Maraia, 2013). Here, we mutated the endogenous rpc37 gene and established that the rpc37-V189D mutant is viable but displays transcription termination defects (Fig 5A). Similarly, in the absence of Sen1 but not in the absence of its close homologue Dbl8, colonies turned white in the presence of the DRT5T construct, indicating that Sen1 but not Dbl8 is required for robust transcription termination at DRT5T (Fig 5A). Consistent with the idea that Sen1 contributes to transcription termination of RNAP3-transcribed genes, we found that Sen1 becomes essential for cell viability when the termination is impaired by the rpc37-V189D mutant (Fig 5B). Figure 5. Lack of Sen1 produces extended read-through tRNA transcripts Cells of the indicated genotypes that carried or not the DRT5T dimeric tRNA construct (schematized on top) were grown either in the presence of the optimum concentration of adenine (left) or in the presence of a limiting concentration of adenine (right). Two independent clones of the same genotype (#1 and #2) were used. See text for details. 3F refers to the 3Flag epitope tag at the C-terminus of Rpc37. Tetrad dissection was used to show that the double-mutant sen1∆ rpc37-V189D is dead. Strand-specific RT–qPCR was used to quantify the levels of read-through transcripts (see Materials and Methods). The mean ± SD from four biological replicates is represented here. P-values were obtained using the Wilcoxon–Mann–Whitney statistical test. Northern blot analysis of the tRNA SPATRNAPRO.02 using an intron-specific probe (TCTAAACTCAGCATACAAGTGGGG). U5 snRNA was used as a loading control. Sequence of the ˜350-nt-long read-through transcript at SPATRNAPRO.02. Residues in blue represent the sequence of the mature tRNA. Residues in red represent potential terminator sequences. Red arrows show the 3′ end nucleotide of the read-through transcripts. The numbers indicate the number of times the sequenced transcripts terminated at the indicated position. Download figure Download PowerPoint To confirm the production of read-through transcripts at endogenous tRNA genes in the absence of Sen1, we used strand-specific RT–qPCR. Using this approach, we detected read-through transcripts in the absence of Sen1 at several tRNA genes (Fig EV3). Importantly, read-through transcripts were not detected in the absence of Dbl8, confirming that Sen1 plays a non-redundant role in RNAP3 transcription (Fig EV3). To rule out that those read-through transcripts only resulted from the defective degradation of naturally occurring longer transcripts (Turowski et al, 2016), we quantified these read-through transcripts in cells deficient for the RNA exosome subunit Dis3. If Sen1 was only involved in the RNA exosome-dependent degradation of naturally occurring read-through transcripts, the amount of read-through transcripts found in RNA exosome mutants should not change upon deletion of Sen1. As shown in Fig 5C, we found that the amount of read-through transcripts detected in RNA exosome mutants increased significantly in the absence of Sen1, suggesting that the accumulation of read-through tRNAs in the absence of Sen1 is independent of RNA exosome activity. Using Northern blots, we detected a predominant ~350-nt-long extended transcript at the intron-containing SPATRNAPRO.02 (tRNAPROCGG) in the
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