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• W2125433175 abstract "Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability. Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability. Nascent RNA forms hybrids with underwound DNA upstream of elongating Pol II Single-stranded DNA so formed is prone to damage which results in genome instability Sen1 helicase acts to remove R loops by resolving RNA:DNA hybrids Sen1 function in Pol II elongation and termination may relate to R loop resolution In S. cerevisiae nascent transcripts formed by RNA polymerase II (Pol II) on protein-coding genes are immediately processed, packaged, and exported to the cytoplasm (Luna et al., 2008Luna R. Gaillard H. González-Aguilera C. Aguilera A. Biogenesis of mRNPs: integrating different processes in the eukaryotic nucleus.Chromosoma. 2008; 117: 319-331Crossref PubMed Scopus (89) Google Scholar, Moore and Proudfoot, 2009Moore M.J. Proudfoot N.J. Pre-mRNA processing reaches back to transcription and ahead to translation.Cell. 2009; 136: 688-700Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Messenger RNA (mRNA) packaging protects transcripts from degradation, but also the DNA template from invasion of nascent RNA into the DNA duplex behind elongating Pol II (Aguilera and Gómez-González, 2008Aguilera A. Gómez-González B. Genome instability: a mechanistic view of its causes and consequences.Nat. Rev. Genet. 2008; 9: 204-217Crossref PubMed Scopus (571) Google Scholar). The resulting RNA:DNA hybrid exposes single stranded (ss) nontemplate DNA, a structure referred to as an R loop. R loop formation has been associated with increased occurrence of transcription-associated mutation (TAM) or recombination (TAR), presumably because both induced and spontaneous lesions are more likely to occur on ssDNA. Thus, deletion of genes encoding the THO (Thp2, Hpr1, Mft1, and Tho2) and THSC or TREX-2 (Thp1, Sac3, Sus1, and Cdc31) complexes required for mRNP formation in S. cerevisiae—or, similarly, the splicing factor ASF/SF2 in metazoans—increase levels of R loop formation and consequently TAM and TAR (Chávez et al., 2000Chávez S. Beilharz T. Rondón A.G. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Lithgow T. Aguilera A. A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae.EMBO J. 2000; 19: 5824-5834Crossref PubMed Scopus (248) Google Scholar, Fischer et al., 2002Fischer T. Strässer K. Rácz A. Rodriguez-Navarro S. Oppizzi M. Ihrig P. Lechner J. Hurt E. The mRNA export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores.EMBO J. 2002; 21: 5843-5852Crossref PubMed Scopus (222) Google Scholar, Gallardo and Aguilera, 2001Gallardo M. Aguilera A. A new hyperrecombination mutation identifies a novel yeast gene, THP1, connecting transcription elongation with mitotic recombination.Genetics. 2001; 157: 79-89PubMed Google Scholar, González-Aguilera et al., 2008González-Aguilera C. Tous C. Gómez-González B. Huertas P. Luna R. Aguilera A. The THP1-SAC3-SUS1-CDC31 complex works in transcription elongation-mRNA export preventing RNA-mediated genome instability.Mol. Biol. Cell. 2008; 19: 4310-4318Crossref PubMed Scopus (118) Google Scholar, Huertas and Aguilera, 2003Huertas P. Aguilera A. Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination.Mol. Cell. 2003; 12: 711-721Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, Li and Manley, 2005Li X. Manley J.L. Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability.Cell. 2005; 122: 365-378Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar). R loop formation in these mutants may also be connected to Pol II stalling, consequently interfering with processive elongation (Mason and Struhl, 2005Mason P.B. Struhl K. Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo.Mol. Cell. 2005; 17: 831-840Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, Rondón et al., 2003Rondón A.G. Jimeno S. García-Rubio M. Aguilera A. Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation.J. Biol. Chem. 2003; 278: 39037-39043Crossref PubMed Scopus (88) Google Scholar) and RNA processing (Libri et al., 2002Libri D. Dower K. Boulay J. Thomsen R. Rosbash M. Jensen T.H. Interactions between mRNA export commitment, 3′-end quality control, and nuclear degradation.Mol. Cell. Biol. 2002; 22: 8254-8266Crossref PubMed Scopus (214) Google Scholar, Rougemaille et al., 2008Rougemaille M. Dieppois G. Kisseleva-Romanova E. Gudipati R.K. Lemoine S. Blugeon C. Boulay J. Jensen T.H. Stutz F. Devaux F. Libri D. THO/Sub2p functions to coordinate 3′-end processing with gene-nuclear pore association.Cell. 2008; 135: 308-321Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Similarly, DNA replication may be compromised when replication forks encounter R loops or a stalled Pol II (Wellinger et al., 2006Wellinger R.E. Prado F. Aguilera A. Replication fork progression is impaired by transcription in hyperrecombinant yeast cells lacking a functional THO complex.Mol. Cell. Biol. 2006; 26: 3327-3334Crossref PubMed Scopus (123) Google Scholar). Although little is known about R loop resolution in yeast, in mammals their formation and resolution play a productive role in the stimulation of class switch recombination (CSR) and somatic hypermutation (SHM) in clonally expanding B cells (Yu et al., 2003Yu K. Chedin F. Hsieh C.L. Wilson T.E. Lieber M.R. R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells.Nat. Immunol. 2003; 4: 442-451Crossref PubMed Scopus (567) Google Scholar). Both processes are initiated by activation induced deaminase (AID) (Muramatsu et al., 1999Muramatsu M. Sankaranand V.S. Anant S. Sugai M. Kinoshita K. Davidson N.O. Honjo T. Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.J. Biol. Chem. 1999; 274: 18470-18476Crossref PubMed Scopus (951) Google Scholar). Double-strand breaks (DSBs) subsequently trigger CSR via nonhomologous end joining (NHEJ) (Yu and Lieber, 2003Yu K. Lieber M.R. Nucleic acid structures and enzymes in the immunoglobulin class switch recombination mechanism.DNA Repair (Amst.). 2003; 2: 1163-1174Crossref PubMed Scopus (72) Google Scholar). Although S. cerevisiae does not express AID, ectopically expressed AID can recognize ssDNA in R loops as a substrate when expressed in mRNA packaging mutants (Gómez-González and Aguilera, 2007Gómez-González B. Aguilera A. Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex.Proc. Natl. Acad. Sci. USA. 2007; 104: 8409-8414Crossref PubMed Scopus (87) Google Scholar, González-Aguilera et al., 2008González-Aguilera C. Tous C. Gómez-González B. Huertas P. Luna R. Aguilera A. The THP1-SAC3-SUS1-CDC31 complex works in transcription elongation-mRNA export preventing RNA-mediated genome instability.Mol. Biol. Cell. 2008; 19: 4310-4318Crossref PubMed Scopus (118) Google Scholar). Many events during transcription are orchestrated by proteins binding to the carboxy-terminal domain (CTD) of the Pol II largest subunit. CTD consists in yeast of 26 hepta-peptide repeats (YSPTSPS) that are dynamically modified during transcription. In particular, serine phosphorylation occurs during early (ser5-, 7-P) and late (ser2-P) elongation phases to allow stage specific binding of elongation and RNA processing factors (Kim et al., 2009Kim M. Suh H. Cho E.J. Buratowski S. Phosphorylation of the yeast Rpb1 C-terminal domain at serines 2, 5, and 7.J. Biol. Chem. 2009; 284: 26421-26426Crossref PubMed Scopus (104) Google Scholar, Komarnitsky et al., 2000Komarnitsky P. Cho E.J. Buratowski S. Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription.Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (802) Google Scholar). Transcription termination is also directed by different CTD-bound proteins that recognize specific sequences on the emerging nascent RNA. For protein-coding genes, this requires polyA (pA) site recognition by a ser2-P CTD bound multicomponent cleavage and polyadenylation complex (CF IA/B and CPF), as well as degradation of the downstream RNA by Rat1 exonuclease (Gross and Moore, 2001Gross S. Moore C.L. Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 3′-end formation.Mol. Cell. Biol. 2001; 21: 8045-8055Crossref PubMed Scopus (75) Google Scholar, Kim et al., 2004bKim M. Krogan N.J. Vasiljeva L. Rando O.J. Nedea E. Greenblatt J.F. Buratowski S. The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II.Nature. 2004; 432: 517-522Crossref PubMed Scopus (377) Google Scholar, Meinhart and Cramer, 2004Meinhart A. Cramer P. Recognition of RNA polymerase II carboxy-terminal domain by 3′-RNA-processing factors.Nature. 2004; 430: 223-226Crossref PubMed Scopus (232) Google Scholar). Termination of many noncoding RNAs requires an additional component, the NRD complex (Sen1, Nab3, and Nrd1), in which Nrd1 is bound to ser5-P CTD (Steinmetz et al., 2001Steinmetz E.J. Conrad N.K. Brow D.A. Corden J.L. RNA-binding protein Nrd1 directs poly(A)-independent 3′-end formation of RNA polymerase II transcripts.Nature. 2001; 413: 327-331Crossref PubMed Scopus (285) Google Scholar, Vasiljeva et al., 2008Vasiljeva L. Kim M. Mutschler H. Buratowski S. Meinhart A. The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain.Nat. Struct. Mol. Biol. 2008; 15: 795-804Crossref PubMed Scopus (210) Google Scholar). NRD-dependent termination also requires recognition of frequent short RNA sequences by Nrd1 and Nab3 (GUAA/G and UCUU respectively) (Carroll et al., 2007Carroll K.L. Ghirlando R. Ames J.M. Corden J.L. Interaction of yeast RNA-binding proteins Nrd1 and Nab3 with RNA polymerase II terminator elements.RNA. 2007; 13: 361-373Crossref PubMed Scopus (104) Google Scholar), although the exact sequence and NRD component requirements may vary for different terminators (Kuehner and Brow, 2008Kuehner J.N. Brow D.A. Regulation of a eukaryotic gene by GTP-dependent start site selection and transcription attenuation.Mol. Cell. 2008; 31: 201-211Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Nrd1 ser5-P CTD specificity confines this termination pathway to transcriptional stages in which ser5-P CTD prevails and leaves promoter distal Nrd1/Nab3 binding sites unrecognized (Arigo et al., 2006aArigo J.T. Carroll K.L. Ames J.M. Corden J.L. Regulation of yeast NRD1 expression by premature transcription termination.Mol. Cell. 2006; 21: 641-651Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, Gudipati et al., 2008Gudipati R.K. Villa T. Boulay J. Libri D. Phosphorylation of the RNA polymerase II C-terminal domain dictates transcription termination choice.Nat. Struct. Mol. Biol. 2008; 15: 786-794Crossref PubMed Scopus (115) Google Scholar). Furthermore, as Nrd1 interacts with the exosome, NRD-terminated RNA is either degraded to protein protected stable transcripts (e.g., snoRNAs) or completely, as is the case with cryptic unstable transcripts (CUTs) (Arigo et al., 2006bArigo J.T. Eyler D.E. Carroll K.L. Corden J.L. Termination of cryptic unstable transcripts is directed by yeast RNA-binding proteins Nrd1 and Nab3.Mol. Cell. 2006; 23: 841-851Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, Thiebaut et al., 2006Thiebaut M. Kisseleva-Romanova E. Rougemaille M. Boulay J. Libri D. Transcription termination and nuclear degradation of cryptic unstable transcripts: a role for the nrd1-nab3 pathway in genome surveillance.Mol. Cell. 2006; 23: 853-864Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, Vasiljeva and Buratowski, 2006Vasiljeva L. Buratowski S. Nrd1 interacts with the nuclear exosome for 3′ processing of RNA polymerase II transcripts.Mol. Cell. 2006; 21: 239-248Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Recent genome-wide transcription profiling studies reveal the wide extent of CUTs produced by Pol II and terminated by NRD. This further emphasizes the biological importance of NRD-dependent termination (Neil et al., 2009Neil H. Malabat C. d'Aubenton-Carafa Y. Xu Z. Steinmetz L.M. Jacquier A. Widespread bidirectional promoters are the major source of cryptic transcripts in yeast.Nature. 2009; 457: 1038-1042Crossref PubMed Scopus (456) 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 (697) Google Scholar). Importantly, both termination pathways can substitute for each other and so provide mutual fail-safe termination mechanisms (Kim et al., 2006Kim M. Vasiljeva L. Rando O.J. Zhelkovsky A. Moore C. Buratowski S. Distinct pathways for snoRNA and mRNA termination.Mol. Cell. 2006; 24: 723-734Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, Rasmussen and Culbertson, 1998Rasmussen T.P. Culbertson M.R. The putative nucleic acid helicase Sen1p is required for formation and stability of termini and for maximal rates of synthesis and levels of accumulation of small nucleolar RNAs in Saccharomyces cerevisiae.Mol. Cell. Biol. 1998; 18: 6885-6896PubMed Google Scholar). Thus, NRD termination is also important to rescue polymerases that fail to terminate at a polyA signal, especially on highly transcribed genes. Interestingly, these genes show a particular requirement for Sen1 (Rondón et al., 2009Rondón A.G. Mischo H.E. Kawauchi J. Proudfoot N.J. Fail-safe transcriptional termination for protein-coding genes in S. cerevisiae.Mol. Cell. 2009; 36: 88-98Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). SEN1 codes for a 240 kDa superfamily I helicase (DeMarini et al., 1992DeMarini D.J. Winey M. Ursic D. Webb F. Culbertson M.R. SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.Mol. Cell. Biol. 1992; 12: 2154-2164Crossref PubMed Scopus (68) Google Scholar), and its S. pombe homolog possesses 3′-5′ nucleic acid unwinding activity (Kim et al., 1999Kim H.D. Choe J. Seo Y.S. The sen1(+) gene of Schizosaccharomyces pombe, a homologue of budding yeast SEN1, encodes an RNA and DNA helicase.Biochemistry. 1999; 38: 14697-14710Crossref PubMed Scopus (86) Google Scholar). The essential C terminus contains the helicase domain, a nuclear localization sequence (NLS), and a domain necessary for interaction with the Glc7 phosphatase component of CPF (Nedea et al., 2008Nedea E. Nalbant D. Xia D. Theoharis N.T. Suter B. Richardson C.J. Tatchell K. Kislinger T. Greenblatt J.F. Nagy P.L. The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes.Mol. Cell. 2008; 29: 577-587Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, Ursic et al., 1995Ursic D. DeMarini D.J. Culbertson M.R. Inactivation of the yeast Sen1 protein affects the localization of nucleolar proteins.Mol. Gen. Genet. 1995; 249: 571-584Crossref PubMed Scopus (39) Google Scholar, Winey and Culbertson, 1988Winey M. Culbertson M.R. Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae.Genetics. 1988; 118: 609-617PubMed Google Scholar). The Sen1 975 N-terminal amino acids, although dispensable for growth, interact with Pol II, RNase III endonuclease Rnt1, and the nucleotide excision repair endonuclease Rad2 (Ursic et al., 2004Ursic D. Chinchilla K. Finkel J.S. Culbertson M.R. Multiple protein/protein and protein/RNA interactions suggest roles for yeast DNA/RNA helicase Sen1p in transcription, transcription-coupled DNA repair and RNA processing.Nucleic Acids Res. 2004; 32: 2441-2452Crossref PubMed Scopus (106) Google Scholar). Mutation of the Sen1 helicase domain results in direct and indirect pleiotropic defects in transcript processing and termination, leading to a perturbed genome-wide profile of Pol II and defective Pol I transcription termination (Kawauchi et al., 2008Kawauchi J. Mischo H. Braglia P. Rondon A. Proudfoot N.J. Budding yeast RNA polymerases I and II employ parallel mechanisms of transcriptional termination.Genes Dev. 2008; 22: 1082-1092Crossref PubMed Scopus (104) Google Scholar, Rasmussen and Culbertson, 1998Rasmussen T.P. Culbertson M.R. The putative nucleic acid helicase Sen1p is required for formation and stability of termini and for maximal rates of synthesis and levels of accumulation of small nucleolar RNAs in Saccharomyces cerevisiae.Mol. Cell. Biol. 1998; 18: 6885-6896PubMed Google Scholar, Steinmetz et al., 2001Steinmetz E.J. Conrad N.K. Brow D.A. Corden J.L. RNA-binding protein Nrd1 directs poly(A)-independent 3′-end formation of RNA polymerase II transcripts.Nature. 2001; 413: 327-331Crossref PubMed Scopus (285) Google Scholar, Steinmetz et al., 2006Steinmetz E.J. Warren C.L. Kuehner J.N. Panbehi B. Ansari A.Z. Brow D.A. Genome-wide distribution of yeast RNA polymerase II and its control by Sen1 helicase.Mol. Cell. 2006; 24: 735-746Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, Ursic et al., 1997Ursic D. Himmel K.L. Gurley K.A. Webb F. Culbertson M.R. The yeast SEN1 gene is required for the processing of diverse RNA classes.Nucleic Acids Res. 1997; 25: 4778-4785Crossref PubMed Scopus (98) Google Scholar). Although the severe character of these phenotypes may be explicable by the limiting presence of Sen1 in NRD (as it is only present at 125 copies/cell) (Ghaemmaghami et al., 2003Ghaemmaghami S. Huh W.K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Global analysis of protein expression in yeast.Nature. 2003; 425: 737-741Crossref PubMed Scopus (3008) Google Scholar), they have not been clearly attributed to a molecular function of Sen1. Employing the temperature sensitive sen1-1 mutant (helicase domain G1747D), we set out to characterize the molecular role of Sen1 in transcription termination. We now identify broad functions for Sen1 during Pol II transcription in reducing R loop formation and consequent prevention of transcription-associated genome instability. Mutation of the Sen1 helicase domain results in genome-wide transcription termination defects of noncoding RNAs, but also of some protein coding genes (Steinmetz et al., 2006Steinmetz E.J. Warren C.L. Kuehner J.N. Panbehi B. Ansari A.Z. Brow D.A. Genome-wide distribution of yeast RNA polymerase II and its control by Sen1 helicase.Mol. Cell. 2006; 24: 735-746Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Thus, when tested by transcription run on (TRO) experiments with the plasmid gene construct KGG (Figure 1A ), with the KanMX4 gene terminated by the weak GAL10 pA signal (Morillon et al., 2003Morillon A. Karabetsou N. O'Sullivan J. Kent N. Proudfoot N. Mellor J. Isw1 chromatin remodeling ATPase coordinates transcription elongation and termination by RNA polymerase II.Cell. 2003; 115: 425-435Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), sen1-1 mutants grown for 150 min at nonpermissive temperature (37°C) show a strong termination defect (Figure 1A, upper panels) (Rondón et al., 2009Rondón A.G. Mischo H.E. Kawauchi J. Proudfoot N.J. Fail-safe transcriptional termination for protein-coding genes in S. cerevisiae.Mol. Cell. 2009; 36: 88-98Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). This indicates either a requirement for Sen1 in Rat-dependent termination or that some transcripts over the weak GAL10 pA signal are terminated by the NRD failsafe termination mechanism. To determine whether Sen1 protein-protein interactions or its helicase function are required for transcription termination, we repeated TRO analysis in WT and sen1-1 cells transformed with additional Sen1 expression constructs. Transcribed from an ACT1 promoter, these either contained the NLS and the Glc7 interaction domain [Sen1(323)] or additionally the C-terminal helicase domain [Sen1(1212)] (Nedea et al., 2008Nedea E. Nalbant D. Xia D. Theoharis N.T. Suter B. Richardson C.J. Tatchell K. Kislinger T. Greenblatt J.F. Nagy P.L. The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes.Mol. Cell. 2008; 29: 577-587Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). As shown in Figure 1A, Sen1(1212) but not Sen1(323) rescued the sen1-1 termination defect, implying that the sen1-1 termination defect is caused by loss of helicase function and not Glc7 mediated recruitment of CPF. We also examined steady-state mRNA produced from the KGG construct (Figure 1B). mRNA levels were reduced in sen1-1 cells and partially complemented by coexpression of Sen1(1212), but not Sen1(323). Similarly, coexpression of Sen1(1212) restored wild-type levels of endogenous PMA1 mRNA, also previously shown to display mild termination defects in sen1-1 (Kawauchi et al., 2008Kawauchi J. Mischo H. Braglia P. Rondon A. Proudfoot N.J. Budding yeast RNA polymerases I and II employ parallel mechanisms of transcriptional termination.Genes Dev. 2008; 22: 1082-1092Crossref PubMed Scopus (104) Google Scholar). The above results indicate that the Sen1 helicase domain is required both for efficient Pol II termination and mRNA accumulation. As these effects could be attributed to defective 3′ end processing, we employed an in vitro cleavage and polyadenylation assay using CYC1 3′ flanking RNA as the pA substrate (Figure 1C). sen1-1 shows no defects in RNA 3′ end processing. Confirmation of this result is provided by reverse transcription analysis of ACT1 pA usage, in which sen1-1 showed WT pA selection (Figure S1 available online). In contrast, a CF IA mutant strain, rna14-1, showed the expected defect in both in vitro 3′ end processing and in vivo pA selection (Figure 1C and Figure S1). Finally, like sen1-1, the rat1-1 termination mutant (or both combined) had no effect on mRNA 3′ end formation but stabilized the 3′ end cleavage product, indicative of loss of exonuclease “torpedo” function (Kim et al., 2004bKim M. Krogan N.J. Vasiljeva L. Rando O.J. Nedea E. Greenblatt J.F. Buratowski S. The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II.Nature. 2004; 432: 517-522Crossref PubMed Scopus (377) Google Scholar, Luo et al., 2006Luo W. Johnson A.W. Bentley D.L. The role of Rat1 in coupling mRNA 3′-end processing to transcription termination: implications for a unified allosteric-torpedo model.Genes Dev. 2006; 20: 954-965Crossref PubMed Scopus (132) Google Scholar). Overall, these combined analyses show that the Sen1 helicase is dispensable for 3′ transcript processing but is required to promote transcriptional termination. Since S. pombe Sen1 can use RNA:DNA hybrids as an in vitro substrate (Kim et al., 1999Kim H.D. Choe J. Seo Y.S. The sen1(+) gene of Schizosaccharomyces pombe, a homologue of budding yeast SEN1, encodes an RNA and DNA helicase.Biochemistry. 1999; 38: 14697-14710Crossref PubMed Scopus (86) Google Scholar), we considered the possibility that Sen1 may remove RNA:DNA hybrids formed by nascent RNA and the template strand. Such hybrids were previously shown to form in THO mutants, causing increased rates of transcription associated mitotic recombination (Huertas and Aguilera, 2003Huertas P. Aguilera A. Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination.Mol. Cell. 2003; 12: 711-721Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar). RNA:DNA hybrids may also be naturally encountered in transcribed regions downstream of pA signals, where THO is undetectable on chromatin (Kim et al., 2004aKim M. Ahn S.H. Krogan N.J. Greenblatt J.F. Buratowski S. Transitions in RNA polymerase II elongation complexes at the 3′ ends of genes.EMBO J. 2004; 23: 354-364Crossref PubMed Scopus (240) Google Scholar, Luna et al., 2005Luna R. Jimeno S. Marín M. Huertas P. García-Rubio M. Aguilera A. Interdependence between transcription and mRNP processing and export, and its impact on genetic stability.Mol. Cell. 2005; 18: 711-722Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). We therefore tested whether sequences downstream of a pA signal elicit TAR in sen1-1. We employed a plasmid borne recombination substrate that carries two truncated regions of LEU2 overlapping by 600 nt of homologous sequence (LNA). Lack of THO elicits TAR in LNA and consequent restoration of LEU2, as previously shown (Figure 2A ) (Prado et al., 1997Prado F. Piruat J.I. Aguilera A. Recombination between DNA repeats in yeast hpr1delta cells is linked to transcription elongation.EMBO J. 1997; 16: 2826-2835Crossref PubMed Scopus (81) Google Scholar). In contrast, when transcription between both repeats is terminated by insertion of the CYC1 38nt pA signal (CYC1t, LNAT), recombination levels in the hpr1Δ strain were reduced to background WT levels, presumably because Pol II termination restricts R loop formation. Similar analysis of LNA and LNAT transformed rat1-1 and rna14-1 showed no detectible increase in recombination, confirming that defects in CPF/Rat1 dependent transcription termination per se do not promote recombination (Luna et al., 2005Luna R. Jimeno S. Marín M. Huertas P. García-Rubio M. Aguilera A. Interdependence between transcription and mRNP processing and export, and its impact on genetic stability.Mol. Cell. 2005; 18: 711-722Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In marked contrast, sen1-1 transformed with either LNA or LNAT showed high levels of recombination, suggesting that RNA:DNA hybrids may form throughout the mRNA coding region irrespective of the CYC1t. This lack of CYC1t suppression reiterates the sen1-1 CYC1 termination defect previously reported (Kawauchi et al., 2008Kawauchi J. Mischo H. Braglia P. Rondon A. Proudfoot N.J. Budding yeast RNA polymerases I and II employ parallel mechanisms of transcriptional termination.Genes Dev. 2008; 22: 1082-1092Crossref PubMed Scopus (104) Google Scholar, Steinmetz et al., 2006Steinmetz E.J. Warren C.L. Kuehner J.N. Panbehi B. Ansari A.Z. Brow D.A. Genome-wide distribution of yeast RNA polymerase II and its control by Sen1 helicase.Mol. Cell. 2006; 24: 735-746Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Moreover, the fact that CYC1t (in LNAT) further stimulated recombination may reflect an increase in R loop formation downstream of pA signals. To determine whether this recombination phenotype was specific to Sen1, we similarly tested other NRD complex components. Although recombination levels were somewhat increased in nab3 and nrd1 CTD-interacting domain mutants (but not the RNA binding domain mutant nrd1-102) transformed with LNA, they were reduced to background levels in LNAT (Figure 2A). This suggests that these NRD mutants still recognize the CYC1t. Why these NRD mutations elicit some recombination is unclear at this point, but may reflect alteration in mRNP biogenesis. The fact that CYC1t abrogates recombination in NRD mutants but stimulates recombination in sen1-1 clearly separates Sen1 function from Nab3 and Nrd1 and argues that Sen1 plays a distinct role outside the NRD complex. Hyperrecombination in THO and THSC/TREX-2 mutants shows clear transcription dependence, as it increases with greater transcript length and transcription rate but decreases when the R loop-forming RNA is removed either by RNase H activity or ribozyme directed RNA cleavage (González-Aguilera et al., 2008González-Aguilera C. Tous C. Gómez-González B. Huertas P. Luna R. Aguilera A. The THP1-SAC3-SUS1-CDC31 complex works in transcription elongation-mRNA export preventing RNA-mediated" @default.
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• W2125433175 title "Yeast Sen1 Helicase Protects the Genome from Transcription-Associated Instability" @default.
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• W2125433175 doi "https://doi.org/10.1016/j.molcel.2010.12.007" @default.
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