Project description:R-loop disassembly by the human helicase Senataxin contributes to genome stability and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin-mediated R-loop disassembly 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 the primary terminator at 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.
Project description:RNA Polymerase II (RNAPII) termination for transcripts containing a polyadenylation signal (PAS) is thought to differ mechanistically from termination for PAS-independent RNAPII transcripts such as sn(o)RNAs. In a screen for factors required for PAS-dependent termination, we identified Sen1, a putative helicase known primarily for its role in PAS-independent termination. We show that Sen1 is required for termination on hundreds of protein-coding genes and suppresses cryptic transcription from nucleosome-free regions on a genomic scale. These effects often overlap with but are also often distinct from those caused by Nrd1 depletion, which also impacts termination of protein-coding and cryptic transcripts, including many genic antisense transcripts. Sen1 controls termination through its helicase activity and stimulates recruitment of factors previously implicated in both PAS-dependent (Rna14, Rat1) and PAS-independent (Nrd1) termination. Thus, RNAPII termination for both protein-coding genes and cryptic transcripts is dependent on multiple pathways. The 2 RNAPII datasets were produced in duplicates and the Sen1 and Nrd1 datasets in triplicates (all IP/Input).
Project description:RNA Polymerase II (RNAPII) termination for transcripts containing a polyadenylation signal (PAS) is thought to differ mechanistically from termination for PAS-independent RNAPII transcripts such as sn(o)RNAs. In a screen for factors required for PAS-dependent termination, we identified Sen1, a putative helicase known primarily for its role in PAS-independent termination. We show that Sen1 is required for termination on hundreds of protein-coding genes and suppresses cryptic transcription from nucleosome-free regions on a genomic scale. These effects often overlap with but are also often distinct from those caused by Nrd1 depletion, which also impacts termination of protein-coding and cryptic transcripts, including many genic antisense transcripts. Sen1 controls termination through its helicase activity and stimulates recruitment of factors previously implicated in both PAS-dependent (Rna14, Rat1) and PAS-independent (Nrd1) termination. Thus, RNAPII termination for both protein-coding genes and cryptic transcripts is dependent on multiple pathways.
Project description:It is currently believed that termination by RNAPII occurs differently depending whether a transcript contains or lacks a polyadenylation signal (PAS). By screening for factors deficient for PAS-dependent termination in an in vivo reporter assay, we identified Sen1, a putative helicase mainly known for its role in PAS-independent termination of snoRNAs. We show for the first time that Sen1 regulates transcription termination at protein-encoding genes genome-wide. As well, we show that Sen1 suppresses cryptic transcription genome-wide, besides being required for termination of most snoRNAs. We provide evidence that Sen1 controls termination through its helicase activity and by effectively recruiting to chromatin, factors implicated in PAS-dependent (Rna14 and Rat1) or PAS-independent termination (Nrd1). Importantly, we demonstrate that the effect on transcription termination of Sen1 and Nrd1, although similar, differ quantitatively and qualitatively. Our results suggest that in yeast, termination by RNAPII at protein encoding-genes makes use of redundant pathways.
Project description:Functional engagement of RNA polymerase II (Pol II) with eukaryotic chromosomes is a fundamental and highly regulated biological process. Here we present the first high-resolution map of Pol II occupancy across the entire yeast genome. We compared a wild-type strain with a strain bearing a substitution in the Sen1 helicase, which is a Pol II termination factor for non-coding RNA genes. The wildtype pattern of Pol II distribution provides unexpected insights into the mechanisms by which genes are repressed or silenced. Remarkably, a single amino acid substitution that compromises Sen1 function causes profound changes in Pol II distribution over both non-coding and protein-coding genes, establishing an important function of Sen1 in the regulation of transcription. Given the strong similarity of the yeast and human Sen1 proteins, our results suggest that progressive neurological disorders caused by substitutions in the human Sen1 homolog, Senataxin, may be due to misregulation of transcription. Keywords: transcription termination, attenuation, silencing, non-coding RNA, Pol II, ChIP-chip
Project description:Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetic, genomic and imaging approaches we found that Rrm3hPif1 and Sen1hSenataxin helicases assist termination at TERs, Sen1 at telomeres. rrm3 and sen1 are synthetic lethal, fail to terminate replication exhibiting lagging chromosomes and fragility at TERs and telomeres. sen1 rrm3 build up RNA-DNA hybrids at TERs, sen1 accumulates RNPII at TERs and telomeres. Double mutants exhibit X-shaped gapped or reversed converging forks. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of fork-associated Top1 and Top2 with those of gene loop-associated Top1 and Top2 by preventing DNA and RNA polymerases slowing down when forks encounter transcription head-on or codirectionally, respectively. Hence Rrm3 and Sen1 are essential to generate permissive topological conditions for replication termination.
Project description:Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetic, genomic and imaging approaches we found that Rrm3hPif1 and Sen1hSenataxin helicases assist termination at TERs, Sen1 at telomeres. rrm3 and sen1 are synthetic lethal, fail to terminate replication exhibiting lagging chromosomes and fragility at TERs and telomeres. sen1 rrm3 build up RNA-DNA hybrids at TERs, sen1 accumulates RNPII at TERs and telomeres. Double mutants exhibit X-shaped gapped or reversed converging forks. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of fork-associated Top1 and Top2 with those of gene loop-associated Top1 and Top2 by preventing DNA and RNA polymerases slowing down when forks encounter transcription head-on or codirectionally, respectively. Hence Rrm3 and Sen1 are essential to generate permissive topological conditions for replication termination.
Project description:Transcription termination is key to gene regulation as it prevents transcription interference with neighboring genes. In Saccharomyces cerevisiae, termination at protein-coding genes is coupled to the cleavage of the nascent transcript, while most non-coding RNA transcription relies on a cleavage-independent termination pathway involving Nrd1, Nab3 and the helicase Sen1 (NNS pathway). In both pathways, the recruitment of termination factors involves phosphorylated forms of the RNA polymerase II C-terminal domain (CTD) but the contribution of individual CTD residues was never systematically investigated. Here, we investigated the impact of mutating phosphorylation sites in the CTD on termination. We observed widespread termination defects at protein-coding genes in mutants for Ser2 or Thr4 but rare defects in Tyr1 mutants for this class of genes. Instead, mutating Tyr1, or its phosphatase Glc7, led to widespread termination defects at non-coding genes known to terminate via the NNS pathway. These defects can be suppressed by slowing down transcription, suggesting that Tyr1 mediates termination via the regulation of elongation or pausing. Our work redefines the role of Tyr1 in termination at protein-coding genes in budding yeast and highlights its key role in termination by the NNS pathway.
Project description:Transcription termination is key to gene regulation as it prevents transcription interference with neighboring genes. In Saccharomyces cerevisiae, termination at protein-coding genes is coupled to the cleavage of the nascent transcript, while most non-coding RNA transcription relies on a cleavage-independent termination pathway involving Nrd1, Nab3 and the helicase Sen1 (NNS pathway). In both pathways, the recruitment of termination factors involves phosphorylated forms of the RNA polymerase II C-terminal domain (CTD) but the contribution of individual CTD residues was never systematically investigated. Here, we investigated the impact of mutating phosphorylation sites in the CTD on termination. We observed widespread termination defects at protein-coding genes in mutants for Ser2 or Thr4 but rare defects in Tyr1 mutants for this class of genes. Instead, mutating Tyr1, or its phosphatase Glc7, led to widespread termination defects at non-coding genes known to terminate via the NNS pathway. These defects can be suppressed by slowing down transcription, suggesting that Tyr1 mediates termination via the regulation of elongation or pausing. Our work redefines the role of Tyr1 in termination at protein-coding genes in budding yeast and highlights its key role in termination by the NNS pathway.
Project description:Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a risk that needs to be controlled to prevent the perturbation of gene expression. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy terminal domain (CTD) of RNA polymerase II and characterize structurally its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires the recognition of the Ser5-phosphorylated form of the CTD by the N-terminal domain of Sen1. Furthermore, we find that the N-terminal and the C-terminal domains of Sen1 can mediate intra-molecular interactions. Our results shed light onto the network of protein-protein interactions that control termination of non-coding transcription by Sen1.