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High-resolution mapping of transcribing RNAPIII by CRAC upon depletion of Nrd1 [Experiment 2]

ABSTRACT: Here we report high-resolution analyses of transcribing RNAPIII in either a wild-type (WT) background or a Nrd1 auxin-inducible degron (AID) strain in which Nrd1 can be rapidly depleted upon addition of the auxin analogue Indole-3-acetic acid (IAA). Nrd1 functions together with Sen1 in transcription termination at RNAPII-dependent non-coding genes. Here we show that depletion of Nrd1 does not affect transcription termination at RNAPIII-dependent genes, indicating that Nrd1 is not an RNAPIII transcription termination factor.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE174699 | GEO | 2022/07/13

REPOSITORIES: GEO

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High-resolution mapping of transcribing RNAPIII by CRAC upon mutation or depletion of Sen1 [Experiment 1]

Project description:Here we report high-resolution analyses of transcribing RNAPIII in either a wild-type (WT) background , a sen1-3 mutant or a Sen1 auxin-inducible degron (AID) strain in which Sen1 can be rapidly depleted upon addition of the auxin analogue Indole-3-acetic acid (IAA). Sen1 is a well-characterized transcription termination factor for RNAPII-dependent genes in budding yeast. Here we show that the presence of three point mutations in sen1-3 that abrogate the interaction of Sen1 with RNAPIII, as well as the depletion of Sen1 provoke global transcription termination defects at RNAPIII-dependent genes. These results indicate that Sen1 is also a transcription termination factor for RNAPIII transcription units.

2022-07-13 | GSE174698 | GEO

High-resolution mapping of transcribing RNAPIII by CRAC upon mutation of Sen1 in the G1 phase of the cell cycle [Experiment 3]

Project description:Here we report high-resolution analyses of transcribing RNAPIII in either a wild-type (WT) background or a sen1-3 mutant in the G1 phase of the cell cycle. The mutations in Sen1-3 prevent the interaction of Sen1 with both RNAPIII and the replisome, raising the question whether the role of Sen1 in RNAPIII transcription termination could depend on the association of Sen1 with the replisome. Our data show that in the G1 phase of the cell cycle, where the replisome is not assembled, the sen1-3 mutant also exhibits transcription termination defects at RNAPIII-dependent genes, as in asynchronous cells. This result indicates that the role of Sen1 in termination at RNAPIII-dependent genes is independent on the association of Sen1 with the replisome.

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High-resolution mapping of transcribing RNAPII by CRAC in the presence of different Sen1 variants

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High-resolution mapping of transcribing RNAPIII by CRAC upon depletion of Nrd1 [Experiment 2]

Project description:High-resolution mapping of transcribing RNAPIII by CRAC upon depletion of Nrd1 [Experiment 2]

| PRJNA731142 | ENA

High-resolution transcriptome-wide maps of Sen1 RNA-binding by CRAC

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High-resolution mapping of transcribing RNAPII by CRAC in a wt or a sen1T1623E mutant

Project description:Manuscript title: Modulated termination of non-coding transcription partakes in the regulation of gene expression Here we report high-resolution analyses of transcribing RNAPII in either a wild-type (WT) background or a sen1T1623E mutant, which harbours a substitution that mimics the phosphorylation at threonine 1623. Sen1 is a well-characterized transcription termination factor for RNAPII-dependent non-coding genes in budding yeast. Here we show that the T1623E phosphomimetic mutation induces moderate but widespread termination defects at target non-coding genes, strongly suggesting that phosphoryation at T1623 modulates negatively Sen1 transcription termination activity.

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Characterization of extended transcripts by RNA-Seq suggests that Rrp6 influences Nrd1-dependent termination

Project description:RNAPII is responsible for transcription of protein-coding genes and short, regulatory RNAs. In Saccharomyces cerevisiae, termination of RNAPII-transcribed RNAs ≤1000 bases requires the NNS complex (comprised of Nrd1, Nab3, and Sen1) processing by the exosome, and the nuclear specific catalytic subunit, Rrp6. It has been shown that Rrp6 interacts directly with Nrd1, but whether or not Rrp6 is required for NNS-dependent termination is unclear. Loss of Rrp6 function may result in extension (or inhibition of termination) of NNS-dependent transcripts, or Rrp6 may only function after the fact to carry out RNA 3’ end processing. Here, we performed in-depth differential expression analyses and compare RNA-sequencing data of transcript length and abundance in cells lacking RRP6 to previously published sequencing data measuring the length of RNAs in Nrd1-depleted cells. We find many transcripts that were defined as unterminated upon loss of Nrd1 activity are of similar length in rrp6Δ, and expression levels of downstream genes are significantly decreased. This suggests a similar transcription interference mechanism occurs in cells lacking either Nrd1 or Rrp6, supporting the hypothesis that Rrp6 activity is required for proper NNS termination in vivo. Four biological replicates each for deletion mutant (RRP6) and reference cells (WT)

2015-04-14 | E-GEOD-57155 | biostudies-arrayexpress

Characterization of extended transcripts by RNA-Seq suggests that Rrp6 influences Nrd1-dependent termination

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Transcriptome-Wide Binding Sites for Components of the Saccharomyces cerevisiae Non-poly(A) termination Pathway: Nrd1, Nab3 and Sen1

Project description:We report here the transcriptome-wide distribution of yeast Rpb2, Sen1, Nrd1 and Nab3 binding sites. These data sets provide highresolution definition of non-poly(A) terminators, identify novel genes regulated by attenuation of nascent transcripts close to the promoter, and demonstrate the widespread occurrence of Nrd1-bound 3'-antisense transcripts on genes that are poorly expressed. In addition, we show that Sen1 does not cross-link to many expected ncRNAs but surprisingly binds to pre-mRNA transcripts suggesting a role in 3' end formation and/or termination. Six samples by adaptation of PAR-ClIP procedure

2011-09-08 | E-GEOD-31764 | biostudies-arrayexpress

The Nrd1-like protein Seb1 coordinates co-transcriptional 3â end processing and polyadenylation site selection

Project description:Termination of RNAPII transcription is associated with RNA 3â end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in S. cerevisiae do not rely on RNA cleavage for termination, but instead terminate via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the S. pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination, but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3â end processing factors, is enriched at the 3â end of genes, and binds RNA motifs downstream of cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3â UTR length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3â end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 co-transcriptionally controls alternative polyadenylation. Two biological replicates of Seb1 and Control (parental strain) CRAC experiments

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