High-resolution mapping of budding yeast Nab3, Nrd1, and RNA polymerase II by CRAC in wild-type and rrp6∆ strains.
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ABSTRACT:
PROVIDER: PRJNA573707 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10166128.fastq.gz | Fastqsanger.gz | |||
| SRR10166129.fastq.gz | Fastqsanger.gz | |||
| SRR10166130.fastq.gz | Fastqsanger.gz | |||
| SRR10166131.fastq.gz | Fastqsanger.gz | |||
| SRR10166132.fastq.gz | Fastqsanger.gz |
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Project description:We obtained Nab3, Nrd1, and RNA polymerase II occupancy profiles across the genome of S.cerevisiae in wild-type and rrp6∆ strains. This allowed us to determine the impact of defective nuclear exosome on NNS-dependent transcription termination.
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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.
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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)
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Project description:Nrd1 and Nab3 are two yeast RNA binding proteins which have been shown to be involved in transcription termination of non poly(A) genes. We have used expression profiling of a Nab3 mutant to discover novel RNA targets of the Nrd1 and Nab3 transcription termination pathway. Failure to terminate RNA polymerase II by Nab3 leads to continued transcription well beyond the correct termination sites, altering the expression of adjacent downstream genes. Using this concept, our microarray uncovered the up-regulation of numerous genes that are located downstream of âcryptic unstable transcriptsâ, transcripts that are transcribed, terminated and rapidly degraded by Nrd1, Nab3, and the nuclear exosome. Experiment Overall Design: Four yeast total RNA samples where analyzed in this study. These RNAs come from two separate strains of yeast, each strain at either the permissive temperature (25C) or the non-permissive temperature (37C). One of these strains is a wild type strain of yeast used as a control. The other strain has a mutation in the Nab3 protein that confers temperature sensitivity at the non-permissive temperature of 37C. After two hours at the non-permissive temperature, we observe disruptions in the Nrd1/Nab3 transcription termination pathway and this is the scheme that we followed for our microarray experiment. Our goal was to observe the global expression changes after 2 hours without Nab3 function.
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Project description:Nrd1 and Nab3 are two yeast RNA binding proteins which have been shown to be involved in transcription termination of non poly(A) genes. We have used expression profiling of a Nab3 mutant to discover novel RNA targets of the Nrd1 and Nab3 transcription termination pathway. Failure to terminate RNA polymerase II by Nab3 leads to continued transcription well beyond the correct termination sites, altering the expression of adjacent downstream genes. Using this concept, our microarray uncovered the up-regulation of numerous genes that are located downstream of “cryptic unstable transcripts”, transcripts that are transcribed, terminated and rapidly degraded by Nrd1, Nab3, and the nuclear exosome. Keywords: yeast temperature sensitive mutant 25C vs 37C
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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
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Project description:The relationship between aging and RNA biogenesis and trafficking is attracting growing interest, yet the precise mechanisms are unknown. The THO complex is crucial for mRNA cotranscriptional maturation and export. Herein, we report that the THO complex is closely linked to maintaining a normal lifespan. Deficiencies in Hpr1 and Tho2, components of the THO complex, reduced replicative lifespan (RLS) and are linked to a novel Sir2-independent RLS control pathway. Although transcript sequestration in hpr1Δ or tho2Δ mutants was countered by exosome component Rrp6, loss of this failed to mitigate RLS defects in hpr1Δ. However, RLS impairment in tho2Δ was counteracted by Nrd1-specific mutants that interacted with Rrp6. This effect relied on the interaction of Nrd1, a transcriptional regulator of aging-related genes, including ribosome biogenesis or RNA metabolism genes, with RNA polymerase II. Nrd1 overexpression reduced RLS in a Tho2-dependent pathway. Intriguingly, THO2 deletion mirrored Nrd1 overexpression effects by inducing arbitrary Nrd1 chromatin binding. Taken together, these findings underscore the importance of Tho2-mediated Nrd1 escorting in maintaining a normal lifespan by transcriptional regulation of aging-related genes.
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