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High-resolution mapping of budding yeast Nab3, Nrd1, and RNA polymerase II by CRAC in wild-type and rrp6∆ strains.

ABSTRACT: High-resolution mapping of budding yeast Nab3, Nrd1, and RNA polymerase II by CRAC in wild-type and rrp6∆ strains.

PROVIDER: PRJNA573707 | ENA |

REPOSITORIES: ENA

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			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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High-resolution mapping of budding yeast Nab3, Nrd1, and RNA polymerase II by CRAC in wild-type and rrp6∆ strains.

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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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.

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Mapping of Nrd1 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

Project description:Using CRAC, we compared the transcriptomic occupancy of Nrd1 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was two-fold: on the one hand, we aimed to verify that the mutations inserted in the Nab3 binding sequences of PIC2 affected the binding of Nrd1 to the PIC2 transcript; on the other hand, we wanted to check whether differential binding of Nab3 to PIC2 would affect how the protein bound other targets in the genome. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.).

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

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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:Whole-proteome profiling enabled us to compare proteomic differences between a Saccharomyces cerevisiae parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. Previous experiments had established that disrupting Nab3 binding to PIC2 while maintaining Nrd1 binding to the same transcript led to severe growth defects, larger cell size, delayed cell cycle and resistance to oxidative stress. Following characterization of a PIC2-overexpressing mutant we established that these phenotypes were not related to Pic2 overabundance. Therefore, the purpose of this experiment was to determine (i) whether the proteomic signature of the mutant lacking Nab3 RNA-binding sites in PIC2 could account for the observed phenotypes and (ii) if the proteins that were differentially expressed in such mutant were targeted by Nab3 and Nrd1.

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Mapping of Nab3 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

Project description:Using CRAC, we compared the transcriptomic occupancy of Nab3 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was two-fold: on the one hand, we aimed to verify that the mutations inserted in the Nab3 binding sequences of PIC2 had indeed abrogated binding of Nab3 to the PIC2 transcript; on the other hand, we wanted to check whether differential binding of Nab3 to PIC2 would affect how the protein bound other targets in the genome. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.).

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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.

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