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Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast - 4tU cordycepin treatment

ABSTRACT: mPAT approach using an anchored oligo-dT (TV12VN) primer to determine changes to 3'UTR length in yeast upon cordycepin treatment and new transcription using 4-thiouracil (4tU) labelling of RNA. This appears as a T to C conversion in sequencing reads.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE160993 | GEO | 2021/06/25

REPOSITORIES: GEO

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Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast - 4tU cordycepin treatment with rpb1 slow mutant

Project description:mPAT approach using an anchored oligo-dT (TV12VN) primer to determine changes to 3'UTR length in yeast with a slow RNA polymerase II mutant rpb1-1, complemented with wild-type RPB1 or a slow rpb1 mutant plasmid, upon cordycepin treatment and new transcription using 4-thiouracil (4tU) labelling of RNA. This appears as a T to C conversion in sequencing reads.

2021-06-25 | GSE160994 | GEO

Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast - 4tU cordycepin treatment

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Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast - cordycepin treatment

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Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast - 4tU cordycepin treatment with rpb1 slow mutant

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Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast - 3'end cleavage factor mPAT

Project description:mPAT approach was used to determine changes to 3'UTR length in yeast upon mutation of cleavage and polyadenylation factor subunits compared to wild type cells

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Architectural Mediator subunits are differentially essential for global transcription in yeast (RNA-seq/4tU-seq ; Sua7-AID)

Project description:This project aims to understand the function of general transcription factor TFIIB in transcription in S. cerevisiae. Thus, we analyzed total and newly synthesized RNA levels in a conditional Sua7 (TFIIB) depletion strain. For this purpose we used an auxin inducible degron system system to specifically induce the degradation Sua7 by addition of auxin into the culture medium. We measured transcript levels after 30 min auxin or DMSO (control) treatment induction of degradation of Sua7 by the addition of auxin (30 min) or with the carrier DMSO (control) followed by a 6 min labelling of newly synthesized RNA with 4-thiouracil (4tU). In parallel wiltype S.pombe cells were labelled with 4tU for 6min during midlog growth phase. Prior to RNA extraction (Ribopure Yeast Kit, Ambion) our labelled S.cerevisiae cells of interest were mixed with labelled S.pombe cells as a spike to allow normalization against an exogenous reference (ratio 3 S .cerevisiae for 1 S.pombe cell. From the total RNA we purified the 4tU labelled fraction of RNAs containing 4tU. Both the total RNA and the purified newly synthesized RNAs will then be subject to RNA-sequencing after ribosomal RNA depletion. In summary, we will provide 8 samples for this project containing RNA from both S.cerevisiae and S.pombe. The sample read counts were normalized with size factors computed by the median-of-ratios method proposed by Anders and Huber (Anders et al. 2010) on S. pombe genes to make these counts comparable between samples.

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Architectural Mediator subunits are differentially essential for global transcription in yeast (RNA-seq/4tU-seq ; Med14-Med17-doubleAID)

Project description:This project aims to understand the function of Mediator and the interplay in transcription in S. cerevisiae. Thus we analyzed total and newly synthesized RNA levels in a conditional Med14 and Med17 depletion strain. For this purpose we used an auxin inducible degron system system to specifically induce the degradation Med14 and Med17 by addition of auxin into the culture medium. We measured transcript levels after 30 min auxin or DMSO (control) treatment induction of degradation of Med14 and Med17 by the addition of auxin (30 min) or with the carrier DMSO (control) followed by a 6 min labelling of newly synthesized RNA with 4-thiouracil (4tU). In parallel wiltype S.pombe cells were labelled with 4tU for 6min during midlog growth phase. Prior to RNA extraction (Ribopure Yeast Kit, Ambion) our labelled S.cerevisiae cells of interest were mixed with labelled S.pombe cells as a spike to allow normalization against an exogenous reference (ratio 3 S .cerevisiae for 1 S.pombe cell. From the total RNA we purified the 4tU labelled fraction of RNAs containing 4tU. Both the total RNA and the purified newly synthesized RNAs will then be subject to RNA-sequencing after ribosomal RNA depletion. In summary, we will provide 8 samples for this project containing RNA from both S.cerevisiae and S.pombe. The sample read counts were normalized with size factors computed by the median-of-ratios method proposed by Anders and Huber (Anders et al. 2010) on S. pombe genes to make these counts comparable between samples.

2020-03-04 | GSE146321 | GEO

Architectural Mediator subunits are differentially essential for global transcription in yeast (RNA-seq/4tU-seq ; Med14-AID, Med17-AID)

Project description:This project aims to understand the function of different mediator subunits (Med14 and Med17) in transcription in S. cerevisiae. Thus we analyzed total and newly synthesized RNA levels in conditional Med14 and Med17 depletion strains. For this purpose we used an auxin inducible degron system system to specifically induce the degradation Mediator subunits by addition of auxin into the culture medium. We measured transcript levels after 30 min auxin or DMSO (control) treatment induction of degradation of Med14 or Med17 by the addition of auxin (30 min) or with the carrier DMSO (control) followed by a 6 min labelling of newly synthesized RNA with 4-thiouracil (4tU). In parallel wiltype S.pombe cells were labelled with 4tU for 6min during midlog growth phase. Prior to RNA extraction (Ribopure Yeast Kit, Ambion) our labelled S.cerevisiae cells of interest were mixed with labelled S.pombe cells as a spike to allow normalization against an exogenous reference (ratio 3 S .cerevisiae for 1 S.pombe cell. From the total RNA we purified the 4tU labelled fraction of RNAs containing 4tU. Both the total RNA and the purified newly synthesized RNAs will then be subject to RNA-sequencing after ribosomal RNA depletion. In summary, we will provide 16 samples for this project containing RNA from both S.cerevisiae and S.pombe. The sample read counts were normalized with size factors computed by the median-of-ratios method proposed by Anders and Huber (Anders et al. 2010) on S. pombe genes to make these counts comparable between samples.

2020-03-04 | GSE146300 | GEO

Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast - cordycepin treatment

Project description:Genetic and pharmacological evidence for kinetic competition between alternative poly(A) sites in yeast - cordycepin treatment

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The molecular effects of cordycepin on RNA synthesis in Saccharomyces cerevisiae

Project description:Cordycepin (3â deoxyadenosine) is a biologically active compound that, when incorporated during RNA synthesis in vitro, provokes chain termination due to the absence of a 3â hydroxyl moiety. We were interested in the effects mediated by this drug in vivo and analysed its impact on RNA metabolism of yeast. Our results support the view that cordycepin-triphosphate (CoTP) is the toxic component that is limiting cell growth through inhibition of RNA synthesis. Unexpectedly, cordycepin treatment modulated 3â end heterogeneity of ACT1 and ASC1 mRNAs and rapidly induced extended transcripts derived from CYH2 and NEL025c loci. Moreover, cordycepin ameliorated the growth defects of poly(A) polymerase mutants and the pap1-1 mutation neutralized the effects of the drug on gene expression. Our observations are consistent with an epistatic relationship between poly(A) polymerase function and cordycepin action and suggest that a major mode of cordycepin activity reduces 3â end formation efficiency independently of its potential to terminate RNA chain elongation. Finally, chemical-genetic profiling revealed genome-wide pathways linked to cordycepin activity and identified novel genes involved in poly(A) homeostasis. Keywords: response to drug treatment Each experiment was performed as triplicate. We analyzed RNA obtained from wild-type cells, from wild-type cells treated with 40 microgram/ml cordycepin for 1 hour, from pap1-1 mutant cells grown at permissive temperature (25Â°C) and from pap1-1 mutant cells grown at permissive temperature (25Â°C) treated with 40 microgram/ml cordycepin for 1 hour.

2010-05-06 | E-GEOD-14619 | biostudies-arrayexpress

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