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

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Schizosaccharomyces pombe Saccharomyces cerevisiae

PROVIDER: GSE146346 | GEO | 2020/03/04

REPOSITORIES: GEO

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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.

2020-03-04 | GSE146318 | GEO

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 Saccharomyces cerevisiae.

Project description:Mediator is a modular coactivator complex involved in the transcription of the majority of RNA polymerase II-regulated genes. However, the degrees to which individual core subunits of Mediator contribute to its activity have been unclear. Here, we investigate the contribution of two essential architectural subunits of Mediator to transcription in Saccharomyces cerevisiae. We show that acute depletion of the main complex scaffold Med14 or the head module nucleator Med17 is lethal and results in global transcriptional downregulation, though Med17 removal has a markedly greater negative effect. Consistent with this, Med17 depletion impairs preinitiation complex (PIC) assembly to a greater extent than Med14 removal. Co-depletion of Med14 and Med17 reduced transcription and TFIIB promoter occupancy similarly to Med17 ablation alone, indicating that the contributions of Med14 and Med17 to Mediator function are not additive. We propose that, while the structural integrity of complete Mediator and the head module are both important for PIC assembly and transcription, the head module plays a greater role in this process and is thus the key functional module of Mediator in this regard.

| S-EPMC8045717 | biostudies-literature

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

Architectural Mediator subunits are differentially essential for global transcription in yeast (ChIP-seq)

Project description:Mediator is a modular complex required for RNA polymerase II transcription in vivo. We show that Mediator destabilization via depletion of the main complex scaffold Med14 or the head module scaffold Med17 is lethal and results in global transcriptional downregulation, though Med17 destruction has a markedly greater negative effect. Depletion of Med14 or Med17 impairs pre-initiation complex (PIC) formation and/or stability to similar degrees, suggesting that the differential transcriptional effects of head module destabilization and overall dissociation of the complex are not due to different extents of defective PIC formation. Co-depletion of Med14 and Med17 reduced transcription and TFIIB promoter occupancy to similar degrees as Med17 loss alone, suggesting that the independent head module can weakly stimulate transcription in vivo, though not at a level that maintains viability. Collectively, our data suggest that the head is the major functional module of Mediator with respect to PIC function in vivo.

2020-03-05 | GSE145494 | GEO

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

Project description:Architectural Mediator subunits are differentially essential for global transcription in yeast (RNA-seq/4tU-seq)

| PRJNA610215 | ENA

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

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

| PRJNA610076 | ENA

Architectural Mediator subunits are differentially essential for global transcription in yeast (ChIP-seq)

Project description:Architectural Mediator subunits are differentially essential for global transcription in yeast (ChIP-seq)

| PRJNA607312 | ENA

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

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

| PRJNA610077 | ENA

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

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

| PRJNA610054 | ENA

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