Unknown,Transcriptomics,Genomics,Proteomics

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Architecture of the RNA polymerase II-Mediator core transcription initiation complex

ABSTRACT: Comparative Dynamic Transcriptome Analysis (cDTA) enables global analysis of newly synthesized RNA as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111) and reveals defects in transcription with much higher sensitivity than conventional steady-state methods. cDTA was carried out as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111), using the S. cerevisiae heterozygous Med17/med17delta strain (Euroscarf) transfected with plasmids pRS315-SRB4 or pRS315-srb4-ts as described in Larivi̬re et al. Nature. 2012 (DOI:10.1038/nature11670), and Y40343-wildtype (Euroscarf) or Med18-FRB-KanMX6 (Euroscarf) strains. Heatshock of SRB4 and srb4-ts strains was applied for 18 or 60 minutes at 37C prior to RNA labeling as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). To deplete the Med18 subunit from the nucleus, anchor-away experiments were performed by rapamycin treatment (1 ug/ml in 200 mL YPD) for 18 or 60 minutes at 30C prior to RNA labeling as described in Sun et al. Mol. Cell. 2013 (DOI:10.1016/j.molcel.2013.09.010). Data analysis was as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111).

ORGANISM(S): Saccharomyces cerevisiae S288c

SUBMITTER: Clemens Plaschka

PROVIDER: E-MTAB-2942 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The rates of mRNA synthesis and degradation determine cellular mRNA levels and can be monitored by comparative Dynamic Transcriptome Analysis (cDTA) that uses non-perturbing metabolic RNA labeling. Here we present cDTA data for 46 yeast strains lacking genes involved in mRNA degradation and metabolism. In these strains, changes in mRNA degradation rates are generally compensated by changes in mRNA synthesis rates, resulting in a buffering of mRNA levels. We show that buffering of mRNA levels requires the RNA exonuclease Xrn1. The buffering is rapidly established when mRNA synthesis is impaired, but is delayed when mRNA degradation is impaired, apparently due to Xrn1-dependent transcription repressor induction. Cluster analysis of the data defines the general mRNA degradation machinery, reveals different substrate preferences for the two mRNA deadenylase complexes Ccr4-Not and Pan2-Pan3, and unveils an interwoven cellular mRNA surveillance network. For this purpose, cDTA was carried out as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). S. cerevisiae cultures were grown at 30¡ãC in 50 ml aliquots of YPD medium. S. pombe cultures were grown at 32¡ãC in YES medium. For inhibitor perturbation, BY4741 cells (OD600=0.1) were inoculated from a saturated overnight culture in two 100 ml aliquots of YPD liquid medium, incubated at 30¡ãC and grown to early-log phase (OD600=0.8). Cells were labeled for 6 minutes (sample 0 min). Then cycloheximide was added and cells were labeled for 10 minutes and 60 minutes. Cells were harvested and treated as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). 1,10-Phenanthroline was added to the culture to a final concentration of 25 _g/ml as described (Dori-Bachash et al., 2012), and labeled for 6 minutes after 18 minutes treatment. Cells were treated as above. For the anchor-away analysis, S. cerevisiae cells (OD600=0.1) were cultured in two 200 ml aliquots of YPD containing 1_g/ml rapamycin and incubated at 30¡ãC untill OD600 reached 0.8. Due to the instability of rapamycin, we added 1_g/ml rapamycin every two hours. Then a 20 ml sample was taken and labeled with 4sU. The cells were then treated as described in Sun et.al. Genome Res. 2012 (DOI:10.1101/gr.130161.111).

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Architecture of the RNA polymerase II-Mediator core transcription initiation complex

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