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

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. Overall design: Experiments were performed using biological replicates (labeled 1 or 2) for each condition. The analyzed cell line contains a knock-in of Tir1 from Oryza sativa and an AID tag on Med14 and Med17 that allows the specific degradation of both proteins in the presence of auxin. Cells were either treated with 3-IAA ("+ auxin") or DMSO ("-auxin") for 30 min followed by a 6 min labelling with 5 mM 4-thiouracil (4tU). total RNA was extracted and analyzed by RNA-seq. Furthermore 4tU labelled RNA (nsRNA) was purified from total RNA extracts and also analyzed by RNA-seq.