Unknown,Transcriptomics,Genomics,Proteomics

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The RNA Exosome Promotes Transcription Termination of backtracked RNA Polymerase II

ABSTRACT: Large fractions of genomes are expressed in most eukaryotic cells, generating a diverse repertoire of protein-coding and non-coding transcripts. With such complexity, and as the number of mRNA molecules per cell are often low, it is clear that errors anywhere in the gene expression process could have profound consequences on cellular functions. The exosome is a conserved RNA degradation machine that plays a fundamental role in monitoring the quality of gene expression in the nucleus. The current view is that the nuclear exosome constantly scrutinizes transcription and contributes to post-transcriptional turnover of faulty mRNAs. Yet, how nuclear RNA surveillance by the exosome is coordinated with transcription is still unknown. Here we show that the exosome can directly target the transcription machinery by terminating transcription events associated with paused and backtracked RNA polymerase II (RNAPII), contrary to the notion that the exosome acts exclusively on RNAs that have been released by RNAPII. Our data support a mechanism by which RNAPII backtracking provides a free RNA 3' end for the core exosome, resulting in transcription termination with the concomitant degradation of the associated transcript. These findings uncover a mechanism of co-transcriptional RNA surveillance whereby termination of transcription by the exosome prevents aberrant read-through RNAs and transcriptional interference at neighboring genes.

INSTRUMENT(S): Illumina MiSeq, Illumina HiSeq 2000

ORGANISM(S): Schizosaccharomyces pombe

SUBMITTER: Samuel Marguerat

PROVIDER: E-MTAB-2237 | 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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The RNA Exosome Promotes Transcription Termination of backtracked RNA Polymerase II

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