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The immediate impact of exoribonucleolysis on nuclear RNA processing, turnover and transcriptional control revealed by rapid depletion of DIS3, EXOSC10 or XRN2 from human cells

ABSTRACT: The immediate impact of exoribonucleolysis on nuclear RNA processing, turnover and transcriptional control revealed by rapid depletion of DIS3, EXOSC10 or XRN2 from human cells

PROVIDER: PRJNA493621 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7909090.fastq.gz	Fastqsanger.gz
	SRR7909091.fastq.gz	Fastqsanger.gz
	SRR7909092.fastq.gz	Fastqsanger.gz
	SRR7909093.fastq.gz	Fastqsanger.gz
	SRR7909094.fastq.gz	Fastqsanger.gz

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The immediate impact of exoribonucleolysis on nuclear RNA processing, turnover and transcriptional control revealed by rapid depletion of DIS3, EXOSC10 or XRN2 from human cells

Project description:Cell-based studies of human ribonucleases traditionally relies on methods that deplete proteins slowly. To assess their immediate roles in nuclear RNA biology, we engineered cells where the 3’->5’ exoribonucleases of the exosome complex, Dis3 and EXOSC10, can be eliminated within 60 minutes. Loss of Dis3 has the greatest impact, causing thousands of enhancer RNAs, intragenic transcripts, promoter upstream transcripts (PROMPTs) and products of premature cleavage and polyadenylation (PCPA) to accumulate. Interestingly, EXOSC10 only targets these substrates when Dis3 is absent, which is explained by its rapid mis-localization to the nucleoplasm following Dis3 loss. Direct detection of EXOSC10 substrates revealed a more specific role in trimming of short 3’ extensions on ribosomal and small nucleolar RNAs. Finally, the 5’-3’ exoribonuclease, Xrn2, has little activity on exosome substrates, but promotes termination following PCPA. Interestingly, Xrn2 is not rate limiting for PROMPT termination providing a distinction between sense and anti-sense transcriptional regulation.

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RNA-seq of shRNA-mediated EXOSC10 and XRN2 knockdown

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RNA-seq of shRNA-mediated EXOSC10 and XRN2 knockdown

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Rapid Depletion of DIS3, EXOSC10, or XRN2 Reveals the Immediate Impact of Exoribonucleolysis on Nuclear RNA Metabolism and Transcriptional Control.

Project description:Cell-based studies of human ribonucleases traditionally rely on methods that deplete proteins slowly. We engineered cells in which the 3'→5' exoribonucleases of the exosome complex, DIS3 and EXOSC10, can be rapidly eliminated to assess their immediate roles in nuclear RNA biology. The loss of DIS3 has the greatest impact, causing the substantial accumulation of thousands of transcripts within 60 min. These transcripts include enhancer RNAs, promoter upstream transcripts (PROMPTs), and products of premature cleavage and polyadenylation (PCPA). These transcripts are unaffected by the rapid loss of EXOSC10, suggesting that they are rarely targeted to it. More direct detection of EXOSC10-bound transcripts revealed its substrates to prominently include short 3' extended ribosomal and small nucleolar RNAs. Finally, the 5'→3' exoribonuclease, XRN2, has little activity on exosome substrates, but its elimination uncovers different mechanisms for the early termination of transcription from protein-coding gene promoters.

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XRN2

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Reducedfemalefertility duetosequestration ofRNA Pol II by pervasive transcription in exosome RNasedepleted oocytes

Project description:We performed mouse single oocyte RNA-seq and bulk oocyte CUT&Tag assays in the current project. In details, SMART-seq based single oocyte RNA-seq was performed at adult GV stage, GV3h stage and MII stage, using control and Dis3 oocyte-speicfic knockout (cKO) oocytes. RiboMinus-seq based single oocyte RNA-seq was performed at adult GV stage using control and Dis3 cKO oocytes, and at p20 GV stage using control, Dis3 cKO, Exosc10 cKO and Dis3/Exosc10 double cKO (dcKO) oocytes. Bulk CUT&Tag of anti-H3K27me3 was done in WT and Dis3 cKO oocytes at adult GV stage, and in WT and Dis3/Exosc10 dcKO oocytes at p20 stage. In addition, CUT&Tag of anti-RNA polymerase II (Ser2+Ser5) was performed in WT and Dis3 cKO oocytes at GV stage. All CUT&Tag experiments share the same rabbit-Igg negative control. All p20 stage oocytes were specified as p20. The non-specified GV oocytes were all adult GV oocytes.

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