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| Status |
Public on Mar 06, 2019 |
| Title |
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 |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
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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| Overall design |
Nuclear RNA-seq was performed on an auxin-inducible degron (AID) tagged cell lines in treated with or without auxin inducing rapid depletion of EXOSC10 or DIS3 protein. HCT116 cells also ectopically express TIR1 from a sleeping beauty transposon integrated cassette. AID incorporated proteins contain in-frame insertion of AID tag, P2A cleavage site, antibiotic resistance marker and SV40 polyA site. Two repair templates expressing different antibiotic resistance markers (neomycin and hygromycin) were used to ensure biallelic intersion of the repair cassette using CRISPR/Cas9. We also performed individual nucleotide cross-linking and immunoprecipitation (iCLIP) on a catalytic mutant of EXOSC10 (D313N), in order to complement a previous iCLIP experiment using EXOSC10 WT. We reasoned that by using a catalytic mutant of EXOSC10 we would be able to stabilize interactions between the exoribonuclease and it global RNA substrates. In the process we identified 30nt footprints of iCLIP density downstream of pre-rRNAs and pre-snoRNAs exclusively seen in EXOSC10 CAT CLIP profiles.
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| Contributor(s) |
Davidson L, Francis L, Cordiner RA, Eaton JD, Estell C, Macias S, Caceres JF, West S |
| Citation(s) |
30840897 |
| Submission date |
Sep 27, 2018 |
| Last update date |
Mar 27, 2019 |
| Contact name |
Steven West |
| E-mail(s) |
s.west@exeter.ac.uk
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| Phone |
0044(0)1392727458
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| Organization name |
University of Exeter
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| Department |
Living Systems Institute
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| Street address |
Stocker Rd
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| City |
Exeter |
| State/province |
Devon |
| ZIP/Postal code |
EX4 4QD |
| Country |
United Kingdom |
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| Platforms (2) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (12)
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| GSM3403542 |
Nuclear RNA-seq EXOSC10-AID Minus auxin rep 1 |
| GSM3403543 |
Nuclear RNA-seq EXOSC10-AID Plus auxin rep 1 |
| GSM3403544 |
Nuclear RNA-seq EXOSC10-AID Minus auxin rep 2 |
| GSM3403545 |
Nuclear RNA-seq EXOSC10-AID Plus auxin rep 2 |
| GSM3403546 |
Nuclear RNA-seq DIS3-AID Minus auxin rep 1 |
| GSM3403547 |
Nuclear RNA-seq DIS3-AID Plus auxin rep 1 |
| GSM3403548 |
Nuclear RNA-seq DIS3-AID Minus auxin rep 2 |
| GSM3403549 |
Nuclear RNA-seq DIS3-AID Plus auxin rep 2 |
| GSM3403550 |
HEK293T EXOSC10CAT rep 1 (iCLip-seq) |
| GSM3403551 |
HEK293T EXOSC10CAT rep 2 (iCLip-seq) |
| GSM3403552 |
HEK293T EXOSC10WT rep 1 (iCLip-seq) |
| GSM3403553 |
HEK293T EXOSC10WT rep 2 (iCLip-seq) |
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| Relations |
| BioProject |
PRJNA493621 |
| SRA |
SRP162755 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE120574_45srDNA.annotation.bed.gz |
217 b |
(ftp)(http) |
BED |
| GSE120574_45srDNA.fa.gz |
4.1 Kb |
(ftp)(http) |
FA |
| GSE120574_EXOSC10-CAT.45srDNA.bw |
158.6 Kb |
(ftp)(http) |
BW |
| GSE120574_EXOSC10-CAT.hg38.antisense.bw |
3.8 Mb |
(ftp)(http) |
BW |
| GSE120574_EXOSC10-CAT.hg38.sense.bw |
5.4 Mb |
(ftp)(http) |
BW |
| GSE120574_EXOSC10-WT.45srDNA.bw |
154.3 Kb |
(ftp)(http) |
BW |
| GSE120574_EXOSC10-WT.hg38.antisense.bw |
3.1 Mb |
(ftp)(http) |
BW |
| GSE120574_EXOSC10-WT.hg38.sense.bw |
4.4 Mb |
(ftp)(http) |
BW |
| GSE120574_RAW.tar |
2.9 Gb |
(http)(custom) |
TAR (of BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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