RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA374881 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5259850.fastq.gz | Fastqsanger.gz | |||
| SRR5259890.fastq.gz | Fastqsanger.gz | |||
| SRR5259900.fastq.gz | Fastqsanger.gz | |||
| SRR5259910.fastq.gz | Fastqsanger.gz | |||
| SRR5259851.fastq.gz | Fastqsanger.gz |
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Similar Datasets
Project description:Genome wide screens identified negative genetic interactions between several cofactors of the exosome nuclease complex and the Bre5-Ubp3 ubiquitin protease complex. RNA-binding was shown for Bre5 with enrichment for sites over exon 2 of spliced pre-mRNAs and close to poly(A) sites. An inducible splicing-reporter showed a requirement for Bre5 in efficient in vivo splicing and for normal RNAPII elongation, specifically on splicing-competent genes. A Bre5-Ubp3 sensitive site of RNAPII ubiquitination was mapped at Lys1246 at the entrance to the active site of the large subunit. Ubiquitinated RNAPII was depleted at the TSS but enriched at the 5’ end of exon 2 and upstream of poly(A) sites, similar to Bre5. Mutation of Lys1246 reduced RNAPII occupancy upstream of the poly(A) site, consistent with reduced pausing at a potential surveillance site, but increased RNAPII residence downstream of the poly(A) site. Strains expressing RNAPII with the Lys1246 mutation showed increased levels of unspliced but poly(A)+ RNA, indicating reduced cotranscriptional splicing efficiency. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript.
2017-10-22 | GSE94941 | GEO
Project description:Numerous links exist between co-transcriptional RNA processing and the transcribing RNAPII. In particular, pre-mRNA splicing was reported to be associated with slowed RNAPII elongation. Here, we identify a site of ubiquitination (K1246) in the catalytic subunit of RNAPII close to the DNA entry path. Ubiquitination was increased in the absence of the Bre5-Ubp3 ubiquitin protease complex. Bre5 binds RNA in vivo, with a preference for exon 2 regions of intron-containing pre-mRNAs and poly(A) proximal sites. Ubiquitinated RNAPII showed similar enrichment. The absence of Bre5 led to impaired splicing and defects in RNAPII elongation in vivo on a splicing reporter construct. Strains expressing RNAPII with a K1246R mutation showed reduced co-transcriptional splicing. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript.
| S-EPMC5673307 | biostudies-literature
Project description:Genome wide screens identified negative genetic interactions between several cofactors of the exosome nuclease complex and the Bre5-Ubp3 ubiquitin protease complex. RNA-binding was shown for Bre5 with enrichment for sites over exon 2 of spliced pre-mRNAs and close to poly(A) sites. An inducible splicing-reporter showed a requirement for Bre5 in efficient in vivo splicing and for normal RNAPII elongation, specifically on splicing-competent genes. A Bre5-Ubp3 sensitive site of RNAPII ubiquitination was mapped at Lys1246 at the entrance to the active site of the large subunit. Ubiquitinated RNAPII was depleted at the TSS but enriched at the 5’ end of exon 2 and upstream of poly(A) sites, similar to Bre5. Mutation of Lys1246 reduced RNAPII occupancy upstream of the poly(A) site, consistent with reduced pausing at a potential surveillance site, but increased RNAPII residence downstream of the poly(A) site. Strains expressing RNAPII with the Lys1246 mutation showed increased levels of unspliced but poly(A)+ RNA, indicating reduced cotranscriptional splicing efficiency. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript.
2017-10-22 | GSE94942 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2017-10-22 | GSE94944 | GEO
Project description:RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit [CRAC]
| PRJNA374885 | ENA
Project description:RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit [RNA-Seq]
| PRJNA374884 | ENA
Project description:Translation elongation rates are regulated to ensure proper conformation and biological function of proteins. Translation of either non-stop mRNA or transcripts coding for poly-basic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control system (RQC). During this process, the stalled ribosome is dissociated into subunits, and the polypeptide is ubiquitinated by the E3 ubiquitin ligase Listerin on the 60S large ribosomal subunit (LSU) leading to subsequent proteasomal degradation. However, it is largely unknown how stalled ribosomes are recognized and dissociated into subunits. Here we report that ubiquitination of the ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 is required for the production of the RQC substrate. RQC-trigger (RQT) factors, a RNA helicase-family protein Slh1/Rqt2, ubiquitin binding protein Cue3/Rqt3 and yKR023W/Rqt4, were also required for the primary steps of RQC, and associated with Hel2-ribosome complexes. Rqt2-4 factors were dispensable for the ubiquitination of uS10 by Hel2/Rqt1 and associated with ribosomes independent of the ubiquitination of uS10. However, the ubiquitin-binding activity of Rqt3 were crucial to trigger RQC. Cryo-electron microscopy (cryo-EM) analysis revealed that Hel2 bound ribosomes are in an rotated state containing hybrid state AP- and PE-tRNAs. Furthermore, ribosome profiling revealed that short footprints, hallmarks of hybrid state ribosomes18, were accumulated at tandem CGA rare codons at the beginning of the poly arginine stalling sequence and long footprints at subsequent codons, respectively. Short footprints at CGA codons were decreased in rqt1 mutant but drastically increased in uS10 mutants defective in the ubiquitination or rqt2 mutant. Collectively, our results demonstrate that Hel2 stabilizes ratcheted ribosomes leading to ubiquitination of uS10. Subsequently, Rqt2-4 factors target these hybrid state ribosomes specifically, allowing subsequent RQC reactions.
2017-07-31 | GSE79622 | GEO
Project description:Translation elongation stalling has the potential to produce toxic truncated protein fragments. Translation of either non-stop mRNA or transcripts coding for poly-basic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. During this process, the stalled ribosome is dissociated into subunits, and the polypeptide is ubiquitinated by the E3 ubiquitin ligase Listerin on the 60S large ribosomal subunit, leading to subsequent proteasomal degradation. However, it is largely unknown how the specific stalled ribosomes are recognized as aberrant to engage the RQC system. Here, we report that ubiquitination of the ribosomal protein uS10 of the 40S small ribosomal subunit, by the E3 ubiquitin ligase Hel2 (or RQC-trigger (Rqt) 1) initiates RQC. We identified a novel RQC-trigger (RQT) complex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlated with sensitivity to anisomycin, which stalls ribosome at the rotated form, suggesting that RQT factors rescue ribosomes stalled by this drug. Our un-biased survey by ribosome profiling revealed that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits. Our results provide mechanistic insight into the surveillance system for aberrant proteins induced by ribosome stalling and mediated by ribosome ubiquitination.
2017-07-31 | GSE90919 | GEO
Project description:The conserved yeast E3 ligase Bre1 and its partner E2 Rad6 monoubiquitinate histone H2B across gene bodies during the transcription cycle. While processive ubiquitination might in principle arise from Bre1/Rad6 traveling with RNA polymerase II, we provide a different explanation. Here we implicate liquid-liquid phase separation as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, whose intrinsically disordered region phase separates via dynamic, multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, accelerating H2B ubiquitination. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone modifying enzymes generate chromatin-associated reaction chambers with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, causing neurological disease when impaired.
2020-01-17 | GSE131639 | GEO
Project description:NguyenLK2011 - Ubiquitination dynamics in
Ring1B-Bmi1 system
This theoretical model investigates the
dynamics of Ring1B/Bmi1 ubiquitination to identify bistable
switch-like and oscillatory behaviour in the
system. Michaelis-Menten (MM) equations are used to formulate
the model. However, the authors show that the dynamics persist even
for Mass-Action kinetics. This SBML file is the MM version of the
model.
This model is described in the article:
Switches,
excitable responses and oscillations in the Ring1B/Bmi1
ubiquitination system.
Nguyen LK, Muñoz-García J,
Maccario H, Ciechanover A, Kolch W, Kholodenko BN.
PLoS Comput. Biol. 2011 Dec; 7(12):
e1002317
Abstract:
In an active, self-ubiquitinated state, the Ring1B ligase
monoubiquitinates histone H2A playing a critical role in
Polycomb-mediated gene silencing. Following ubiquitination by
external ligases, Ring1B is targeted for proteosomal
degradation. Using biochemical data and computational modeling,
we show that the Ring1B ligase can exhibit abrupt switches,
overshoot transitions and self-perpetuating oscillations
between its distinct ubiquitination and activity states. These
different Ring1B states display canonical or multiply branched,
atypical polyubiquitin chains and involve association with the
Polycomb-group protein Bmi1. Bistable switches and oscillations
may lead to all-or-none histone H2A monoubiquitination rates
and result in discrete periods of gene (in)activity. Switches,
overshoots and oscillations in Ring1B catalytic activity and
proteosomal degradation are controlled by the abundances of
Bmi1 and Ring1B, and the activities and abundances of external
ligases and deubiquitinases, such as E6-AP and USP7.
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2024-09-02 | BIOMD0000000622 | BioModels