Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control [I]
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE114415 | GEO | 2019/01/18
REPOSITORIES: GEO
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Project description:Ribosome-associated quality control (RQC) pathways monitor and respond to stalling of translating ribosomes. Using a newly developed technique based on in vivo UV crosslinking and mass spectrometry, we identify a C-terminal region in Hel2/Rqt1 as an RNA binding domain, with amino acids L501/K502 directly interacting with RNA. In vivo crosslinking of Hel2 revealed binding to 18S rRNA and translating mRNAs. Consistent with the 18S binding site located between mRNA entrance and exit channels, Hel2 preferentially bound mRNA both upstream and downstream of the termination codon. A C-terminal truncation that deleted L501/K502, abolished crosslinking to 18S rRNA, altered mRNA binding patterns, and reduced Hel2 function comparable to hel2∆. Asc1, also participates in RQC and ASC1 deletion impaired Hel2 18S and mRNA binding. We conclude that Hel2 is recruited or stabilized on translating 40S ribosomal subunits by interactions with 18S rRNA and Asc1. Ribosome-bound Hel2 interacts with mRNA, predominately during translation termination.
2019-01-18 | GSE114420 | GEO
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:Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified the SDD1 mRNA from S. cerevisiae as an endogenous RQC substrate and reveal its mRNA and nascent peptide dependent stalling mechanism by mutational and cryo-EM analyses. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent poly-ubiquitination of collided di- and preferentially tri-ribosomes. Distinct trisome architecture was visualized by cryo-EM and provides the structural basis for more efficient recognition by Hel2 over disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled poly-ubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.
2020-01-27 | GSE131214 | GEO
Project description:Box C/D snoRNAs snR4 and snR45, whose role was unknown, guide 18S rRNA acetylations by Kre33 in yeast. UV-crosslinking (CRAC) was used to determine binding sites of Kre33 on RNA.
2017-07-25 | GSE87480 | GEO
Project description:Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by targeting of the faulty nascent peptides for degradation. However, endogenous RQC inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified the SDD1 mRNA from S. cerevisiae as an endogenous RQC substrate and reveal its mRNA and nascent peptide dependent stalling mechanism by mutational and cryo-EM analyses. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent poly-ubiquitination of colliding di- and preferentially tri-ribosomes. Their distinct architecture is visualized by cryo-EM and provides the structural basis for more efficient recognition by Hel2 over disomes. Subsequently, the Slh1/Rqt2 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled poly-ubiquitinated ribosome in an ATP dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in dealing with endogenous substrates to maintain cellular protein homeostasis.
2020-02-24 | PXD017196 | Pride
Project description:Tumor recurrence is main pattern of treatment failure for early-stage hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying disease recurrence remain poorly understood. Here, we showed that 18S rRNA N6-methyladenosine (m6A1832) modification and its methyltransferase complex METTL5/TRMT112 were upregulated in HCC and correlated with poor prognosis. Loss-of-function and gain-of-function assays demonstrated that METTL5/TRMT112 mediated 18S rRNA m6A1832 modification promotes HCC tumorigenesis in vitro and in vivo. Mechanistically, 18S rRNA m6A1832 modification selectively regulated the translation of mRNAs with long 5’UTR and short 3’UTR through affecting the assembly of 80S subunit at translation initiation and its dissociation at translation termination which was executed by weakening the interaction of ABCE1 with eRF1 and eRF3. Moreover, METTL5-mediated 18S rRNA m6A1832 modification regulated β-oxidation of long-chain fatty acid through ACSL4 to promote HCC progression. Our work uncovered a novel layer of mRNA translation regulation mechanism at ribosome 80S subunit assembly and dissociation step mediated by 18S rRNA m6A1832 modification and revealed a new crosslink between RNA epigenetic modification and fatty acid metabolism in HCC.
2021-06-01 | GSE175827 | GEO
Project description:Human cells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and protein products. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and rapid decay of the truncated nascent polypeptide via the ribosome-associated quality control (RQC). Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at continuous A-tracts during RQC. Using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we show that MKRN1 is positioned upstream of A-rich stretches and poly(A) tails in mRNAs through an interaction with the cytoplasmic poly(A)-binding protein (PABP). Ubiquitin remnant profiling uncovered PABP and ribosomal protein RPS10 as well as additional translational regulators as main substrates of MRKN1. We propose that MKRN1 serves as a first line of poly(A) recognition at the mRNA level to prevent production of erroneous proteins, thus maintaining proteome integrity.
2019-08-19 | GSE122869 | GEO
Project description:Purpose: Pre-ribosomal RNA is cleaved at defined sites, but many endonucleases involved in 18S rRNA release are not known. We apply an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between a yeast candidate pre-rRNA endonuclease (Utp24) and its targets in a living cell. Methods: We apply CRAC to an HTP-tagged Utp24 protein (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A). At least two independent experiments were performed and analyzed separately. Results: We found that yeast Utp24 UV-crosslinked in vivo to the U3 snoRNA and all (pre-)rRNA elements that form the central pseudoknot in the 18S rRNA. The pseudoknot is an evolutionarily highly conserved structure that is required to ensure pre-rRNA processing at three cleavage sites (A0, A1 and A2) and still present in the mature rRNA. According to our crosslinking data, the endonuclease Utp24 is placed in close proximity to site A1 at the 5'-end of the 18S rRNA. Conclusion: Our study strongly supports the hypothesis that Utp24 cleaves pre-rRNA at sites A1 and A2. Examination of targets for pre-rRNA endonucleases in yeast cells.
2016-04-01 | E-GEOD-75991 | biostudies-arrayexpress
Project description:The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome or higher order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3’UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not trigger degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2alpha, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs and such events may be important for cellular homeostasis.
2020-05-26 | GSE139036 | 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