Detection and Degradation of Stalled Nascent Chains via Ribosome-Associated Quality Control.
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ABSTRACT:
SUBMITTER: Sitron CS
PROVIDER: S-EPMC8258965 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ribosome stalling during translation can potentially be harmful, and is surveyed by a conserved quality control pathway that targets the associated mRNA and nascent polypeptide chain (NC). In this pathway, the ribosome-associated quality control (RQC) complex promotes the ubiquitylation and degradation of NCs remaining stalled in the 60S subunit. NC stalling is recognized by the Rqc2/Tae2 RQC subunit, which also stabilizes binding of the E3 ligase, Listerin/Ltn1. Additionally, Rqc2 modifies stalled NCs with a carboxy-terminal, Ala- and Thr-containing extension-the 'CAT tail'. However, the function of CAT tails and fate of CAT tail-modified ('CATylated') NCs has remained unknown. Here we show that CATylation mediates formation of detergent-insoluble NC aggregates. CATylation and aggregation of NCs could be observed either by inactivating Ltn1 or by analyzing NCs with limited ubiquitylation potential, suggesting that inefficient targeting by Ltn1 favors the Rqc2-mediated reaction. These findings uncover a translational stalling-dependent protein aggregation mechanism, and provide evidence that proteins can become specifically marked for aggregation.
| S-EPMC4805532 | biostudies-literature
Project description:Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex 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 correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals 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.Several protein quality control mechanisms are in place to trigger the rapid degradation of aberrant polypeptides and mRNAs. Here the authors describe a mechanism of ribosome-mediated quality control that involves the ubiquitination of ribosomal proteins by the E3 ubiquitin ligase Hel2/RQT1.
| S-EPMC5534433 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2017-07-31 | GSE90920 | 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: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:Ubiquitination of Stalled Ribosome Triggers Ribosome-associated Quality Control
| PRJNA356322 | ENA
Project description:The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.
| S-EPMC3534965 | biostudies-literature
Project description:Folding of proteins usually involves intermediates, of which an important type is the molten globule (MG). MGs are ensembles of interconverting conformers that contain (non-)native secondary structure and lack the tightly packed tertiary structure of natively folded globular proteins. Whereas MGs of various purified proteins have been probed to date, no data are available on their presence and/or effect during protein synthesis. To study whether MGs arise during translation, we use ribosome-nascent chain (RNC) complexes of the electron transfer protein flavodoxin. Full-length isolated flavodoxin, which contains a non-covalently bound flavin mononucleotide (FMN) as cofactor, acquires its native ?/? parallel topology via a folding mechanism that contains an off-pathway intermediate with molten globular characteristics. Extensive population of this MG state occurs at physiological ionic strength for apoflavodoxin variant F44Y, in which a phenylalanine at position 44 is changed to a tyrosine. Here, we show for the first time that ascertaining the binding rate of FMN as a function of ionic strength can be used as a tool to determine the presence of the off-pathway MG on the ribosome. Application of this methodology to F44Y apoflavodoxin RNCs shows that at physiological ionic strength the ribosome influences formation of the off-pathway MG and forces the nascent chain toward the native state.
| S-EPMC5207065 | biostudies-literature
Project description:Cdc37 is a molecular chaperone that functions with Hsp90 to promote protein kinase folding. Analysis of 65 Saccharomyces cerevisiae protein kinases ( approximately 50% of the kinome) in a cdc37 mutant strain showed that 51 had decreased abundance compared with levels in the wild-type strain. Several lipid kinases also accumulated in reduced amounts in the cdc37 mutant strain. Results from our pulse-labeling studies showed that Cdc37 protects nascent kinase chains from rapid degradation shortly after synthesis. This degradation phenotype was suppressed when cdc37 mutant cells were grown at reduced temperatures, although this did not lead to a full restoration of kinase activity. We propose that Cdc37 functions at distinct steps in kinase biogenesis that involves protecting nascent chains from rapid degradation followed by its folding function in association with Hsp90. Our studies demonstrate that Cdc37 has a general role in kinome biogenesis.
| S-EPMC1857360 | biostudies-literature
Project description:Ribosomal surveillance pathways scan for ribosomes that are transiently paused or terminally stalled owing to structural elements in mRNAs or nascent chain sequences1, 2. Some stalls in budding yeast are sensed by the GTPase Hbs1, which loads Dom34, a catalytically inactive member of the archaeo-eukaryotic release factor 1 superfamily. Hbs1-Dom34 and the ATPase Rli1 dissociate stalled ribosomes into 40S and 60S subunits. However, the 60S subunits retain the peptidyl-tRNA nascent chains, which recruit the ribosome quality control complex that consists of Rqc1-Rqc2-Ltn1-Cdc48-Ufd1-Npl4. Nascent chains ubiquitylated by the E3 ubiquitin ligase Ltn1 are extracted from the 60S subunit by the ATPase Cdc48-Ufd1-Npl4 and presented to the 26S proteasome for degradation3-9. Failure to degrade the nascent chains leads to protein aggregation and proteotoxic stress in yeast and neurodegeneration in mice10-14. Despite intensive investigations on the ribosome quality control pathway, it is not known how the tRNA is hydrolysed from the ubiquitylated nascent chain before its degradation. Here we show that the Cdc48 adaptor Vms1 is a peptidyl-tRNA hydrolase. Similar to classical eukaryotic release factor 1, Vms1 activity is dependent on a conserved catalytic glutamine. Evolutionary analysis indicates that yeast Vms1 is the founding member of a clade of eukaryotic release factor 1 homologues that we designate the Vms1-like release factor 1 clade.
| S-EPMC6226276 | biostudies-literature