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Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection

ABSTRACT: Viral infection both activates stress signaling pathways and redistributes ribosomes away from host mRNAs to translate viral mRNAs. The intricacies of this ribosome shuffle from host to viral mRNAs are poorly understood Here, we uncover a role for the ribosome associated quality control (RQC) factor, ZNF598, during vaccinia virus mRNA translation. ZNF598 acts on collided ribosomes to ubiquitylate 40S subunit proteins uS10 and eS10 initiating RQC-dependent nascent chain degradation and ribosome recycling We show that vaccinia infection in human cells enhances uS10 ubiquitylation indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells which either lack ZNF598 or express a ubiquitylation deficient version of uS10 Using SILAC-based proteomics and concurrent RNAseq analysis, we determine that translation and not transcription of vaccinia virus mRNAs is compromised in cells with deficient RQC activity. Additionally, vaccinia virus infection reduces cellular RQC activity, suggesting that co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE165732 | GEO | 2021/04/14

REPOSITORIES: GEO

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Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection

Project description:Ribosomes are highly abundant cellular machines that perform the essential task of translating the genetic code into proteins. Cellular translation activity is finely tuned and proteostasis insults, such as those incurred upon viral infection, activate stress signaling pathways that result in translation reprogramming. Viral infection selectively shuts down host mRNA while redistributing ribosomes for selective translation of viral mRNAs. The intricacies of this selective ribosome shuffle from host to viral mRNAs are poorly understood. Here, we uncover a role for the ribosome associated quality control (RQC) factor ZNF598, a sensor for collided ribosomes, as a critical factor for vaccinia virus mRNA translation. Collided ribosomes are sensed by ZNF598, which ubiquitylates 40S subunit proteins uS10 and eS10 and thereby initiates RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection in human cells enhances uS10 ubiquitylation indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells which either lack ZNF598 or contain a ubiquitylation deficient version of uS10. Using SILAC-based proteomics and concurrent RNAseq analysis, we determine that host translation of vaccinia virus mRNAs is compromised in cells that lack RQC activity as compared to control cells whereas there was little evidence of differences in host or viral transcription. Additionally, vaccinia virus infection resulted in a loss of cellular RQC activity, suggesting that ribosomes engaged in viral protein production recruit ZNF598 away from its function in host translation. Thus, co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.

2021-01-05 | MSV000086660 | MassIVE

Translation of zinc finger domains induces ribosome collision and Znf598-dependent mRNA decay in zebrafish [Ribo-seq]

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Translation of zinc finger domains induces ribosome collision and Znf598-dependent mRNA decay in zebrafish [RNA-seq]

2024-10-03 | GSE236143 | GEO

The RNA-binding ubiquitin ligase MKRN1 acts as a sensor for ribosome-associated quality control of poly(A)-translation

Project description:Human cells have evolved multiple quality control mechanisms to ensure protein homeostasis by detecting aberrant mRNAs and protein products which are condemned for rapid decay. Translating ribosomes that run into poly(A) tails 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) acts as a novel sensor of poly(A) sequences to initiate ribosome stalling in 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 via interaction with the cytoplasmic poly(A)-binding protein (PABP). Ubiquitin remnant profiling uncovers PABP and ribosomal protein RPS10 as well as multiple translational regulators as main targets of MRKN1-mediated ubiquitylation. The modified lysine in RPS10 is distinct from the main target sites of ZNF598, the second E3 ubiquitin ligase involved in initial ribosome stalling, indicating that multiple modifications need to converge to prevent promiscuous ubiquitylation of lysine-translating ribosomes. We propose that MKRN1 serves as a first line of poly(A) recognition at the RNA level to prevent erroneous protein products and maintain proteome integrity.

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Disome and trisome profiling reveal targets of ribosome quality control

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2020-05-26 | GSE139036 | GEO

Ribosome collisions trigger cis-acting feedback inhibition of translation initiation

Project description:Translation of aberrant mRNAs can cause ribosomes to stall, leading to collisions with trailing ribosomes. Collided ribosomes are specifically recognized by ZNF598 to initiate protein and mRNA quality control pathways. Here we found using quantitative proteomics of collided ribosomes that EDF1 is a ZNF598-independent sensor of ribosome collisions. EDF1 recruits and stabilizes GIGYF2 at collisions to inhibit translation initiation in cis via 4EHP. The GIGYF2 axis acts independently of the ZNF598 axis, but each pathway’s output is more pronounced without the other. We propose that the widely conserved and highly abundant EDF1 monitors the transcriptome for excessive ribosome density, then triggers a GIGYF2-mediated response to locally and temporarily reduce ribosome loading. Only when collisions persist is translation abandoned to initiate ZNF598-dependent quality control. This tiered response to ribosome collisions would allow cells to dynamically tune translation rates while ensuring fidelity of the resulting protein products.

2020-07-14 | PXD020239 | Pride

Cue2 and Slh1 define parallel pathways to rescue stalled ribosomes

Project description:Ribosome stalling at problematic sequences in mRNAs leads to collisions that trigger a collection of quality control events including ribosome rescue, targeting the nascent polypeptide for decay (Ribosome-mediated Quality Control or RQC), and targeting of the mRNA for decay (No Go Decay or NGD). Using a reverse genetic screen in yeast, we identify Cue2 as the endonuclease that is recruited to stalled ribosomes to promote NGD. Following Cue2-mediated cleavage, ribosomes upstream of the cleavage site translate to the end of the truncated mRNA and are rescued by the Dom34:Hbs1 complex. We also show that the putative helicase Slh1 (part of the RQC Trigger or RQT complex) removes collided ribosomes behind the lead stalled ribosome and thereby reduces endonucleolytic cleavage by Cue2. The synergistic activities of Cue2 and Slh1 define two parallel pathways that allow cells to recognize and respond to ribosomes trapped on problematic mRNAs.

2019-06-25 | GSE129128 | GEO

Ribosome collisions trigger subunit splitting in E. coli

Project description:Although many clinically important antibiotics inhibit bacterial ribosomes, the mechanisms by which bacterial cells rescue ribosomes stalled by antibiotics remain poorly understood. Ribosome stalling leads to collisions that recruit ribosome quality control (RQC) factors that recycle the ribosome subunits and target nascent proteins for degradation. Surprisingly, loss of known RQC factors in E. coli does not lead to significant antibiotic sensitivity, even though antibiotics stall ribosomes and induce collisions, suggesting the existence of additional, uncharacterized RQC mechanisms. Here we report a novel mechanism for ribosome quality control (RQC) in bacteria in which the DExH-box ATPase HrpA splits stalled ribosomes into subunits. HrpA selectively acts on collided ribosomes and its activity is dependent on ATP hydrolysis. The cryo-EM structure of HrpA bound to collided ribosomes reveals insight into its selectivity and mechanism: the C-terminal domain of HrpA senses the collision and its helicase domain bind mRNA downstream of the ribosomes, where it likely exerts a pulling force that destabilizes the stalled ribosome. These studies highlight the importance of ribosome splitting as a highly conserved RQC mechanism across all three domains of life and identify an important pathway in proteobacteria that allows proteobacteria to tolerate ribosome-targeting antibiotics.

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Ubiquitination of Stalled Ribosome Triggers Ribosome-associated Quality Control [SET2]

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.

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