Translation of zinc finger domains induces ribosome collision and Znf598-dependent mRNA decay in zebrafish [Ribo-seq]
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ABSTRACT:
ORGANISM(S): Danio rerio
PROVIDER: GSE236144 | GEO | 2024/10/03
REPOSITORIES: GEO
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Project description:Quality control of translation is crucial for maintaining cellular and organismal homeostasis. Obstacles in translation elongation induce ribosome collision, which is monitored by multiple sensor mechanisms in eukaryotes. The E3 ubiquitin ligase Znf598 recognizes collided ribosomes, triggering ribosome-associated quality control (RQC) to rescue stalled ribosomes and no-go decay (NGD) to degrade stall-prone mRNAs. However, the impact of RQC and NGD on maintaining the translational homeostasis of endogenous mRNAs has remained unclear. In this study, we investigated the endogenous substrate mRNAs of NGD during the maternal-to-zygotic transition (MZT) of zebrafish development. RNA-Seq analysis of zebrafish znf598 mutant embryos revealed that Znf598 downregulates mRNAs encoding the C2H2-type zinc finger domain (C2H2-ZF) during the MZT. Reporter assays and disome profiling indicated that ribosomes stall and collide while translating tandem C2H2-ZFs, leading to mRNA degradation by Znf598. Our results suggest that NGD maintains the quality of the translatome by mitigating the risk of ribosome collision at the abundantly present C2H2-ZF sequences in the vertebrate genome.
2024-10-03 | GSE236143 | GEO
Project description: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.
2021-04-14 | GSE165732 | GEO
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.
2024-10-10 | GSE270401 | GEO
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
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: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
Project description:Maintaining the integrity of nucleic acids is an essential feature of all organisms. Unwanted modification of DNA, if left unrepaired, is deleterious to cellular homeostasis and could have far-reaching consequences that include genomic instability and accumulation of mutations. Similarly, accumulation of damaged RNA has been correlated with various neurodegenerative diseases. Given their inherent reactivity, nucleic acids are susceptible to damage from both endogenous and exogenous reactive oxygen species (ROS) as well as alkylation agents. We have recently begun to address how some of these modifications on mRNA impact the function of the ribosome and in particular the decoding process. We find that most modifications severely change the speed and accuracy of translation. These observations revealed that oxidation of RNA is most likely to stall the ribosome and necessitates the presence of quality-control processes to handle damaged mRNA. To this end, we have uncovered a connection between the process of no-go decay (NGD), which degrades mRNAs that stall translation, and chemical insults. In the absence of key NGD factors in yeast, the levels of damaged mRNA significantly increase and cells are rendered sensitive to oxidizing and alkylation agents. Furthermore, these agents activate ribosome quality control (RQC) of nascent peptides. Deletion of the E3 ligase responsible for the ubiquitination of the nascent peptides resulted in the accumulation of protein aggregates in the presence of oxidizing and alkylating agents. These observations suggest that chemical damage stalls translation, activating NGD and RQC. Interestingly, the addition of nucleic-acids-damaging agents was also found to result in K63-linked ubiquitination of ribosomal proteins. This signaling-mode of ubiquitination precedes DNA-damage marks, indicating that cells respond to RNA damage due to stalled ribosomes before DNA damage. Collectively our data highlights the burden of chemically-damaged mRNA on cellular homeostasis and suggests that organisms evolved ribosome-based mRNA-surveillance processes to rapidly degrade it.
2019-11-07 | PXD013834 | Pride
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
Project description:The control of mRNA stability plays a central role in regulating gene expression patterns. While much is known about the roles of 5´ and 3´ untranslated regions in the mRNA stability control, the impact of protein-coding sequences on mRNA stability had been obscure. Recently, several groups reported that codon composition in the ORF affects mRNA deadenylation and degradation rates in a translation-dependent manner. Hence, codons define not only the amino acid sequences to be synthesized but also the stability of mRNAs. However, how 61 codons differently affect mRNA stability remains unclear. Besides, aberrant stalling of the ribosome induces ribosome quality control (RQC) and No-go decay. The relationship between the two co-translational mRNA decay pathways is not systematically analyzed. To precisely characterize the effects of 61 codons on mRNA stability, we developed a simplified reporter system that allows detection of the effect of every single codon on mRNA stability in zebrafish embryos. Using this system, we show that the effect of codons on mRNA stability is partially but significantly correlated with the translation elongation rate and tRNA abundance. Interestingly, the codon effect is still maintained in zebrafish embryos lacking Znf598, an essential mediator of RQC and NGD. Znf598-dependent NGD targets a particular type of ribosome stalling but has limited impact on endogenous mRNA stability. Our study thus defines two related co-translational mRNA decay pathways during animal development.
2020-05-05 | GSE133392 | GEO
Project description:Key protein adapters couple translation to mRNA decay on specific classes of problematic mRNAs in eukaryotes. Slow decoding on nonoptimal codons leads to codon-optimality-mediated decay (COMD) and prolonged arrest at stall sites leads to no-go decay (NGD). The identities of the decay factors underlying these processes and the mechanisms by which they respond to translational distress remain open areas of investigation. We use carefully-designed reporter mRNAs to perform genetic screens and functional assays in S. cerevisiae. We characterize the roles of Hel2 and Syh1 in coordinating translational repression and mRNA decay on NGD reporter mRNAs, finding that Syh1 acts as the primary link to mRNA decay in NGD. Importantly we, observe that these NGD factors are not involved in the degradation of mRNAs enriched in nonoptimal codons. Further, we establish that key factors previously implicated in COMD, Not5 and Dhh1 , contribute modestly to the degradation of an NGD-targeted mRNA. Finally, we use ribosome profiling to reveal distinct ribosomal states associated with each reporter mRNA that readily rationalize the contributions of NGD and COMD factors to degradation of these reporters. Taken together, these results provide new mechanistic insight into the role of Syh1 in NGD and define the molecular triggers that determine how distinct pathways target mRNAs for degradation in yeast.
2022-08-08 | PXD030076 | Pride