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RqcH and RqcP catalyze processive poly-alanine synthesis in a reconstituted ribosome-associated quality control system.

ABSTRACT: In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP. However, the mechanistic details of this process remain unclear. Here we demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Additionally, we have reconstituted polyalanine-tailing in vitro and can demonstrate that RqcH and RqcP are necessary and sufficient for processivity in a minimal system. Moreover, the in vitro reconstituted system recapitulates our in vivo findings by reproducing the importance of conserved residues of RqcH and RqcP for functionality. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway.

SUBMITTER: Takada H

PROVIDER: S-EPMC8373112 | biostudies-literature |

REPOSITORIES: biostudies-literature

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RqcH and RqcP catalyze processive poly-alanine synthesis

Project description:In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. We demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway. the synthesis of C-terminal poly-Alanine ‘tail’. While it is well-established that RqcP and RqcH are essential for poly-Alanine-tailing in living cells, here, by reconstituting the RQC elongation biochemically we provide a direct proof that the two factors are sufficient. We demonstrate that while RqcH is responsible for tRNAAla selection during RQC elongation, RqcP lacks the tRNA specificity. Through mutational mapping we show that stable interaction of RqcP with 23S rRNA – but not with tRNA – is crucial for RQC, supporting the role of the E-site bound RqcP as a pawl of RQC elongation ratchet. We show that ribosomal protein uL11 is crucial for RqcH – but nor RqcP – recruitment to 50S, and B. subtilis lacking uL11 is RQC-deficient. Finally, we show that proteins encoded by non-stop mRNAs are not universally efficiently targeted by RQC, and that simultaneous disruption of RQC and trans-translation results in bacterial filamentation

2021-08-19 | GSE174254 | GEO

RqcH and RqcP catalyze processive poly-alanine synthesis

Project description:RqcH and RqcP catalyze processive poly-alanine synthesis

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Alanine Tails Signal Proteolysis in Bacterial Ribosome-Associated Quality Control.

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Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection

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Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination.

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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 (RPS20) and eS10 (RPS10), initiating RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection 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 that either lack ZNF598 or express a ubiquitylation-deficient version of uS10. Using SILAC-based proteomics and concurrent RNA-seq analysis, we determine that translation, but 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.

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Eukaryotic ribosome quality control system: a potential therapeutic target for human diseases.

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Ubiquitination of stalled ribosome triggers ribosome-associated quality control.

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The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation.

Project description:BackgroundCells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. 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 decay of the truncated nascent polypeptide via ribosome-associated quality control.ResultsHere, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during ribosome-associated quality control. We show that MKRN1 directly binds to the cytoplasmic poly(A)-binding protein (PABPC1) and associates with polysomes. MKRN1 is positioned upstream of poly(A) tails in mRNAs in a PABPC1-dependent manner. Ubiquitin remnant profiling and in vitro ubiquitylation assays uncover PABPC1 and ribosomal protein RPS10 as direct ubiquitylation substrates of MKRN1.ConclusionsWe propose that MKRN1 mediates the recognition of poly(A) tails to prevent the production of erroneous proteins from prematurely polyadenylated transcripts, thereby maintaining proteome integrity.

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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

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