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Unresolved stalled ribosome complexes restrict cycle progression after genotoxic stress

ABSTRACT: Translation of damaged mRNA can lead to ribosome stalling, thereby producing incomplete proteins toxic to the cell. The mechanism of ribosome-associated quality control (RQC) disassembles stalled ribosomes through the actions of the ASC-1 complex (ASCC). Here, we show that some reagents that chemically damage RNA, such as ultraviolet light (UV), cause ribosome stalling, which leads to accumulation of the ASC-1 complex (ASCC) on stalled ribosomes and stable interaction of the ASCC3 helicase with RNA. In contrast, the ASCC was not similarly affected by emetine or anisomycin-induced ribosome stalling. Our work identified two different types of stalled ribosome. Ribosomes arrested by emetine or anisomycin are transient as they are resolved by the ASCC. Whereas the ASCC fails to split some stalled ribosomes, such as those induced by UV, resulting in long-lived stalled ribosome complexes. We show that ribosome stalling activates the G1/S and G2/M cell cycle checkpoints with long-lived stalled ribosomes causing prolonged checkpoint activation. Thus, the cell adjusts this adaptive survival response to match the nature of the stalled ribosome.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Rebecca Jukes

LAB HEAD: Prof. Anne Willis

PROVIDER: PXD023878 | Pride | 2022-02-07

REPOSITORIES: Pride

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			Action	DRS
	MS_-UV_Harry_A_260914001.mzid.gz	Mzid
	MS_-UV_Harry_A_260914001.mzid_MS_-UV_Harry_A_260914001.MGF	Mzid
	MS_-UV_Harry_A_260914001.raw.zip	Raw
	MS_-UV_Harry_A_260914002.mzid.gz	Mzid
	MS_-UV_Harry_A_260914002.mzid_MS_-UV_Harry_A_260914002.MGF	Mzid

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Unresolved stalled ribosome complexes restrict cycle progression after genotoxic stress

Project description:Translation of damaged mRNA can lead to ribosome stalling, thereby producing incomplete proteins toxic to the cell. The mechanism of ribosome-associated quality control (RQC) disassembles stalled ribosomes through the actions of the ASC-1 complex (ASCC). Here, we show that some reagents that chemically damage RNA, such as ultraviolet light (UV), cause ribosome stalling, which leads to accumulation of the ASC-1 complex (ASCC) on stalled ribosomes and stable interaction of the ASCC3 helicase with RNA. In contrast, the ASCC was not similarly affected by emetine or anisomycin-induced ribosome stalling. Our work identified two different types of stalled ribosome. Ribosomes arrested by emetine or anisomycin are transient as they are resolved by the ASCC. Whereas the ASCC fails to split some stalled ribosomes, such as those induced by UV, resulting in long-lived stalled ribosome complexes. We show that ribosome stalling activates the G1/S and G2/M cell cycle checkpoints with long-lived stalled ribosomes causing prolonged checkpoint activation. Thus, the cell adjusts this adaptive survival response to match the nature of the stalled ribosome.

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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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