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Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control.

ABSTRACT: Ribosome-associated quality control (RQC) pathways monitor and respond to ribosome stalling. Using in vivo UV-crosslinking and mass spectrometry, we identified a C-terminal region in Hel2/Rqt1 as an RNA binding domain. Complementary crosslinking and sequencing data for Hel2 revealed binding to 18S rRNA and translated mRNAs. Hel2 preferentially bound mRNAs upstream and downstream of the stop codon. C-terminal truncation of Hel2 abolished the major 18S crosslink and polysome association, and altered mRNA binding. HEL2 deletion caused loss of RQC and, we report here, no-go decay (NGD), with comparable effects for Hel2 truncation including the RNA-binding site. Asc1 acts upstream of Hel2 in RQC and asc1? impaired Hel2 binding to 18S and mRNA. In conclusion: Hel2 is recruited or stabilized on translating 40S ribosomal subunits by interactions with 18S rRNA and Asc1. This 18S interaction is required for Hel2 function in RQC and NGD. Hel2 probably interacts with mRNA during translation termination.

SUBMITTER: Winz ML

PROVIDER: S-EPMC6362110 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control.

Winz Marie-Luise ML Peil Lauri L Turowski Tomasz W TW Rappsilber Juri J Tollervey David D

Nature communications 20190204 1

Ribosome-associated quality control (RQC) pathways monitor and respond to ribosome stalling. Using in vivo UV-crosslinking and mass spectrometry, we identified a C-terminal region in Hel2/Rqt1 as an RNA binding domain. Complementary crosslinking and sequencing data for Hel2 revealed binding to 18S rRNA and translated mRNAs. Hel2 preferentially bound mRNAs upstream and downstream of the stop codon. C-terminal truncation of Hel2 abolished the major 18S crosslink and polysome association, and alter ...[more]

PMID: 30718516

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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.

Dataset Information

Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control.

Publications

Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control.

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