Ubiquitination of stalled ribosome triggers ribosome-associated quality control

Yoshitaka Matsuo,Shintaro Iwasaki,Keiji Tanaka,Tsuyoshi Udagawa,Thomas Becker,Fumiya Sato,Yasushi Saeki,Toshifumi Inada,Nicholas T Ingolia,Christian Schmidt,Hikaru Tsuchiya,Ken Ikeuchi,Roland Beckmann

doi:10.1038/s41467-017-00188-1

Abstract

Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex 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 correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals 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.

Highlights

Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system
R(CGN)[12] reporter assay showed that full-length products were significantly increased in hel[2] deletion mutant, and more interestingly, arrest products were completely disappeared in the hel2Δltn1Δ double mutant (Fig. 1a), indicating that the ribosome could read through the R12 arrest sequence in the hel2Δ mutant owing to defective induction of RQC, so that it was not detected even in the absence of Ltn[1]; that is, as Hel[2] apparently sorted the stalled ribosome from normal translation to RQC pathway, here we referred this protein as “RQC-trigger factor 1” (Rqt1)
Recent study demonstrated that the ubiquitination of ribosome plays a crucial role in translation arrest by poly(A) sequence K(AAA)[24] in mammalian cells[33, 34]

Summary

Introduction

Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. The yeast Ltn[1] was identified by genetic screen as a factor that is involved in the repression of aberrant nonstop products with poly-lysine sequence produced by translation of a poly(A) tail of nonstop mRNA27. A search for genetic interactions with Ltn[1] and mRNA decay pathways uncovered Rqc[1] and Rqc[2] in yeast[2, 4, 19] These factors together comprise the 60S-associated RQC machinery that is required for the efficient degradation of stalled protein products. Genetic screen identifies two key factors, Asc[1] and Hel[2] that are required for RQC by poly-lysine or poly-arginine sequences in yeast. It is still unknown how the ubiquitinated ribosome is recognized and dissociated into the subunits for Ltn1-dependent ubiquitination of peptidyl-tRNA on 60S subunit

Methods

Results

Conclusion