Ubiquitination of stalled ribosome triggers ribosome-associated quality control

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.

Collided ribosomes form a unique structural interface to induce Hel2‐driven quality control pathways

“…We have previously described an interesting phenotype of sensitivity to the anisomycin translation elongation inhibitor in RQC-deficient cells (Matsuo et al, 2017). Intriguingly, Hel2 mutants that were unable to trigger remained sensitive to anisomycin, whereas RQC-competent Hel2 mutants (1-539, 61-539) were not susceptible to this drug (Appendix Fig S1).…”

“…The 5 0 ends of 3 0 NGD-IMs were determined with a fluorescence-labelled primer that hybridized to the region of the FLAG-encoding mRNA sequence (Fig 1A and E). Our previous study demonstrated that the ribosome is stalled mainly at positions R2(CGA) and R3(CGA) of the R(CGN) 12 sequence in P-and A-sites, respectively, and subjected to RQC (Matsuo et al, 2017), indicating that the X1 cleavage site is located in the P-site of the stalled ribosome. Our previous study demonstrated that the ribosome is stalled mainly at positions R2(CGA) and R3(CGA) of the R(CGN) 12 sequence in P-and A-sites, respectively, and subjected to RQC (Matsuo et al, 2017), indicating that the X1 cleavage site is located in the P-site of the stalled ribosome.…”

“…To investigate the relation between the two quality control pathways induced by ribosome stalling, NGD and RQC, we now investigated the role of Hel2 in the endoribonucleolytic cleavage of mRNA, a triggering step of the NGD quality control using several stalling reporter mRNAs ( Fig EV1A). Notably, this sequence contains codon pairs (CGA-CGA) which have been described to efficiently cause ribosomal stalling in yeast (Gamble et al, 2016;Matsuo et al, 2017). Notably, this sequence contains codon pairs (CGA-CGA) which have been described to efficiently cause ribosomal stalling in yeast (Gamble et al, 2016;Matsuo et al, 2017).…”

“…We generated a translation extract from ski2ΔuS10-3HA cells and programmed it with a mRNA containing the previously reported stalling dicodon CGA-CCG (Gamble et al, 2016;Matsuo et al, 2017) and a sequence coding for an N-terminal tag for affinity purification ( Fig 3A). We generated a translation extract from ski2ΔuS10-3HA cells and programmed it with a mRNA containing the previously reported stalling dicodon CGA-CCG (Gamble et al, 2016;Matsuo et al, 2017) and a sequence coding for an N-terminal tag for affinity purification ( Fig 3A).…”

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

“…We recently reported that ribosome profiling using a cocktail of elongation inhibitors can trap ribosomes in their different functional states, distinguished by the size of ribosome-protected footprints (RPFs). Building on an earlier study that showed an enrichment in ribosome density when the 17 inhibitory codon pairs are aligned (Gamble et al, 2016;Matsuo et al, 2017), we generated libraries using CHX and TIG to better distinguish the functional state of the paused ribosomes. Building on an earlier study that showed an enrichment in ribosome density when the 17 inhibitory codon pairs are aligned (Gamble et al, 2016;Matsuo et al, 2017), we generated libraries using CHX and TIG to better distinguish the functional state of the paused ribosomes.…”