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

Abstract

Ribosome stalling triggers quality control pathways targeting the mRNA (NGD: no‐go decay) and the nascent polypeptide (RQC: ribosome‐associated quality control). RQC requires Hel2‐dependent uS10 ubiquitination and the RQT complex in yeast. Here, we report that Hel2‐dependent uS10 ubiquitination and Slh1/Rqt2 are crucial for RQC and NGD induction within a di‐ribosome (disome) unit, which consists of the leading stalled ribosome and the following colliding ribosome. Hel2 preferentially ubiquitinated a disome over a monosome on a quality control inducing reporter mRNA in an in vitro translation reaction. Cryo‐EM analysis of the disome unit revealed a distinct structural arrangement suitable for recognition and modification by Hel2. The absence of the RQT complex or uS10 ubiquitination resulted in the elimination of NGD within the disome unit. Instead, we observed Hel2‐mediated cleavages upstream of the disome, governed by initial Not4‐mediated monoubiquitination of eS7 and followed by Hel2‐mediated K63‐linked polyubiquitination. We propose that Hel2‐mediated ribosome ubiquitination is required both for canonical NGD (NGDRQC+) and RQC coupled to the disome and that RQC‐uncoupled NGD outside the disome (NGDRQC−) can occur in a Not4‐dependent manner.

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

“…We processed the data using the 80S extension approach as described previously and segregated classes of ribosomal particles stalled in the POST and PRE states (Appendix Fig S9). These collided disomes, which were composed of two POST state ribosomes, are thus strikingly different from previously characterized disomes in both mammalian and yeast systems (Juszkiewicz et al, 2018;Ikeuchi et al, 2019). These collided disomes, which were composed of two POST state ribosomes, are thus strikingly different from previously characterized disomes in both mammalian and yeast systems (Juszkiewicz et al, 2018;Ikeuchi et al, 2019).…”

“…Interestingly, when studying ribosomal collisions as a consequence of poly(A)-mediated stalling, we found a large fraction of the disomes in a novel POST-POST state that was distinct from the previously characterized disome structures in both mammalian and yeast systems (Fig 6A and B; Juszkiewicz et al, 2018;Ikeuchi et al, 2019). Interestingly, when studying ribosomal collisions as a consequence of poly(A)-mediated stalling, we found a large fraction of the disomes in a novel POST-POST state that was distinct from the previously characterized disome structures in both mammalian and yeast systems (Fig 6A and B; Juszkiewicz et al, 2018;Ikeuchi et al, 2019).…”

“…In particular, recent work has suggested that ribosomal collisions with the leading stalled ribosome are a key event that triggers the quality control responses that include decay of the mRNA ("No Go Decay" or NGD) and the nascent peptide (ribosome-associated quality control or RQC; Simms et al, 2017;Juszkiewicz et al, 2018;Ikeuchi et al, 2019). Considerable attention has been paid to the molecular consequences of the translating ribosomes encountering such mRNA sequences (i.e., the downstream quality control events that are triggered).…”

Ubiquitin—a beacon for all during quality control on the ribosome

“…Here, Ikeuchi et al (2019) demonstrated that Hel2-mediated ubiquitination of collided ribosomes act as a master regulator for both processes of NGD and RQC. Here, Ikeuchi et al (2019) demonstrated that Hel2-mediated ubiquitination of collided ribosomes act as a master regulator for both processes of NGD and RQC.…”

“…How these two processes are triggered by the same molecular signal is not obvious. The new results from the Inada and Beckmann groups in this issue (Ikeuchi et al, 2019) now provide compelling evidence suggesting that ribosomal protein ubiquitination by Hel2 is required for both processes. It is thus likely that ubiquitination of ribosomal proteins is the first reaction to proceed after stalling has occurred, and is utilized by all downstream events to manifest the different facets of NGD and RQC.…”