Identification of a novel trigger complex that facilitates ribosome-associated quality control in mammalian cells

Abstract

Ribosome stalling triggers the ribosome-associated quality control (RQC) pathway, which targets collided ribosomes and leads to subunit dissociation, followed by proteasomal degradation of the nascent peptide. In yeast, RQC is triggered by Hel2-dependent ubiquitination of uS10, followed by subunit dissociation mediated by the RQC-trigger (RQT) complex. In mammals, ZNF598-dependent ubiquitination of collided ribosomes is required for RQC, and activating signal cointegrator 3 (ASCC3), a component of the ASCC complex, facilitates RQC. However, the roles of other components and associated factors of the ASCC complex remain unknown. Here, we demonstrate that the human RQCtrigger (hRQT) complex, an ortholog of the yeast RQT complex, plays crucial roles in RQC. The hRQT complex is composed of ASCC3, ASCC2, and TRIP4, which are orthologs of the RNA helicase Slh1(Rqt2), ubiquitin-binding protein Cue3(Rqt3), and zinc-finger type protein yKR023W(Rqt4), respectively. The ATPase activity of ASCC3 and the ubiquitin-binding activity of ASCC2 are crucial for triggering RQC. Given the proposed function of the RQT complex in yeast, we propose that the hRQT complex recognizes the ubiquitinated stalled ribosome and induces subunit dissociation to facilitate RQC. Cells have evolved various quality control mechanisms to guarantee accurate gene expression 1-4. Ribosome stalling induces quality control mechanisms for mRNA, referred to as No-go decay (NGD) 5-7 , as well as for protein, referred to as ribosome-associated quality control (RQC) 8-11. RQC is conserved throughout species and consists primarily of four steps: (i) recognition of abnormal ribosome stalling; (ii) ubiquitination of specific residue(s) on the stalled ribosome; (iii) dissociation of ribosome into 40S and 60S subunits; and (iv) degradation of the nascent polypeptide on the 60S subunit 2,4. In the first step of RQC, the stalling of a ribosome at a specific sequence results in the formation of a di-ribosome (disome), which consists of the leading stalled ribosome and the following collided ribosome 5,6,10. Cryo-EM structural analysis has shown that the leading stalled ribosome is in the POST-state, with an empty A-site, whereas the colliding ribosome is in a rotated state with hybrid tRNAs 6,8,10. In the second step, the RING-type E3 ubiquitin ligase Hel2 recognizes the ribosome collision and ubiquitinates ribosomal protein uS10 (in yeast, at residue(s) K6/8) 8. In the third step, ubiquitinated ribosomes are dissociated into 40S subunits and 60S ribosome-nascent chain complexes (60S-RNCs), leading to subsequent RQC reactions. We recently proposed a model in which these ubiquitinated ribosomes are targeted by the RQT complex 8. The RQT complex is composed of three proteins: RNA helicase Slh1(Rqt2), ubiquitin-binding protein Cue3(Rqt3), and zinc-finger domain-containing protein yKR023W(Rqt4). The ubiquitin-binding activity of Cue3 and the ATPase activity of Slh1 are crucial for triggering RQC 8. After subunit dissociation, Rqc2 binds to tRNA at the subunit interface of 6...

The ASC-1 Complex Disassembles Collided Ribosomes

“…No effect on the RFP:GFP ratio was seen for a (K) 0 control reporter lacking the stalling sequence ( Figure 1 B, middle graph) and the effect was almost completely lost in cells knocked out for ZNF598 ( Figure 1 B, right graph). Thus, like ZNF598 ( Garzia et al., 2017 , Juszkiewicz and Hegde, 2017 , Sundaramoorthy et al., 2017 ), ASCC3 is needed to terminally abort translation at a poly(A)-mediated stall similar to other stalls ( Hashimoto et al., 2020 ).

Figure 1 ASCC Is Required for Aborting Translation at a Ribosome Stall (A) The dual-fluorescence reporter used to study translational stalling.

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“…Hel2 and Slh1 are therefore needed to abort translation and avoid readthrough at sites of ribosome slowing. Three of four subunits of the ASC-1 complex (ASCC) are homologous to the yeast RQT complex and seem to show similar phenotypes when disrupted in cultured cells ( Hashimoto et al., 2020 , Matsuo et al., 2017 ).…”

Ribosome collisions trigger cis-acting feedback inhibition of translation initiation

“…ASCC is a conserved complex containing the ASCC3 helicase (Slh1 in yeast) involved in resolving collided ribosomes ( D'Orazio et al, 2019 ; Hashimoto et al, 2020 ; Ikeuchi et al, 2019 ; Juszkiewicz et al, 2020 ; Matsuo et al, 2017 ; Matsuo et al, 2020 ). Recent experiments suggest that by directly dissociating the lead ribosome into subunits, ASCC is the factor that irreversibly aborts translation and initiates RQC on the stalled ribosome ( Juszkiewicz et al, 2020 ; Matsuo et al, 2020 ).…”

“…The steps downstream of ZNF598 are incompletely understood and appear to involve nuclease ( D'Orazio et al, 2019 ), a helicase complex ( Hashimoto et al, 2020 ; Matsuo et al, 2017 ; Sitron et al, 2017 ), and possibly ribosome rescue factors ( Tsuboi et al, 2012 ). Although the roles and order of action of these factors is only partially resolved ( Juszkiewicz et al, 2020 ; Matsuo et al, 2020 ), the requirement for ZNF598-mediated 40S ubiquitination ( Garzia et al, 2017 ; Juszkiewicz and Hegde, 2017 ; Matsuo et al, 2017 ; Sundaramoorthy et al, 2017 ) together with the specificity of ZNF598 for ribosome collisions ( Juszkiewicz et al, 2018 ) strongly argues that the collided ribosome is a key proxy of aberrant translation.…”

The interaction of DNA repair factors ASCC2 and ASCC3 is affected by somatic cancer mutations

“…Finally, a genome-wide CRISPRi screen showed that ASCC2 and ASCC3 are the two most potent modifiers of cell fitness in the presence of a translation inhibitor, suggesting that the proteins affect stalled ribosomes 22 . Indeed, ASCC3 has been suggested to resolve stalled ribosomes on poly-A sequences in a ribosome quality control pathway [23][24][25] . Likewise, its yeast homolog, Slh1p, has been implicated in ribosomeassociated quality control to prevent the accumulation of aberrant proteins 23,26,27 and in non-functional ribosomal RNA decay 28 .…”