Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.During the canonical termination and recycling steps of translation, stop codon recognition triggers factor-mediated hydrolysis of the nascent peptidyl-tRNA conjugate, nascent chain release, and ribosome splitting (1–3). Conversely, translation of aberrant mRNA, such as mRNA lacking stop codons (“nonstop mRNA”), renders 80S ribosomes stalled with nascent polypeptides (1–3). Furthermore, “nonstop proteins” cannot be corrected by quality control chaperones and have the potential to interfere with cellular function (3, 4). Not surprisingly, defective termination and recycling are under surveillance by a variety of mechanisms (1–3). In eukaryotes, “rescue factors” homologous to termination factors promote dissociation of translationally halted ribosomes in a stop codon-independent manner (5). However, because rescue factors lack peptidyl-tRNA hydrolase activity, their action results in nascent chains remaining stalled on the released 60S subunit.Ltn1 is the critical E3 ligase mediating ubiquitylation of aberrant proteins that become stalled on ribosomes during translation (4). Mutation of the Ltn1 mouse ortholog, listerin, causes neurodegeneration (6), suggesting an important function for this process. Ltn1 works together with several cofactors as part of the ribosome-associated quality control complex (RQC) (7–9) and appears to first associate with nascent chain-stalled 60S subunits together with two proteins of unknown function, Tae2 and Rqc1 (7, 9). Ltn1-mediated ubiquitylation of the stalled polypeptide then results in the recruitment of the AAA ATPase Cdc48/p97/VCP. This recruitment also requires Rqc1 and Tae2, and is followed by Cdc48-mediated nascent chain extraction and delivery to the proteasome for degradation (7–9).Exactly how RQC recognizes stalled ribosomes and performs its functions has not been understood, and the only structure available in this system is that of free Ltn1 at 40-Å resolution (10). To elucidate mechanisms underlying RQC function, we set out to solve the structure of the endogenous complex purified from yeast. To reduce sample heterogeneity so as to facilitate structural characterization, RQC assembly was blocked in a preubiquitylation step by using cells expressing endogenous Ltn1 with a deletion of the E3-catalytic RING domain (Ltn1-∆R). This Ltn1 mutant is competent for binding to 60S subunits but fails to ubiquitylate substrates (4), thus preventing downstream events from being triggered.