Ribosome stalling on eukaryotic mRNAs triggers cotranslational RNA and protein degradation through conserved mechanisms. For example, mRNAs lacking a stop codon are degraded by the exosome in association with its cofactor, the SKI complex, whereas the corresponding aberrant nascent polypeptides are ubiquitinated by the E3 ligases Ltn1 and Not4 and become proteasome substrates. How translation arrest is linked with polypeptide degradation is still unclear. Genetic screens with SKI and LTN1 mutants allowed us to identify translation-associated element 2 (Tae2) and ribosome quality control 1 (Rqc1), two factors that we found associated, together with Ltn1 and the AAA-ATPase Cdc48, to 60S ribosomal subunits. Translation-associated element 2 (Tae2), Rqc1, and Cdc48 were all required for degradation of polypeptides synthesized from NonStop mRNAs (Non-Stop protein decay; NSPD). Both Ltn1 and Rqc1 were essential for the recruitment of Cdc48 to 60S particles. Polysome gradient analyses of mutant strains revealed unique intermediates of this pathway, showing that the polyubiquitination of Non-Stop peptides is a progressive process. We propose that ubiquitination of the nascent peptide starts on the 80S and continues on the 60S, on which Cdc48 is recruited to escort the substrate for proteasomal degradation.quality control | Saccharomyces cerevisiae

Ski2-Ski3-Ski8 (Ski) is a helicase complex functioning with the RNA-degrading exosome to mediate the 3'-5' messenger RNA (mRNA) decay in turnover and quality-control pathways. We report that the Ski complex directly associates with 80S ribosomes presenting a short mRNA 3' overhang. We determined the structure of an endogenous ribosome-Ski complex using cryo-electron microscopy (EM) with a local resolution of the Ski complex ranging from 4 angstroms (Å) in the core to about 10 Å for intrinsically flexible regions. Ribosome binding displaces the autoinhibitory domain of the Ski2 helicase, positioning it in an open conformation near the ribosomal mRNA entry tunnel. We observe that the mRNA 3' overhang is threaded directly from the small ribosomal subunit to the helicase channel of Ski2, primed for ongoing exosome-mediated 3'-5' degradation.

Protein homeostasis is maintained by quality control mechanisms that detect and eliminate deficient translation products. Cytosolic defective proteins can arise from translation of aberrant mRNAs lacking a termination codon (NonStop) or containing a sequence that blocks translation elongation (No-Go), which results in translational arrest. Stalled ribosomes are dissociated, aberrant mRNAs are degraded by the cytoplasmic exosome, and the nascent peptides remaining in stalled 60S exit tunnels are detected by the ribosome-bound quality control complex (RQC) composed of Ltn1, Rqc1, Rqc2, and Cdc48. Whereas Ltn1 polyubiquitylates these nascent peptides, Rqc2 directs the addition of C-terminal alanine-threonine tails (CATtails), and a Cdc48 hexamer is recruited to extract the nascent peptides, which are addressed to the proteasome for degradation. Although the functions of most RQC components have been described, the role of Rqc1 in this quality control process remains undetermined. In this article we show that the absence of Rqc1 or Ltn1 results in the aggregation of aberrant proteins, a phenomenon that requires CAT-tail addition to the nascent peptides by Rqc2. Our results suggest that aberrant CAT-tailed protein aggregation results from a defect in Cdc48 recruitment to stalled 60S particles, a process that requires both Rqc1 and Ltn1. These protein aggregates contain Ltn1-dependent polyubiquitin chains and are degraded by the proteasome. Finally, aggregate characterization by proteomics revealed that they contain specific chaperones including Sis1, Sgt2, Ssa1/2, and Hsp82, suggesting that these protein aggregates may be addressed to aggresome-like structures when the RQC complex fails to deliver aberrant nascent peptides to the proteasome for degradation.