In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing

Abstract

Ribosomes can stall during translation due to defects in the mRNA template or translation machinery, leading to the production of incomplete proteins. The Ribosome-associated Quality control Complex (RQC) engages stalled ribosomes and targets nascent polypeptides for proteasomal degradation. However, how each RQC component contributes to this process remains unclear. Here we demonstrate that key RQC activities—Ltn1p-dependent ubiquitination and Rqc2p-mediated Carboxy-terminal Alanine and Threonine (CAT) tail elongation—can be recapitulated in vitro with a yeast cell-free system. Using this approach, we determined that CAT tailing is mechanistically distinct from canonical translation, that Ltn1p-mediated ubiquitination depends on the poorly characterized RQC component Rqc1p, and that the process of CAT tailing enables robust ubiquitination of the nascent polypeptide. These findings establish a novel system to study the RQC and provide a framework for understanding how RQC factors coordinate their activities to facilitate clearance of incompletely synthesized proteins.Cells make proteins by reading instructions encoded in molecules called messenger RNAs. Structures called ribosomes move along the messenger RNAs and translate the coded instructions to build new proteins from building blocks known as amino acids. Normally, a ribosome will encounter a stop signal on the messenger RNA, which ends translation and allows the newly built protein to be released. Sometimes, however, ribosomes stall before they reach the genuine stop signal, which can happen due to defects in the messenger RNAs or ribosomes.To prevent incomplete proteins from accumulating and causing damage, cells contain a group of other proteins called the Ribosome-associated Quality-control Complex (or RQC for short). This quality-control complex is composed of three components that assemble on stalled ribosomes and attach two different tags to the incomplete protein. One component adds a degradation tag called ubiquitin. A second component works with the ribosome to tag the incomplete protein with a ‘tail’ that contains the amino acids alanine and threonine. These amino acids are abbreviated to A and T, and are added to the end of the protein known as the ‘C-terminus’, so this tag is named a ‘CAT tail’. Although all three RQC components are needed to degrade incomplete proteins, little was known about why the CAT tails are added, or what the third component – a protein called Rqc1p – actually does.Osuna et al. have now investigated how the apparently unrelated activities of RQC components are coordinated to destroy incomplete proteins. Ribosomes and RQC components from yeast cells were extracted and mixed in the laboratory with a messenger RNA that stalls ribosomes. In this cell-free system, the RQC components could still tag incomplete proteins with both ubiquitin and CAT tails. Osuna et al. then used this system to show that the way the ribosome added amino acids to form a CAT tail was different from how it normally builds proteins. The experiments also showed that in order for the ubiquitin tags to be added efficiently, Rqc1p must be present, and in some cases, the incomplete proteins also need to be ‘CAT tailed’.When either were missing, very few ubiquitin tags could be added to the incomplete proteins. The results show that the three core RQC components need to work together to degrade incomplete proteins. This quality-control complex is also found in mice and humans, and mice with mutations in the genes that encode RQC components often have damaged nervous systems. In the future, researchers building upon these findings and other studies of the RQC may eventually understand the relationship between the RQC and neurodegenerative diseases in humans.