Quality Control of Translation Factors

Abstract

Protein synthesis is a remarkably error prone process, necessitating protein quality control pathways to recognize and resolve these abnormal events.

In chapters 2 and 3, I examine downstream steps in the ribosome-associated protein quality control (RQC) pathway. Ribosome stalling during translation triggers the RQC pathway to degrade the incompletely synthesized nascent chain. However, the fate of other components of the stalled ribosome, including the peptidyl-tRNA, is unclear. During RQC, the stalled ribosome is first dissociated into 40S and 60S subunits. The nascent chain-tRNA (NC-tRNA) remains trapped on the 60S subunit due to the tRNA and folded polypeptide on both sides of the ribosome exit tunnel. Previous studies have identified ANKZF1 as the release factor that releases the nascent chain from the tRNA, allowing the nascent chain to be degraded by the proteasome. I show that ANKZF1 is a peptidyl-tRNA endonuclease that cleaves the invariant 3’CCA of peptidyl-tRNA on the 60S-NC-tRNA complex. This tRNA can be recycled in mammalian cytosol in a two-step pathway involving removal of a 2’,3’-cyclic phosphate followed by CCA-readdition by the CCA-adding enzyme TRNT1. Through biochemical fractionation, I identified the 2’3’- cyclic phosphatase as ELAC1, and it is both necessary and sufficient for 2’,3’-cyclic phosphate removal. These studies reveal that all tRNAs arising from stalled ribosomes must undergo CCA-readdition before they can be reused in translation, and suggest an over-arching hypothesis that all components of stalled translational complexes are quality-checked before they can be reused, thus ensuring that defective components are not recycled.

In chapter 4, I study the quality control of unassembled cytosolic proteins by the enzyme UBE2O. About half of all cytosolic proteins exist as protein complexes. Protein expression must be carefully regulated to ensure that excess, unassembled proteins do not accumulate. One mechanism by which this occurs is the selective degradation of unassembled, ‘orphan’ proteins. The quality control enzyme UBE2O, that possess both ubiquitin-conjugating and ubiquitin ligase activities, has previously been implicated in directly recognizing and ubiquitylating these ‘orphan’ proteins that fail to assemble into their native complexes. However, the mechanism of client selection by UBE2O has remained unclear. In this study, I identified two determinants of UBE2O client selection – a ‘priming’ ubiquitin modification on clients, and a substrate adaptor NAP1L1. A cryogenic electron microscopy structure of UBE2O in complex with NAP1L1 and ubiquitinated client reveals a client-binding cleft capped by the ubiquitin- conjugating (UBC) domain of UBE2O, and a previously un-annotated ubiquitin-binding site on UBE2O. Our study reveals how multi-valency and a feed forward mechanism determine client selection by UBE2O.