Protein ubiquitylation plays a critical role in shaping proteome dynamics and responding to proteostasis dysfunction. Activation of the integrated stress response (ISR) or the ribosome-associated quality control (RQC) pathway stimulates regulatory ribosomal ubiquitylation (RRub) on distinct 40S ribosomal proteins, yet the cellular role for these ubiquitylation events remains unclear. We previously demonstrated that conserved monoubiquitylation events are required for downstream RQC events following the translation of poly(A) sequences. We identified the E3 ubiquitin ligase, ZNF598, which is responsible for initiating RQC by catalyzing the ubiquitylation of eS10 and uS10. An additional set of ubiquitylation events on uS5 and uS3 are triggered upon activation of the ISR and appear to function outside of the RQC pathway, however the critical regulators remained unknown. In this dissertation I establish that RRub events diminish over time following exposure to UV stress, implicating a role for deubiquitylating enzymes (Dubs) within the RQC pathway. I identified the Dubs OTUD3 and USP21 that, when overexpressed, result in read-through of poly(A)-mediated stalls, and directly antagonize ZNF598. USP21 or OTUD3 knockout cell lines revealed that loss of expression for either Dub results in enhanced stalling on poly(A) sequences and prolonged site-specific RRub following UV exposure. Additionally, I establish a hierarchical structure for the ribosome ubiquitin code by demonstrating that eS10 and uS3 ubiquitylation is necessary for subsequent uS10 and uS5 ubiquitylation, respectively, suggesting a specific order of ribosome ubiquitylation events occurs to ensure optimal resolution of RQC nucleating events. These results demonstrate that Dubs can constrain RQC activation and may serve to remove ubiquitin from 40S subunits to allow for subunit recycling.
uS5 and uS3 ubiquitylation events operate outside of the canonical RQC pathway. Here I demonstrate that translation initiation inhibition, either through moderate ISR activation which produces low levels of eIF2a phosphorylation, or overt pharmacological inhibition of translation initiation trigger these site-specific modifications. I identify the E3 ubiquitin ligase RNF10 and the Dub USP10 as the regulators of uS3 and uS5 ubiquitylation. Additionally, I show that prolonged ubiquitylation results in 40S but not 60S ribosomal protein degradation in an autophagy-independent manner. This study identifies and characterizes a discrete ribosome-associated quality control pathway that surveys preinitiation complex status during mRNA translation initiation.