UC Santa Cruz

Ribosomes build and maintain life. It is essential that ribosomes promptly and faithfully decode mRNAs to produce proteins, so as to avoid the development of disease phenotypes such as neurodegeneration.

In the last two decades, major advances have been made in the discovery and characterization of quality control pathways centered around the ribosome. Two of these pathways are No-Go mRNA Decay (NGD) and Nonstop mRNA Decay (NSD). NGD acts on ribosomes at a strong obstacle to elongation, such as a stable secondary structure or a stretch of poly-basic codons. NSD is triggered by ribosomes elongated to the 3’-most edge of an mRNA as a result of premature polyadenylation or mRNA cleavage. Despite their distinct mRNA species, NGD and NSD share a team of cellular machinery specialized to detect the ribosome state, free the ribosome, and destroy the aberrant mRNA. While a number of these players are now identified, the early steps of stall detection and the links that connect factors remained untested. Here, we illustrate mechanisms of mRNA repression prompted by stalled ribosomes.

In Chapter 1, we review the understanding of ubiquitin signals on an array of ribosomal proteins. Some of these ubiquitination events are thought to play a part in NGD and/or NSD, but the vast majority of ribosomal ubiquitination events are uncharacterized. In our review, we put forth a hypothesis that widespread ribosomal ubiquitination participates in a myriad of cellular signaling to swiftly alter translation.

In Chapter 2, we delineate mechanistic links between an E3 ubiquitin ligase (ZNF-598), a ribosome rescue factor (HBS-1), and an endonuclease (NONU-1) that collaborate to carry out NGD. We demonstrate that ubiquitination of two ribosomal proteins (uS10 and eS10) by ZNF-598 is necessary for NGD, and that inhibition of ubiquitination on these sites produces ribosomes that are immune to NGD. We proceed to characterize a function for these ubiquitination events in NONU-1 recruitment, and we uncover a surprising requirement for HBS-1 in promoting the NONU-1 cleavage reaction. These findings reveal a novel order of events during NGD which will inform future studies, suggesting a model in which ribosome rescue precedes mRNA cleavage.

In Chapter 3, we report a phenomenon present in metazoan ribosome footprint profiling data which is of great importance to the study of NGD. We thoroughly interrogate the dynamics of ribosomes genome-wide and on our genetically validated NGD reporter, revealing an absence of NGD-dependent effects. Key to our studies is the expectation of an effect on our NGD reporter, which was lacking in previous work and limited interpretations. Our findings are consistent with the existence of a fleeting and specialized ribosome state during NGD that evades current ribosome capture protocols. We expect our future work and that of others to enable a greater understanding of these ribosome species.

Overall, this dissertation significantly advances the understanding of the cellular response to ribosomal stalls. In Chapter 4, we reflect on our findings and highlight outstanding questions for future research.