RESEARCH STRATEGY NIGMS administrative equipment supplement for MIRA GM 1R35GM137954 PI: Gustavo Silva - Dissecting the roles of ubiquitin in translation control I am requesting funds for the acquisition of a multicolor flow cytometer (BD FACSCelesta) capable of quantifying molecular fluorescence at the cellular level. Aligned with the goals of this award, our lab has made great strides in understanding the role of protein ubiquitination in the regulation of gene expression at the translational level. Our recent fortuitous discoveries on sequence-specific translation pausing and mitochondria physiology opened up unexpected opportunities to further advance the goals within the scope of this research program. However, we currently lack instrumentation to perform assays that will elucidate the mechanisms by which ubiquitination of ribosomes controls translation and its arrest under stress. These new capabilities will allow us to advance the goals of this award, providing faster, quantitative, and high throughput data to dissect the roles of ubiquitin in translation control and beyond. Scientific Justification The main goal of our R35 parental proposal is to understand the role of protein ubiquitination in translation control, particular during stress conditions. This award was based on our discovery of a new pathway in which K63 ubiquitination regulates translation in response to oxidative stress. Recently, we have shown that K63-linked polyubiquitin accumulates rapidly in ribosomal proteins after stress induction and controls the elongation stage of translation (Zhou et al., PNAS 2020). We also identified that ribosome ubiquitination is regulated by a triad of enzymes (Ubp2, Rad6, and Bre1), however, the mechanisms by which ribosome ubiquitination impacts translation remains completely unknown. During the development of this R35 award, we have discovered that the ubiquitin conjugating enzyme Rad6 is critical for the regulation of protein synthesis, mitochondria physiology, and cellular resistance to a variety of stresses (manuscript in the final stages of revision in Cell Reports, IF=9.4). Using state-of-the-art ribosome profiling methods, we observed that ribosomes from wild-type cells show a strong pausing (arrest) effect on selective tripeptide motifs under oxidative stress. Impressively, these redox pausing signatures are completely abolished in yeast cells lacking RAD6. Moreover, cells deleted for Rad6 show dysregulation of mitochondria physiology and of the integrated stress response, an important pathway that represses translation globally at the initiation level during environmental stresses and nutrient starvation. These findings are the backbone of a new manuscript currently under preparation exploring the role of Rad6 and ribosomes ubiquitination in translational control. From these discoveries, two critical questions arose in our research program: 1. What is the mechanism by which ubiquitin induces redox pausing signatures under stress?...