Dissecting the roles of ubiquitin in translation control

Duke University, Durham NC

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Abstract

Project Summary This research program will investigate new mechanisms by which eukaryotic cells regulate gene expression at the protein level during exposure to stress. Failure to reprogram protein expression and inadequate management of unneeded and damaged proteins are the underlying causes of a variety of neurodegenerative diseases, tumor progression, and the process of aging. Understanding and modulating stress defense pathways, such as translation reprogramming, would provide new tools to promote healthier lives and fight diseases. Therefore, this proposal will focus on deciphering new molecular mechanisms by which a series of ubiquitin- mediated processes regulate translation through the control of ribosome structure and function during stress. My laboratory discovered in budding yeast that ribosomes are heavily modified by K63-linked polyubiquitin chains in response to oxidative stress, as part of a new pathway named Redox control of Translation by Ubiquitin (RTU). In the last funding cycle, we identified key enzymes involved in ubiquitination and deubiquitination of ribosomes in the RTU (Rad6, Bre1, and Ubp2) and their mechanisms of regulation in the presence of reactive oxygen species (ROS). We further showed that ubiquitination of ribosomes arrests them in a sequence-specific manner at the elongation stage of translation. Finally, we developed new proteomics tools to characterize the dynamics of ribosome ubiquitination during stress progression with amino acid resolution. Inactivation of the RTU leads to translation dysregulation and sensitivity to stress, but the mechanisms controlling ribosome pausing in the RTU remain largely elusive. This proposal will explore four key questions in our research program: 1) How does unique sites of ubiquitination regulate ribosome fate and function during oxidative stress and how do they differ from other quality control systems? 2) Why and how do ribosomes pause at selective mRNA motifs in the RTU? 3) What is the molecular composition and the nature of cofactors regulating redox ribosome pausing? 4) How does ubiquitin regulate translation in human cells during oxidative stress? My research uses a combination of molecular, cellular, biochemical, computational, and high-throughput approaches in proteomics and genomics, which has provided a solid and rigorous foundation for a continued exploration of the mechanisms driving gene expression at the translational level. The overarching goal of my research laboratory is to leverage the power of the ubiquitin proteasome system (UPS) in regulating the proteostasis network to design new treatments and interventions for stress-related diseases.