Regulation of Translation by O-GlcNAc - Resubmission 03-05-2020

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The cycling of N-acetylglucosamine on Ser(Thr) residues (O-GlcNAcylation; OGN) on nuclear, cytoplasmic and mitochondrial proteins serves as a nutrient sensor to regulate signaling, transcription, and cellular physiology. Abnormal OGN underlies the etiology of diabetes, cancer and Alzheimer’s disease. OGN regulates nearly every aspect of transcription, including RNA polymerase II, histones, DNA methyltransferases, and nearly all transcription factors. Recent findings by us and others indicate that O- GlcNAcylation also regulates protein translation and mRNA utilization, but much less is known. Understanding how nutrients and stress regulate protein translation via OGN is not only critical to our basic understanding of one of the cell’s most vital processes, but also is key to understanding mechanisms underlying chronic diseases of aging, such as diabetes, cancer and neurodegeneration. We hypothesize that O-GlcNAc cycling on proteins in the translational machinery regulates proteostasis by mediating communication between the proteasome and ribosomal machinery, and that nutrients regulate translation rates and mRNA selection by dynamic O-GlcNAc cycling on many ribosome-associated proteins. We propose three specific aims to advance our understanding of OGN’s roles in nutrient regulation of translation: Aim 1 will use state-of-the-art mass spectrometric methods to identify both nascent and mature ribosome associated proteins and translation factors that are modified by OGN, and we will specifically focus on those that appear to be involved in ribosome:proteasome communication. We will then determine the functions of OGN at the site level on selected OGN translation proteins. Aim 2 will elucidate how high glucose alters the OGN of the translation machinery. Using both live HEK293 cells and a rabbit reticulocyte translation system, we will determine if OGN plays a role in mRNA selection by performing RNA seq analyses of polysome preparations. Aim 3 will determine the mechanisms by which the O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are rapidly targeted to ribosomes in response to proteasome inhibition. These studies are not only elucidating molecular mechanisms of how nutrients regulate protein synthesis, but they also are key to revealing how hyperglycemia, as occurs in diabetes, abnormally alters protein expression in many tissues. Molecular mechanisms revealed in these studies will likely lead to totally novel targets for the treatment of chronic diseases of aging, particularly diabetes.