Novel mechanisms of ubiquitin signaling coordinating ribosome maturation and function

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Cell signaling through ubiquitination plays critical roles in virtually all biological processes of eukaryotic organisms. This reaction occurs in three sequential steps involving the action of E3 ubiquitin ligases. Moreover, ubiquitination is reversible and specific proteases, i.e. deubiquitinases (DUBs), deubiquitinate protein substrates. A vast repertoire of ubiquitin ligases (>500 genes) and DUBs (~100 genes) regulate ubiquitin-mediated processes. Although, we now appreciate the importance of ubiquitin ligases and DUBs, their biological functions and mechanisms of action remain incompletely understood. USP16 (UBP-M) was initially implicated in the regulation of chromatin-associated processes. However, previous findings provided evidence that this DUB is also present in the cytoplasm. Thus, we sought to investigate how USP16 nucleocytoplasmic trafficking is regulated. With the previously funded NSERC grant, we demonstrated that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for USP16 cytoplasm localization. We also challenged previously established findings and further show that this DUB does not accumulate in the nucleus following DNA damage. Thus, in contrast to the prevailing view, our data indicate that USP16 is a cytoplasmic and suggest new functions of this DUB in the cytoplasm. Indeed, we and others present evidence that USP16 is associated with ribosomes and regulate their biogenesis and function. Moreover, we identified several post-translational modifications (including ubiquitination and phosphorylation) and interacting partners of USP16 that might regulate its function. HYPOTHESIS: USP16 deubiquitinase functions in the cytoplasm to orchestrate ribosome maturation and other cellular processes, and this is required for proper cell growth and proliferation. This DUB targets multiple substrates and is regulated by interacting partners and signaling pathways. SPECIFIC AIMS: To elucidate the roles of USP16 in ribosome maturation and other cellular process, we plan to: 1) Determine the importance of USP16-interacting partners for 40S ribosome maturation. 2) Investigate the roles of USP16 post-translational modifications in regulating 40S ribosome maturation. 3) Identify the full spectrum of USP16 substrates in the cytoplasm These studies will be accomplished using model cell lines as well as primary cells. RNAi and pharmacological inhibitors will be used to determine the requirement of specific signaling molecules in regulating USP16 function. Expression of various mutants of USP16 and other relevant proteins will be conducted to determine their effects on ribosome composition, maturation and function. This investigation will not only shed new light on the importance of the ubiquitin system in regulating critical cellular processes such as protein synthesis, but will also help in furthering our understanding of the mechanisms of cell signaling.