Project Summary Ribosome biogenesis, a complex and coordinated cellular process for making the ribosome in the nucleolus, is essential for cell growth and proliferation. Defects in ribosome biogenesis are associated with a group of diseases called ribosomopathies. The goal of our research program is to understand how ribosome biogenesis is regulated during normal cell homeostasis and how it is deregulated in human diseases. Emerging evidence, including our own work, suggests that SUMOylation plays a critical role in ribosome biogenesis. We identified the ubiquitin-specific protease USP36 as a novel SUMO ligase that promotes the SUMOylation of snoRNP components Nop58, Nhp2, NOP56 and DKC1, the RNA exosome component EXOSC10, and the endonuclease Las1L, and plays a key role in multiple steps of rRNA processing. Meanwhile, deSUMOylation of the key nucleolar proteins by SENP3 is also essential for ribosome biogenesis. Thus, the proper nucleolar protein SUMOylation dynamics controlled by the coordinated action of USP36-mediated SUMOylation and SENP3-mediated deSUMOylation might be essential for ribosome biogenesis. However, there are several key gaps in understanding the SUMOylation dynamics in ribosome biogenesis, including the unclear mechanistic insights into the role of USP36 and SENP3, the limited scope of SUMO regulation of ribosome biogenesis identified, and the lack of dissection of the nucleolar SUMOylation dynamics. Our laboratory is well equipped in investigating ribosome biogenesis and protein posttranslational modifications. The goals of our research program for the next five years are to address these key gaps. We will investigate the mechanisms underlying the roles of USP36 and SENP3 using molecular imaging, structural biology, and molecular biology approaches. We will identify novel SUMOylation/deSUMOylation substrates of USP36 and SENP3 and dissect their roles in specific steps of ribosome biogenesis. We will then investigate the SUMO dynamics and its role in ribosome biogenesis and cell growth as well as the role of USP36 and SENP3 in cells in response to stress. Achieving these goals will provide critical insights into how USP36 and SENP3 properly coordinate to regulate nucleolar SUMOylation and ribosome biogenesis. The long-term vision of our research program is to understand how ribosome biogenesis is deregulated in various human diseases.