Regulation of the nucleolar RNA exosome in cancer

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Project Summary Deregulated over-activation of ribosome biogenesis and protein translation is tightly linked to human cancers. Thus, ribosome biogenesis must be tightly regulated during normal cell homeostasis. Of many regulatory accessory factors, the nucleolar RNA exosome plays a key role in ribosome biogenesis. It contains a 9-subunit core and a distributive 3ʹ to 5ʹ exonuclease called hRrp6 (human EXOSC10) and mediates the processing and degradation of nucleolar RNAs including pre-ribosomal RNA (rRNA) and snoRNAs. Currently it is largely unknown how the RNA exosome is regulated in the nucleolus. We recently found that USP36, a nucleolar deubiquitinating enzyme (Dub), binds to the exosome through direct interaction with hRrp6. Interestingly, USP36 does not regulate the levels of hRrp6 and other exosome subunits. Instead, it acts as a novel SUMO E3 and mediates SUMOylation of hRrp6 at Lys (K) 583 in cells and in vitro. Knockdown of either USP36 or hRrp6 impairs rRNA processing and significantly inhibited cell proliferation. Intriguingly, mutating K583 impaired the binding of hRrp6 to pre-rRNAs and the K583R mutant failed to rescue the rRNA processing defects caused by knockdown of endogenous hRrp6, indicating that hRrp6 SUMOylation is critical for hRrp6 binding to and the processing of pre-rRNA. These preliminary data lead to a novel hypothesis that USP36 regulates the nucleolar RNA exosome by acting as a novel SUMO E3, thus being critical for pre-rRNA processing and ribosome biogenesis and implicated in cancer. To gain further insight into the role of USP36 in regulating the nucleolar RNA exosome and ribosome biogenesis, we will investigate the molecular and biochemical mechanisms underlying USP36 association with the nucleolar RNA exosome and the SUMOylation of hRrp6 in Aim 1, including how USP36 interacts with the nucleolar RNA exosome, whether it SUMOylates other exosome subunits and adaptor proteins, and how USP36 acts as a SUMO E3 for exosome and its adaptor proteins. We will then focus on the functional role of USP36 regulation of RNA exosome in ribosome biogenesis in Aim 2, including the investigation of the role of USP36-exosome regulation in ribosome biogenesis, translation, nuclear RNA quality control as well as cell growth and proliferation. As USP36 is frequently overexpressed in human cancers including breast cancers, we will elucidate whether USP36's SUMO E3 activity contributes to tumorigenesis in Aim 3, including the investigation of if USP36 and its SUMO E3 contribute to cell transformation in culture and mammary tumorigenesis in vivo using transgenic mouse models, whether deletion of USP36 suppresses tumor formation and growth using knockout mouse models and whether inhibiting USP36 suppresses breast cancer cell proliferation and this inhibition correlates with the levels of SUMOylation. Achieving these goals will provide critical insight into how USP36 properly regulates the RNA exosome SUMOylation in the nucleolus, how deregulation of this activity contributes to tumorigenesis, and would also reveal that USP36 SUMO E3 activity could be a therapeutic target.