Regulation of the nucleolar RNA exosome in cancer

项目摘要 核糖体生物发生和蛋白质翻译的非调控过度激活与人类癌症密切相关。 因此，在正常的细胞动态平衡期间，核糖体的生物发生必须受到严格的调控。在众多监管机构中 作为辅助因子，核仁RNA外切体在核糖体的生物发生中起着关键作用。它含有一个9亚基 核心和分布的3ʹ到5ʹ外切酶hRrp6(人EXOSC10)，并介导加工和 核仁RNA的降解，包括核糖体前RNA(RRNA)和snoRNAs。目前在很大程度上 未知核糖核酸外切体是如何在核仁中调节的。我们最近发现核仁USP36 去泛素酶(DUB)是一种通过与hRrp6直接相互作用而与外体结合的酶。有趣的是， USP36不调节hRrp6和其他外体亚基的水平。相反，它就像一场新奇的相扑 E3，并在细胞内和体外介导hRrp6在Lys(K)583位的SUMO化。击倒USP36或 HRrp6抑制rRNA加工，显著抑制细胞增殖。有趣的是，突变的K583 破坏hRrp6与前rRNA的结合，K583R突变体未能挽救rRNA加工 内源性hRrp6基因敲除引起的缺陷，表明hRrp6 SUMO化对hRrp6起关键作用 Pre-rRNA的结合和加工。这些初步数据导致了一种新的假设，即USP36 通过作为新的相扑E3来调节核仁rna外体，从而对前rrRNA至关重要。 加工和核糖体的生物发生，并与癌症有关。为了进一步了解USP36在 调控核仁RNA外糖体和核糖体的生物发生，我们将研究分子和 USP36与核仁RNA外切体相关的生化机制 目标1中hRrp6的苏莫化，包括USP36如何与核仁RNA外体相互作用，无论是它 SUMO化其他外体亚基和接头蛋白，以及USP36如何作为外体的相扑E3 和它的接头蛋白。然后我们将重点介绍USP36对RNA外切体的调节在 AIM 2中的核糖体生物发生，包括USP36-外糖体调节在核糖体中的作用的研究 生物发生、翻译、核RNA质量控制以及细胞生长和增殖。和USP36一样 在包括乳腺癌在内的人类肿瘤中经常过表达，我们将阐明USP36‘S 相扑E3活性在AIM 3肿瘤发生中的作用，包括对IF USP36及其相扑的研究 E3对培养细胞转化和转基因小鼠体内乳腺成瘤的作用 利用基因敲除小鼠模型研究USP36缺失是否抑制肿瘤的形成和生长 以及抑制USP36是否抑制乳腺癌细胞的增殖，这种抑制与 SUMO化水平。实现这些目标将为USP36如何适当地监管 核仁中的RNA外切体SUMO化，这种活性的放松如何促进肿瘤的发生， 并将揭示USP36相扑E3活性可能成为治疗的靶点。

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.