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Regulation of the nucleolar RNA exosome in cancer

核仁 RNA 外泌体在癌症中的调控

基本信息

批准号：
10458409
负责人：
Mu-Shui Dai
金额：
$ 46.61万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10458409
关键词：
Adaptor Signaling Protein Affect Affinity Animals Binding Biochemical Biogenesis Breast Cancer Cell Cell Culture Techniques Cell Cycle Progression Cell Nucleolus Cell Proliferation Cell physiology Cells Cellular Stress Complex DNA Repair DNA biosynthesis Data Defect Deubiquitinating Enzyme Endoribonucleases Ensure Exonuclease Genetic Transcription Goals Growth Homeostasis Human Impairment In Vitro Investigation Knockout Mice Lead Ligase Link Malignant Neoplasms Mammary Tumorigenesis Mass Spectrum Analysis Mediating Modification Molecular Mutate Normal Cell Nuclear Export Nuclear RNA Pathway interactions Physiological Play Proteins Quality Control RNA RNA Degradation RNA Processing Regulation Reporting Ribosomal RNA Ribosomes Role Side Signal Transduction Small Nucleolar RNA Sumoylation Pathway Transgenic Mice Translations Untranslated RNA cancer therapy cell growth cell transformation design exosome in vivo in vivo Model insight knock-down malignant breast neoplasm mouse model mutant novel overexpression protein complex response therapeutic target tumor tumorigenesis ubiquitin-protein ligase

项目摘要

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.

项目概要核糖体生物合成和蛋白质翻译的过度激活失调与人类癌症密切相关。因此，在正常细胞稳态过程中，核糖体生物发生必须受到严格调控。众多监管中作为辅助因素，核仁 RNA 外泌体在核糖体生物合成中发挥着关键作用。它含有9个亚基核心和分配的 3ʹ 至 5ʹ 核酸外切酶，称为 hRrp6（人 EXOSC10），并介导加工和核仁 RNA 的降解，包括核糖体前 RNA (rRNA) 和 snoRNA。目前主要是尚不清楚RNA外泌体在核仁中是如何调节的。我们最近发现USP36，一种核仁去泛素化酶 (Dub) 通过与 hRrp6 直接相互作用与外泌体结合。有趣的是， USP36 不调节 hRrp6 和其他外泌体亚基的水平。相反，它充当新颖的 SUMO E3 并介导细胞内和体外 hRrp6 Lys (K) 583 处的 SUMO 化。 USP36 或的击倒 hRrp6 损害 rRNA 加工并显着抑制细胞增殖。有趣的是，K583 发生突变削弱 hRrp6 与前 rRNA 的结合，并且 K583R 突变体未能挽救 rRNA 加工内源性 hRrp6 敲低引起的缺陷，表明 hRrp6 SUMOylation 对 hRrp6 至关重要与 pre-rRNA 的结合和加工。这些初步数据得出了一个新的假设，即 USP36 通过充当新型 SUMO E3 来调节核仁 RNA 外泌体，因此对于 pre-rRNA 至关重要加工和核糖体生物发生并与癌症有关。进一步了解 USP36 在调节核仁 RNA 外泌体和核糖体生物发生，我们将研究分子和 USP36 与核仁 RNA 外泌体关联的生化机制目标 1 中 hRrp6 的 SUMO 化，包括 USP36 如何与核仁 RNA 外泌体相互作用，是否 SUMO 化其他外泌体亚基和接头蛋白，以及 USP36 如何充当外泌体的 SUMO E3 及其衔接蛋白。然后我们将重点关注 USP36 对 RNA 外泌体的调节的功能作用目标 2 中的核糖体生物合成，包括研究 USP36-外泌体在核糖体中的调节作用生物发生、翻译、核 RNA 质量控制以及细胞生长和增殖。由于 USP36 是在包括乳腺癌在内的人类癌症中经常过度表达，我们将阐明 USP36 是否 SUMO E3 活性有助于目标 3 中的肿瘤发生，包括 if USP36 及其 SUMO 的研究使用转基因小鼠，E3 有助于培养细胞转化和体内乳腺肿瘤发生模型，使用敲除小鼠模型删除 USP36 是否会抑制肿瘤形成和生长以及抑制 USP36 是否会抑制乳腺癌细胞增殖，并且这种抑制与 SUMO化水平。实现这些目标将为 USP36 如何正确监管核仁中 RNA 外泌体 SUMO 化，这种活性的失调如何导致肿瘤发生，并且还将揭示 USP36 SUMO E3 活性可能是一个治疗靶点。