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：
10589142
负责人：
Mu-Shui Dai
金额：
$ 45.68万
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/10589142
关键词：
Ablation Adaptor Signaling Protein Affect Affinity Chromatography 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 Genetic Transcription Goals Growth Homeostasis Human Impairment In Vitro Inhibition of Cell Proliferation 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 Phosphodiesterase I Physiological Play Proteins Quality Control RNA RNA Degradation RNA Processing Regulation Reporting Ribosomal RNA Ribosomes Role Shapes 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 isopeptidase

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.

项目摘要核糖体生物发生和蛋白质翻译的过度激活与人类癌症紧密相关。这是在正常细胞稳态期间必须严格调节核糖体生物发生。许多监管辅助因子，核仁RNA外泌体在核糖体生物发生中起关键作用。它包含9份核心和一个称为HRRP6（人exosc10）的3°至5ʹ外核酸酶的分配，并介导了处理和包括核糖体RNA（RRNA）和SNORNA在内的核RNA的降解。目前主要是未知在核仁中如何调节RNA外泌体。我们最近发现usp36，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，它充当新颖的相扑 E3并介导在细胞和体外的LYS（K）583处HRRP6的Sumoylation。 USP36或 HRRP6会损害RRNA的加工并显着抑制细胞增殖。有趣的是，突变K583 HRRP6与前RNA和K583R突变体的结合损害未能营救RRNA处理由内源性HRRP6敲低引起的缺陷，表明HRRP6 sumoylation对HRRP6至关重要与前rNNA的结合和处理。这些初步数据导致了一个新的假设，即USP36 通过充当新型SUMO E3来调节核RNA外泌体，因此对于rrNA至关重要加工和核糖体生物发生，并在癌症中隐含。为了进一步了解usp36在调节核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外泌体和 AIM 1中HRRP6的sumoylation，包括USP36如何与核仁RNA外泌体相互作用，是否是 Sumoylate其他外泌体亚基和衔接蛋白，以及USP36如何充当外来的E3 及其衔接蛋白。然后，我们将重点介绍USP36 RNA外泌体调节的功能作用 AIM 2中的核糖体生物发生，包括研究核糖体中usp36-异位调节的作用生物发生，翻译，核RNA质量控制以及细胞生长和增殖。如USP36 我们将在包括乳腺癌在内的人类癌症中过表达，我们将阐明USP36是否 SUMO E3活性在AIM 3中有助于肿瘤发生，包括对IF USP36及其相扑的研究 E3使用转基因小鼠在体内培养和乳腺肿瘤发生的细胞转化有助于细胞转化模型，删除USP36是否会使用基因敲除鼠标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 sumoylation的水平。实现这些目标将提供有关USP36如何正确调节的关键见解 RNA外泌体在核仁中，该活性的放松管制如何导致肿瘤发生，并且还表明USP36 SUMO E3活性可能是一个治疗靶点。