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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：
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合成和蛋白质翻译的过度激活与人类癌症密切相关。因此，核糖体生物合成必须在正常细胞内稳态期间受到严格调控。许多监管机构作为辅助因子，核仁RNA外泌体在核糖体生物合成中起着关键作用。它含有一个9亚基核心和称为hRrp 6（人EXOSC 10）的分布性3 - 5 bp外切核酸酶，并介导加工和核仁RNA包括前核糖体RNA（rRNA）和snoRNA的降解。目前，它在很大程度上目前还不清楚RNA外泌体在核仁中是如何调节的。我们最近发现，USP 36，一个核仁去泛素化酶（Dub）通过与hRrp 6的直接相互作用与外泌体结合。有趣的是， USP 36不调节hRrp 6和其他外泌体亚基的水平。相反，它作为一个新的相扑 E3在细胞内和体外介导hRrp 6在Lys（K）583处的SUMO化。USP 36或 hRrp 6损害rRNA加工并显著抑制细胞增殖。有趣的是，突变的K583 K583 R突变体破坏了hRrp 6与前体rRNA的结合，不能挽救rRNA的加工内源性hRrp 6敲低导致的缺陷，表明hRrp 6 SUMO化对于hRrp 6 与前体rRNA的结合和加工。这些初步数据导致了一个新的假设，即USP 36 作为一个新的SUMO E3调节核仁RNA外泌体，因此对前体rRNA至关重要。加工和核糖体生物合成，并与癌症有关。为了进一步了解USP 36的作用，调节核仁RNA外泌体和核糖体的生物合成，我们将研究分子和 USP 36与核仁RNA外泌体和细胞外基质结合的生化机制 Aim 1中hRrp 6的SUMO化，包括USP 36如何与核仁RNA外泌体相互作用， SUMO化其他外泌体亚基和衔接蛋白，以及USP 36如何作为外泌体的SUMO E3 及其衔接蛋白。然后，我们将重点关注USP 36调节RNA外泌体在细胞凋亡中的功能作用。 Aim 2中的核糖体生物发生，包括USP 36-外泌体调控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。USP 36是在包括乳腺癌在内的人类癌症中经常过表达，我们将阐明USP 36是否 SUMO E3活性有助于Aim 3中的肿瘤发生，包括USP 36及其SUMO的研究 E3促进转基因小鼠体内细胞转化和乳腺肿瘤发生使用基因敲除小鼠模型，USP 36的缺失是否抑制肿瘤形成和生长以及抑制USP 36是否抑制乳腺癌细胞增殖，这种抑制与乳腺癌的发生有关。 SUMO化水平。实现这些目标将为USP 36如何正确监管核仁中RNA外泌体SUMO化，这种活性的失调如何有助于肿瘤发生，并且还将揭示USP 36 SUMO E3活性可能是治疗靶点。