Novel roles for USP36 in ribosome biogenesis

USP36 在核糖体生物发生中的新作用

• 批准号: 9978841
• 负责人: Mu-Shui Dai
• 金额: $ 30.57万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-16 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978841
• 关键词: Affinity Chromatography; Amino Acids; Animals; Binding; Biochemical; Biogenesis; Cell Nucleolus; Cell Proliferation; Cell physiology; Cells; Complex; Data; Defect; Deubiquitinating Enzyme; Disease; Goals; Homeostasis; Human; In Vitro; Knock-out; Knockout Mice; Lead; Ligase; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Methylation; Modeling; Modification; Molecular; Mus; N-terminal; Natural regeneration; Normal Cell; Nuclear Export; Nucleolar Proteins; Physiological; Play; Post-Translational Protein Processing; Proteins; Regulation; Reporting; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Small Nucleolar RNA; Small Nucleolar Ribonucleoproteins; System; Testing; Tissues; Translations; Ubiquitin; cell growth; design; enzyme activity; human disease; in vitro activity; in vivo; insight; knock-down; mouse model; novel; overexpression; reconstitution; therapeutic target

Project Summary Ribosome biogenesis, a complex cellular process for making the ribosome in the nucleolus, is essential for normal cell growth and proliferation. Defects in ribosome biogenesis are associated with a group of diseases called ribosomopathies. Thus, it is crucial to understand how ribosome biogenesis is properly regulated during normal cell homeostasis. It has been shown that SUMOylation plays a key role in ribosome biogenesis in the nucleolus, including rRNA synthesis and processing, ribosome subunit assembly, maturation and nuclear export. Yet, a SUMO ligase (E3) mediating nucleolar SUMOylation has not been identified. We recently discovered that the deubiquitinating enzyme USP36 is a novel nucleolar SUMO E3. Overexpression of USP36 promotes SUMOylation, mainly in the nucleolus, whereas knockdown of USP36 drastically reduces the levels of SUMOylation in cells. We show that USP36 directly binds to Ubc9 (SUMO E2) and SUMO, and possesses SUMO E3 activity in vitro in reconstituted systems, demonstrating that USP36 is a novel bona fide SUMO E3. The SUMO E3 activity is mapped to an N-terminal region (amino acids 120-300). We further found that USP36 mediates the SUMOylation of Nop58 and Nhp2, components of the box C/D and box H/ACA small nucleolar ribonucleoprotein (snoRNP) complexes, respectively, and regulates their binding to snoRNAs. Together, these data lead to a novel hypothesis that USP36 functions as a crucial SUMO E3 mediating nucleolar SUMOylation and thus being critical for ribosome biogenesis. To gain further insight into the role of USP36 in the regulation of nucleolar SUMOylation and ribosome biogenesis, we will investigate the molecular and biochemical mechanisms underlying the SUMO E3 activity of USP36 in Aim 1, including how the N-terminal SUMO E3 domain contributes to USP36's SUMO E3 activity, how USP36 promotes poly-SUMO chain formation and SUMO transfer to substrates, and how its SUMO E3 and Dub activity are determined. We will then elucidate the nucleolar functions of USP36 as a SUMO E3 in snoRNP and ribosome biogenesis in Aim 2, including whether USP36 regulates snoRNP and ribosome biogenesis-related protein group SUMOylation, whether it regulates rRNA modifications and rRNA processing, and whether it also regulates ribosome subunit maturation and nuclear export. Finally, we will elucidate whether USP36's nucleolar SUMO E3 activity is critical for cell growth and proliferation as well as tissue homeostasis and regeneration in vivo using inducible USP36 knockout mice models in Aim 3. Achieving these goals will provide critical insight into how USP36 properly regulates SUMOylation in the nucleolus and how it regulates ribosome biogenesis.

项目摘要 核糖体生物发生是使核仁中核糖体的复杂细胞过程，对于 正常的细胞生长和增殖。核糖体生物发生缺陷与一组疾病有关 称为核糖体病。因此，了解如何正确调节核糖体生物发生在 正常的细胞体内平衡。已经表明，sumoylation在核糖体生物发生中起关键作用 核仁，包括rRNA合成和加工，核糖体亚基组装，成熟和核 出口。然而，尚未鉴定出介导核仁烯酰基化的相扑酶（E3）。我们最近 发现去泛素化酶USP36是一种新型的核仁E3。 USP36的过表达 促进Sumoylation，主要是在核仁中，而USP36的敲低大幅降低了水平 细胞中的sumoylation。我们表明USP36直接与Ubc9（Sumo E2）和Sumo结合，并拥有 Sumo E3在重构系统中的体外活性，表明USP36是一种新颖的善意Sumo E3。 SUMO E3活性映射到N末端区域（氨基酸120-300）。我们进一步发现USP36 介导NOP58和NHP2的sumoylation，盒子C/D和盒子H/ACA小核仁的组件 核糖核蛋白（SNORNP）复合物分别调节它们与snornas的结合。在一起，这些 数据导致了一个新的假设，即USP36起着至关重要的SUMO E3介导核酸 Sumoylation，因此对于核糖体生物发生至关重要。为了进一步了解usp36在 核仁和核糖体生物发生的调节，我们将研究分子和 AIM 1中USP36的SUMO E3活性的生化机制，包括N端如何 Sumo E3域有助于USP36的Sumo E3活动，USP36如何促进多苏链 形成和相扑转移至底物，以及如何确定其ESO E3和DUB活性。我们将 然后阐明snornp中的usp36的核仁函数为E3和核糖体生物发生，在AIM 2中， 包括USP36是否调节SNORNP和核糖体生物发生相关的蛋白质组， 它是否调节rRNA修改和rRNA处理，以及是否还调节核糖体亚基 成熟和核出口。最后，我们将阐明USP36的核酸Sumo E3活性是否至关重要 使用诱导usp36进行细胞生长和增殖以及组织体内平衡和体内再生 AIM 3中的淘汰小鼠模型。实现这些目标将为USP36正确地提供关键的见解 调节核仁中的sumoylation及其如何调节核糖体生物发生。