Novel roles for USP36 in ribosome biogenesis

USP36 在核糖体生物合成中的新作用

• 批准号: 10413038
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10413038
• 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; Sumoylation Pathway; 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.

项目概要 核糖体生物发生是在核仁中制造核糖体的复杂细胞过程，对于 正常的细胞生长和增殖。核糖体生物发生的缺陷与一组疾病相关 称为核糖体病。因此，了解核糖体生物发生如何在 正常的细胞稳态。研究表明，SUMO 化在核糖体生物合成中发挥着关键作用。 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，尚未确定介导核仁 SUMO 化的 SUMO 连接酶 (E3)。我们最近 发现去泛素化酶USP36是一种新型核仁SUMO E3。 USP36 的过度表达 促进 SUMO 化，主要是在核仁中，而 USP36 的敲除则大大降低了 SUMO 化水平 细胞中的 SUMO 化。我们发现 USP36 直接与 Ubc9 (SUMO E2) 和 SUMO 结合，并具有 SUMO E3 在重组系统中的体外活性，证明 USP36 是一种新型的真正的 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（盒 C/D 和盒 H/ACA 小核仁的组成部分）的 SUMOylation 核糖核蛋白（snoRNP）分别复合，并调节它们与 snoRNA 的结合。在一起，这些 数据得出了一个新的假设，即 USP36 充当关键的 SUMO E3 介导核仁 SUMOylation 因此对于核糖体生物发生至关重要。进一步了解 USP36 在 核仁 SUMO 化和核糖体生物发生的调节，我们将研究分子和 目标 1 中 USP36 SUMO E3 活性的生化机制，包括 N 末端如何 SUMO E3 结构域有助于 USP36 的 SUMO E3 活性，USP36 如何促进聚 SUMO 链 形成和 SUMO 转移到底物，以及如何测定其 SUMO E3 和 Dub 活性。我们将 然后阐明 USP36 作为 snoRNP 中的 SUMO E3 的核仁功能和目标 2 中的核糖体生物合成， 包括USP36是否调节snoRNP和核糖体生物发生相关蛋白组SUMO化， 是否调节 rRNA 修饰和 rRNA 加工，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的核仁SUMO E3活性是否至关重要 使用诱导型 USP36 进行体内细胞生长和增殖以及组织稳态和再生 目标 3 中的基因敲除小鼠模型。实现这些目标将为了解 USP36 如何正确发挥作用提供重要见解 调节核仁中的 SUMOylation 及其如何调节核糖体生物发生。