Novel roles for USP36 in ribosome biogenesis

项目摘要 核糖体生物发生是一个复杂的细胞过程，在细胞核中产生核糖体， 正常的细胞生长和增殖。核糖体生物合成的缺陷与一组疾病有关 叫做核糖体病因此，了解核糖体生物合成在细胞周期中是如何被适当调节的是至关重要的。 正常的细胞稳态已经表明，SUMO化在核糖体的生物合成中起关键作用。 核仁，包括rRNA的合成和加工，核糖体亚基组装，成熟和核 导出.然而，SUMO连接酶（E3）介导的核仁SUMO化尚未确定。我们最近 发现去泛素化酶USP 36是一种新的核仁SUMO E3。USP 36的过表达 促进SUMO化，主要是在核仁中，而USP 36的敲低大大降低了 细胞中的SUMO化。我们发现USP 36直接与Ubc 9（SUMO E2）和SUMO结合，并具有 复溶系统中的体外SUMO E3活性，证明USP 36是一种新型真正的SUMO E3。 SUMO E3活性定位于N-末端区域（氨基酸120-300）。我们进一步发现，USP 36 介导Nop 58和Nhp 2的SUMO化，它们是盒C/D和盒H/ACA小核仁的组分。 核糖核蛋白（snoRNP）复合物，并调节它们与snoRNA的结合。所有这些 数据导致了一个新的假设，即USP 36作为一个重要的SUMO E3介导的核仁 SUMO化，因此对核糖体生物合成至关重要。为了进一步了解USP 36的作用， 核仁SUMO化和核糖体生物合成的调控，我们将研究分子和 Aim 1中USP 36的SUMO E3活性的生化机制，包括N-末端 SUMO E3结构域有助于USP 36的SUMO E3活性，USP 36如何促进聚SUMO链 形成和SUMO转移到底物，以及如何确定其SUMO E3和Dub活性。我们将 然后阐明USP 36在snoRNP中作为SUMO E3的核仁功能和Aim 2中的核糖体生物合成， 包括USP 36是否调节snoRNP和核糖体生物发生相关蛋白组SUMO化， 它是否调节rRNA修饰和rRNA加工，以及它是否也调节核糖体亚基 成熟和核出口。最后，我们将阐明USP 36的核仁SUMO E3活性是否是关键的 使用诱导型USP 36进行体内细胞生长和增殖以及组织稳态和再生 Aim 3中的基因敲除小鼠模型。实现这些目标将为USP 36如何正确地 调节核仁中的SUMO化以及它如何调节核糖体生物发生。

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.