Novel roles for USP36 in ribosome biogenesis

项目摘要 核糖体生物发生是在核仁中制造核糖体的一个复杂的细胞过程，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 称为核糖体病症。因此，了解核糖体的生物发生是如何在过程中被适当调控是至关重要的。 正常的细胞动态平衡。已证明SUMO化在核糖体的生物发生中起着关键作用。 核仁，包括rRNA的合成和加工，核糖体亚单位的组装，成熟和核 出口。然而，介导核仁SUMO化的相扑连接酶(E3)还没有被确定。我们最近 发现脱泛素酶USP36是一种新的核仁相扑E3。USP36基因的过表达 促进SUMO化，主要是在核仁中，而USP36的敲除显著降低了水平 在细胞中的SUMO化。我们发现USP36直接与Ubc9(相扑E2)和相扑结合，并拥有 在重组系统中的体外相扑E3活性，证明USP36是一种新的真正的相扑E3。 相扑E3活性被映射到一个N-末端区域(氨基酸120-300)。我们进一步发现，USP36 介导盒C/D和盒H/ACA小核仁的Nop58和Nhp2的SUMO化 核糖核蛋白(SnoRNP)复合体，并调节它们与snoRNAs的结合。加在一起，这些 数据导致了一种新的假设，即USP36作为关键的相扑E3介导核仁的功能 和甲基化，因此对核糖体的生物发生至关重要。为了进一步了解USP36在 核仁糖基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相扑E3活性的生化机制，包括N-末端如何 相扑E3结构域参与USP36 S相扑E3活性，USP36如何促进多相扑链 相扑形成和相扑转移到底物，以及相扑E3和DUB活性是如何确定的。我们会 然后阐明了USP36作为snoRNP中的相扑E3的核仁功能和目标2中的核糖体生物发生， 包括USP36是否调节snoRNP和核糖体生物发生相关蛋白基团SUMO化， 它是否调节rRNA修饰和rRNA加工，是否也调节核糖体亚单位 成熟和核出口。最后，我们将阐明USP36的S核仁相扑E3活性是否至关重要 使用可诱导的USP36在体内用于细胞生长和增殖以及组织动态平衡和再生 AIM 3中的基因敲除小鼠模型。实现这些目标将为USP36如何正确地 调节核仁中的SUMO化，以及它如何调节核糖体的生物发生。

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.