Functional and Structural Analysis of Eukaryotic snoRNP Complexes Catalysing rRNA Ribose Methylation

在前rRNA转录本的生物合成和加工过程中，核糖核苷酸的转录后修饰发生在功能区，包括亚基间接口、解码和肽转移酶中心。在可能的修饰中，2(质数)-O-核糖甲基化被证明可以保护RNA免受核糖核溶解切割，稳定单碱基对，充当伴侣，并在高温下冲击折叠。RRNA甲基化对于前rRNA加工和核糖体组装都是必不可少的，完全抑制甲基化导致细胞死亡。在真核生物中，核糖甲基化是由Box C/D小核仁RNA-蛋白质复合体(SnoRNP)执行的，其中引导snoRNAs将修饰机制靶向修饰部位，因为它们与待修饰的RNA序列互补。除了rRNA，snoRNAs还可以引导少数mRNAs的核糖甲基化，产生有趣的结果。5-羟色胺2C受体mRNA中特定腺苷残基的核糖甲基化失败最近被认为与人类神经遗传学Prader-Willi综合征(PWS)有关。这表明，RNA编辑，特别是核糖甲基化，可能涉及比目前认识到的更多的疾病。此外，已发现RNA甲基化通过提供区分病毒和内源RNA的机制，在病毒复制和宿主免疫防御中发挥作用。真核snoRNP复合体的酶学、rRNA修饰的特殊功能和分子结构仍然是一个科学挑战。有关snoRNPs的最新信息来自对重组古菌sRNPs的研究。然而，真核snoRNPs在许多方面不同于古生物sRNPs，并表现出更高的复杂性和调节性。因此，古生菌rRNPs获得的许多知识不能直接转移到真核系统中。在这里，我们提出了一个竞争性的工作计划，旨在提供对真核生物中核糖甲基化的分子理解。在SSP1784中，我们的目标是确定真核生物snoRNPs的结构和酶机制，以及调控原则。我们将回答这样一个问题，即甲基化水平是否以及如何在不同的位点被调节，以及这一调控起到了什么功能作用。从方法学的角度来看，我们将使用一种跨学科的方法，包括分子工程，基于体内核糖甲基化的高效液相色谱/质谱仪定量，以及重组和天然分离的酵母snoRNPs的结构、生物物理和生化分析。除了对揭示细胞生命周期的基本原理的影响外，我们将在这个项目中赢得的知识将有助于理解核糖甲基化在疾病中的作用，并可能有助于发现涉及RNA甲基化缺陷的新的病理。

During the biosynthesis and processing of the pre-rRNA transcripts post-transcriptional modifications of ribonucleotides occur in functional regions, including intersubunit interfaces, decoding and peptidyltransferase centers. Among the possible modifications, 2(prime)-O-ribose methylation was shown to protect RNA from ribonucleolytic cleavage, stabilize single base pairs, serve as chaperone and impact folding at high temperatures. rRNA methylation is essential for both pre-rRNA processing and ribosome assembly, with complete suppression of methylation leading to cell death. In eukaryotes, ribose methylation is carried out by the Box C/D small nucleolar RNA-protein complex (snoRNP), where the guide snoRNAs target the modification machinery to the sites of modification, due to their complementarity to the RNA sequences to be modified. In addition to rRNA, snoRNAs can guide ribose-methylation of a few mRNAs with intriguing consequences. Failure to methylate the ribose of a specific adenosine residue within the serotonin 2C receptor mRNA has been recently linked to human neurogenetic Prader-Willi syndrome (PWS). This suggests that RNA editing, and in particular ribose methylation, might be involved in many more diseases than currently recognized. In addition, RNA methylation has been found to play a role in viral replication and in the host immunologic defense, by providing a mechanism to distinguish the viral from the endogenous RNA. The enzymology, the particular function of rRNA modifications, and the molecular structure of eukaryotic snoRNP complexes remain a scientific challenge. Most recent information on snoRNPs has been obtained from studies with reconstituted archaeal sRNPs. However, eukaryotic snoRNPs differ from archaeal sRNPs in many aspects and display a higher level of complexity and regulation. Thus, much of the knowledge achieved for archaeal sRNPs cannot be directly transferred to the eukaryotic system. Here we propose a competitive work plan that aims at providing a molecular understanding of ribose-methylation in eukaryotes. Within SSP1784 we aim at determining the structure and the enzymatic mechanism of eukaryotic snoRNPs, as well as the principles of regulation. We will answer the question about whether and how the levels of methylation are modulated at the different sites and which functional purpose this regulation serves. From the methodological point of view, we will use an interdisciplinary approach consisting of molecular engineering, HPLC/MS-based quantification of ribose methylation in vivo as well as structural, biophysical and biochemical analysis of reconstituted and natively isolated yeast snoRNPs. Besides the impact on revealing basic principles of the cell life-cycle, the knowledge that we will win in this project will help understanding the role of ribose methylation in disease and possibly contribute to the discovery of new pathologies where defects in RNA methylation are involved.