Mechanisms and Biological functions of SPOUT methyltransferases

项目摘要/摘要 转移RNAs(TRNAs)是转化以核酸为基础的基因所必需的通用接头分子 核糖体在蛋白质合成(翻译)过程中对蛋白质序列的编码。这个过程是普遍存在的 对所有生命来说都是保守的和基本的，因此，翻译的分子参与者中的缺陷，包括 TRNA，会导致多种人类疾病。甲基化等特殊的化学修饰在 TRNA，但对结合它们的酶及其对tRNA功能的贡献有详细的了解 (和疾病中的功能障碍)只是最近才出现的几个精选例子。自从发现了 酿酒酵母中的tRNA甲基转移酶(Trm10)，这是一个积累的证据，包括 酵母的表型和一种与人类突变相关的多症状疾病，已经建立了一个 Trm10在tRNA生物学中的重要作用。为了更好地理解Trm10修改的含义， Trm10识别和作用于tRNA的机制需要解决。该项目旨在确定 用多学科方法研究Trm10机制和功能的分子基础。遗传、生化 分子酶学方法将与酶-tRNA复合体的结构分析相结合， 和天然甲基供体S-腺苷-L-蛋氨酸的合成类似物，以唯一确定Trm10的作用 以维持高质量的tRNA池。这些研究将在三个相辅相成的 独立的目标将：1)利用生物物理来确定Trm10甲基化的分子机制 和X射线晶体结构分析，通过一种基于新机制(SAM模拟)的方法来实现 TRAP酶-tRNA复合体，并辅以Trm10变异体的生化分析和研究 确定Trm10酶的替代底物、细胞定位和天然修饰状态；2)确定 酵母和人Trm10同源基因tRNA底物选择性的分子基础 考虑tRNA的结构和稳定性；以及，3)评估m1G9在酵母中Trm10靶tRNA中的作用 以及斑马鱼脊椎动物模型。总的来说，拟议的研究将推动酶学领域的发展， 通过提供对tRNA修饰的机制和生物学见解来实现RNA生物化学和tRNA生物学 一种在真核生物中普遍保守并对人类生物学至关重要的酶，但其 其分子机制和生物学功能目前尚不清楚。这些结果还将提供新的 洞察多细胞真核生物中tRNA修饰的动态图景。

PROJECT SUMMARY/ ABSTRACT Transfer RNAs (tRNAs) are the universal adaptor molecules necessary to convert the nucleic acid-based genetic code to protein sequence during protein synthesis (translation) by the ribosome. This process is universally conserved and fundamental to all life, and, as such, defects in the molecular players of translation, including tRNAs, result in diverse human diseases. Specific chemical modifications such as methylation are common in tRNA, but a detailed understanding of the enzymes that incorporate them and their contributions to tRNA function (and disfunction in disease) have only recently emerged for a few select examples. Since the discovery of the tRNA methyltransferase (Trm10) in Saccharomyces cerevisiae, an accumulating body of evidence, including phenotypes in yeast and a multisymptomatic disease associated with human mutations, has established a significant role for Trm10 in tRNA biology. To better understand the implications of Trm10 modification, the mechanism by which Trm10 recognizes and acts on tRNA needs to be addressed. This project aims to determine the molecular basis for Trm10 mechanism and function using a multi-disciplinary approach. Genetic, biochemical and molecular enzymology approaches will be combined with structural analyses of enzyme-tRNA complexes, and synthetic analogs of the native methyl donor, S-adenosyl-L-methionine, to uniquely identify the role of Trm10 in the maintenance of a high quality pool of tRNA. The studies will be performed in three complementary but independent aims that will: 1) Determine the molecular mechanism of methylation by Trm10, using biophysical and x-ray crystallographic structural analysis enabled by a novel mechanism (SAM analog)-based approach to trap enzyme-tRNA complexes, and complemented by biochemical analyses of Trm10 variants and studies to identify alternative substrates for Trm10 enzymes, cellular localization and native modification status; 2) Identify the molecular basis for tRNA substrate-selectivity of yeast and human Trm10 orthologs through detailed consideration of tRNA structure and stability; and, 3) Assess the roles of m1G9 in Trm10 target tRNAs in yeast and the zebrafish vertebrate model. Collectively, the proposed studies will advance the fields of enzymology, RNA biochemistry and tRNA biology by providing mechanistic and biological insight into a tRNA modification enzyme that is universally conserved among eukaryotes and critically important for human biology, yet whose molecular mechanism and biological functions are not at all understood. These results will also provide new insight into the dynamic landscape of tRNA modifications in multicellular eukaryotes.