Mechanisms and Biological functions of SPOUT methyltransferases

项目总结/摘要 转移RNA（transfer RNA，tRNA）是将基于核酸的遗传物质转化为RNA所必需的通用接头分子。 在核糖体合成（翻译）蛋白质的过程中，将编码转化为蛋白质序列。这个过程是普遍的。 保守的和基本的所有生命，并因此，缺陷的分子球员的翻译，包括 tRNA导致多种人类疾病。特定的化学修饰，如甲基化， tRNA，而是对整合它们的酶及其对tRNA功能的贡献的详细了解 (and疾病中的功能障碍）只是最近才出现的几个选择的例子。自从发现了 酿酒酵母中的tRNA甲基转移酶（Trm 10），一个不断积累的证据，包括 酵母的表型和与人类突变相关的多症状疾病，已经建立了一个 Trm 10在tRNA生物学中的重要作用。为了更好地理解Trm 10修改的含义， Trm 10识别和作用于tRNA的机制需要解决。该项目旨在确定 Trm 10机制和功能的分子基础，使用多学科的方法。遗传的，生化的 分子酶学方法将与酶-tRNA复合物的结构分析相结合， 和天然甲基供体S-腺苷-L-甲硫氨酸的合成类似物，以独特地鉴定Trm 10的作用 维持高质量的tRNA库。这些研究将在三个互补的，但 独立的目标，将：1）确定甲基化的分子机制Trm 10，使用生物物理 和X射线晶体学结构分析，通过基于新机制（SAM类似物）的方法实现， 捕获酶-tRNA复合物，并辅以Trm 10变体的生化分析和研究， 鉴定Trm 10酶的替代底物、细胞定位和天然修饰状态; 2）鉴定 酵母和人Trm 10直系同源物的tRNA底物选择性的分子基础，通过详细的 考虑tRNA结构和稳定性; 3）评估m1 G9在酵母中Trm 10靶tRNA中的作用 和斑马鱼脊椎动物模型。总的来说，拟议的研究将推进酶学领域， RNA生物化学和tRNA生物学，通过提供对tRNA修饰的机制和生物学见解 一种在真核生物中普遍保守的酶，对人类生物学至关重要，但其 分子机制和生物学功能完全不清楚。这些结果也将提供新的 深入了解多细胞真核生物中tRNA修饰的动态景观。

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.