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）蛋白质的过程中编码成蛋白质序列。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家族酶特异性识别并作用于其底物tRNA的机制， tRNA修饰对重要细胞过程的影响需要解决。该项目将决定 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 使用天然甲基供体S-腺苷-L-甲硫氨酸的合成类似物，以独特地鉴定 Trm 10在维持高质量的tRNA库中。一种新的脊椎动物Trm 10功能模型 这将使以前具有挑战性的问题的调查Trm 10的生物功能的作用， 多细胞真核生物这些研究将在三个互补但独立的目标下进行，这些目标将： 1)确定酵母和脊椎动物Trm 10酶如何选择特定的tRNA底物进行修饰 使用结构、生物化学和遗传学方法; 2）评估生物学和生物学的分子基础 独特保守的脊椎动物m1 A9修饰的重要性，开发了Trm 10的新脊椎动物模型 功能，以及3）使用互补的方法鉴定酵母和斑马鱼中G9修饰的tRNA特异性功能。 两种模式物种的遗传学方法。总的来说，拟议的研究将推进以下领域： 酶学、RNA生物化学和tRNA生物学，通过提供对tRNA的机制和生物学见解， 修饰酶在真核生物中普遍保守并且对人类健康至关重要， 但其分子机制和生物学功能还完全不清楚。这些结果还将提供 对多细胞真核生物中tRNA修饰动态景观的新见解。

PROJECT SUMMARY/ ABSTRACT Transfer RNAs (tRNAs) are the universal adaptor molecules necessary to convert the nucleic acid-based genetic code into 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 mechanisms by which Trm10 family enzymes specifically recognize and act on their substrate tRNA, and the impact of tRNA modifications on important cellular processes need to be addressed. This project will 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 using 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. A newly developed vertebrate model for Trm10 function will enable investigation of previously challenging questions on Trm10's role in the biological function of multicellular eukaryotes. The studies will be performed in three complementary but independent aims that will: 1) Determine how specific tRNA substrates are selected for modification by yeast and vertebrate Trm10 enzymes using structural, biochemical and genetic approaches; 2) Assess the molecular basis for and biological significance of the uniquely conserved vertebrate m1A9 modification exploiting a new vertebrate model for Trm10 function, and 3) Identify tRNA-specific functions for G9 modification in yeast and zebrafish using complementary genetic approaches in both model species. 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 is critically important for human health, 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.