Mechanisms and Biological functions of SPOUT methyltransferases

SPOUT甲基转移酶的机制和生物学功能

• 批准号: 10218211
• 负责人: Graeme L Conn
• 金额: $ 27.21万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-14 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10218211
• 关键词: Address; Adenosine; Affect; Amino Acid Sequence; Anticodon; Archaea; Binding; Biochemical; Biological; Biological Assay; Biological Process; Biology; Biophysics; Catalysis; Cells; Chemicals; Chimera organism; Complement; Complex; Crystallization; Defect; Deuterium; Disease; Endocrine; Ensure; Enzymatic Biochemistry; Enzymes; Eukaryota; Exhibits; Family; Fluorouracil; Genetic; Genetic Code; Genetic Transcription; Guanosine; Health; Human; Human Biology; Hydrogen; Individual; Kinetics; Life; Link; Maintenance; Mass Spectrum Analysis; Methylation; Methyltransferase; Modeling; Modification; Molecular; Molecular Conformation; Mutation; Neurologic; Nucleic Acids; Nucleotides; Orthologous Gene; Pathway interactions; Phenotype; Play; Positioning Attribute; Process; Production; Protein Biosynthesis; Purine Nucleotides; RNA; RNA Biochemistry; RNA metabolism; Reaction; Ribosomes; Roentgen Rays; Role; S-Adenosylhomocysteine; S-Adenosylmethionine; Saccharomyces cerevisiae; Structure; Substrate Specificity; Surface; Syndrome; Synthesis Chemistry; Transfer RNA; Translations; Variant; Vertebrates; Yeasts; Zebrafish; analog; base; biological adaptation to stress; dimer; disease phenotype; drug sensitivity; flexibility; human disease; in vitro activity; in vivo; insight; interdisciplinary approach; new therapeutic target; novel; paralogous gene; structural biology; tRNA Methyltransferases

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.

项目概要/摘要 转移 RNA (tRNA) 是转换基于核酸的遗传物质所必需的通用接头分子。 在核糖体合成（翻译）蛋白质过程中编码蛋白质序列。这个过程是通用的 对所有生命来说都是保守的和基础的，因此，翻译分子参与者的缺陷，包括 tRNA 会导致多种人类疾病。特定的化学修饰（例如甲基化）常见于 tRNA，但详细了解整合它们的酶及其对 tRNA 功能的贡献 （以及疾病中的功能障碍）直到最近才出现了一些选定的例子。自从发现 酿酒酵母中的 tRNA 甲基转移酶 (Trm10)，证据不断积累，包括 酵母表型和与人类突变相关的多症状疾病，已经建立了 Trm10 在 tRNA 生物学中发挥着重要作用。为了更好地理解 Trm10 修饰的影响， 需要解决 Trm10 识别 tRNA 并作用于 tRNA 的机制。该项目旨在确定 使用多学科方法研究 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复合物的结构分析相结合， 以及天然甲基供体 S-腺苷-L-甲硫氨酸的合成类似物，以独特地识别 Trm10 的作用 维持高质量的 tRNA 库。这些研究将在三个互补但 独立目标将： 1) 利用生物物理学确定 Trm10 甲基化的分子机制 以及基于新机制（SAM 模拟）的 X 射线晶体结构分析 捕获酶-tRNA 复合物，并辅以 Trm10 变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）识别 通过详细的研究，了解酵母和人类 Trm10 直系同源物 tRNA 底物选择性的分子基础 考虑 tRNA 结构和稳定性； 3) 评估 m1G9 在酵母 Trm10 靶 tRNA 中的作用 和斑马鱼脊椎动物模型。总的来说，拟议的研究将推动酶学领域的发展， RNA 生物化学和 tRNA 生物学，提供对 tRNA 修饰的机制和生物学见解 这种酶在真核生物中普遍保守，对人类生物学至关重要，但其 分子机制和生物学功能根本不被了解。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。