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.

项目摘要/摘要 转移RNA（TRNA）是转化基于核酸的遗传所需的通用衔接子分子 核糖体蛋白质合成期间的蛋白质序列代码。这个过程是普遍的 对所有生活的保守和基础 TRNA，导致各种人类疾病。特定的化学修饰（例如甲基化）在 tRNA，但对将其纳入酶及其对tRNA功能的贡献的酶有详细的理解 （疾病中的失功）直到最近才出现了一些精选的例子。由于发现 酿酒酵母中的tRNA甲基转移酶（TRM10），一种积累的证据，包括 酵母中的表型和与人类突变相关的多肿瘤疾病已经建立了 TRM10在TRNA生物学中的重要作用。为了更好地了解TRM10修改的含义 需要解决TRM10识别和作用在tRNA上的机制。该项目旨在确定 使用多学科方法的TRM10机制和功能的分子基础。遗传，生化 和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，以唯一识别TRM10的作用 在维持高质量的tRNA中。这些研究将以三个互补性进行，但 将使用生物物理的独立目标：1）确定TRM10的甲基化分子机制 和X射线晶体学结构分析通过新机制（SAM类似）的方法来实现 陷阱酶-TRNA复合物，并通过TRM10变体的生化分析和研究补充 确定TRM10酶，细胞定位和天然修饰状态的替代底物； 2）识别 通过详细的 考虑tRNA结构和稳定性； 3）评估M1G9在TRM10靶标在酵母中的作用 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生物学通过提供机械和生物学洞察力来修饰tRNA 在真核生物中普遍保守的酶，对人类生物学至关重要，却至关重要 分子机制和生物学功能根本不了解。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。