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Mechanisms and biological functions of SPOUT methyltransferases

SPOUT甲基转移酶的机制和生物学功能

基本信息

批准号：
10736306
负责人：
Graeme L Conn
金额：
$ 31.01万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-14 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10736306
关键词：
Address Adopted Amino Acid Sequence Archaea Aspartic Acid-Specific tRNA Award Biochemical Biological Biological Assay Biological Models Biological Process Biology Cell physiology Cells Chemicals Complement Complex Cryoelectron Microscopy Defect Disease Endocrine Ensure Enzymatic Biochemistry Enzymes Eukaryota Exhibits Family Functional disorder Generations Genetic Genetic Code Genetic Models Health Homologous Gene Human Individual Investigation Knowledge Life Ligation Maintenance Methylation Methyltransferase Modeling Modification Molecular Mutation Neurologic Nucleic Acids Nucleotides Organism Orthologous Gene Pathway interactions Pattern Phenotype Positioning Attribute Process Production Protein Biosynthesis Proteins Purine Nucleotides Quality Control RNA RNA Biochemistry Ribosomes Role S-Adenosylhomocysteine S-Adenosylmethionine Saccharomyces cerevisiae Specificity Structure Substrate Specificity Syndrome System Time Tissues Transfer RNA Translations Yeasts Zebrafish analog base biochemical tools biological adaptation to stress cofactor dimer disease phenotype drug sensitivity fitness genetic approach human disease in vitro Assay in vivo insight interdisciplinary approach loss of function member monomer mutant new therapeutic target posttranscriptional tRNA Methyltransferases yeast genetics

项目摘要

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.

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)过程中编码成蛋白质序列。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家族酶特异性识别和作用于底物tRNA的机制，以及需要解决tRNA修饰对重要细胞过程的影响。这个项目将决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利用合成的天然甲基供体S-腺苷-L-蛋氨酸的类似物来唯一地鉴定 Trm10在维持高质量的tRNA池中。一种新的Trm10功能的脊椎动物模型将使以前关于Trm10的挑战性问题的调查成为可能，S在Trm10的生物学功能中的作用多细胞真核生物。这些研究将以三个相辅相成但独立的目标进行，它们将： 1)确定酵母和脊椎动物Trm10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)评估生物的分子基础唯一保守的脊椎动物m1A9基因修饰的意义利用新的脊椎动物模型研究trm10 功能，以及3)鉴定酵母和斑马鱼中针对G9修饰的tRNA特异性功能在两个模式物种中都有遗传方法。总的来说，拟议的研究将推动以下领域的发展酶学、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，对人类健康至关重要，然而，其分子机制和生物学功能尚不清楚。这些结果还将提供对多细胞真核生物中tRNA修饰的动态格局的新见解。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Tied up in knots: Untangling substrate recognition by the SPOUT methyltransferases.

DOI：
10.1016/j.jbc.2022.102393
发表时间：
2022-10
期刊：
JOURNAL OF BIOLOGICAL CHEMISTRY
影响因子：
4.8
作者：
Strassler, Sarah E.;Bowles, Isobel E.;Dey, Debayan;Jackman, Jane E.;Conn, Graeme L.
通讯作者：
Conn, Graeme L.

Insights into Catalytic and tRNA Recognition Mechanism of the Dual-Specific tRNA Methyltransferase from Thermococcus kodakarensis.

深入了解柯达热球菌双特异性 tRNA 甲基转移酶的催化和 tRNA 识别机制。