Determining how 2'-O-methylations in the eukaryotic anticodon loop region of tRNA are formed and how they affect translation

确定 tRNA 真核反密码子环区域中 2-O-甲基化的形成方式以及它们如何影响翻译

• 批准号: 10438971
• 负责人: Michael P. Guy
• 金额: $ 41.65万
• 依托单位: NORTHERN KENTUCKY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10438971
• 关键词: Affect; Amino Acids; Animal Model; Animals; Anticodon; Award; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biomedical Research; Candidate Disease Gene; Cell physiology; Cells; Chemicals; Codon Nucleotides; Defect; Disease; Drosophila genus; Enzymes; Eukaryota; Exposure to; Fission Yeast; Funding; Gene-Modified; Generations; Genes; Genetic; Grant; Growth; Health; Homologous Gene; Human; Individual; Institution; Intellectual functioning disability; Kentucky; Knowledge; Link; Malignant Neoplasms; Mammalian Cell; Methylation; Methyltransferase; Modernization; Modification; Molecular; Molecular Biology; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organism; Phenotype; Plants; Polycystic Ovary Syndrome; Positioning Attribute; Process; Proteins; RNA; RNA Binding; RNA-Binding Proteins; Reporter; Research; Research Personnel; Research Project Grants; Role; Saccharomyces cerevisiae; Saccharomycetales; Science; Sequence Alignment; Site-Directed Mutagenesis; Stress; Students; Techniques; Testing; Training; Transfer RNA; Translations; United States National Institutes of Health; Universities; Variant; Work; X-linked mental retardation 9; Yeasts; career; cell growth; experimental study; human disease; insight; knock-down; mutant; nervous system disorder; overexpression; protein function; protein protein interaction; tool; undergraduate student

ABSTRACT Translation is a key step in the overall process that cells use to convert genes into proteins, and defects in translation are linked to many human diseases. A critical component of translation is transfer RNA (tRNA), which must be extensively chemically modified by numerous cellular enzymes to function properly. Many of these enzymes and the tRNA modifications that they form are conserved in eukaryotic organisms ranging from single-celled budding yeast to multi-cellular organisms such as humans, thus making yeast a powerful model organism for studying the roles of tRNA modifications. Importantly, defects in tRNA modifications cause diverse neurological disorders such as intellectual disability (ID), and are linked to many other diseases. This NIH R15 AREA proposal seeks to expose undergraduate students to modern biomedical research by advancing the field of tRNA modification research in three ways. First, we propose to study yeast Trm732, a protein which binds to the methyltransferase Trm7 to form a highly conserved modification at position 32 located in the critical anticodon loop (ACL) region of the tRNA. In addition, we will study yeast Trm734, which also binds to Trm7 to form a conserved modification at tRNA position 34 in the ACL. Defects in these modifications due to mutations in human TRM7 (known as FTSJ1) cause ID. Moreover, human TRM732 (THADA) is linked to type 2 diabetes, obesity, and polycystic ovary syndrome, and human TRM734 (WDR6) also has links to human health. Thus, in Aim 1 of the proposal, students will perform biochemical experiments to determine the roles of Trm732 and Trm734 in tRNA modification by Trm7. In our previous round of funding, students identified Trm732 and Trm734 variant proteins lacking tRNA modification activity, which will be valuable tools to understand how each protein functions in tRNA modification. Second, although the cumulative role of these modifications at 32 and 34 have been studied previously, little is known about the individual role of each modification by itself. Thus, in Aim 2 we propose to determine how each modification individually affects translation in yeast, as well as in cultured human and fruit fly cells. Third, we propose to identify the gene required to form a human tRNA ACL modification not found in yeast, and to study its role in translation. In Aim 3 we will finish our work to identify the enzyme that adds a modification to position 39 of certain human, animal, and plant tRNAs. Students will continue to test candidate genes for the modification activity using approaches that we developed previously to identify other tRNA modification genes. Once the gene is identified, we will use reporter assays to determine the role of this modification in translation. The research proposed herein meets the criteria of the NIH R15 AREA award because it gives undergraduate students the opportunity to fully participate in research that will increase knowledge of the underlying molecular causes of disease. Students will be trained in genetic, molecular biological, and biochemical techniques, helping to prepare a new generation of biomedical researchers.

抽象的 翻译是细胞用于将基因转化为蛋白质的整体过程的关键步骤，并在 翻译与许多人类疾病有关。翻译的关键成分是转移RNA（tRNA）， 必须通过大量细胞酶进行广泛的化学修饰才能正常运行。许多 这些酶及其形成的tRNA修饰是在真核生物中保守的 单细胞发芽的酵母菌对人类等多细胞生物，从而使酵母成为强大的模型 研究tRNA修饰的作用的生物体。重要的是，tRNA修饰的缺陷原因 多种神经系统疾病，例如智力障碍（ID），与许多其他疾病有关。这 NIH R15地区提案旨在使本科生接受现代生物医学研究 通过三种方式推进tRNA修饰研究领域。首先，我们建议研究酵母TRM732，a 与甲基转移酶TRM7结合的蛋白质在位置32处形成高度保守的修饰 位于tRNA的临界反密码子环（ACL）区域。此外，我们将学习酵母TRM734，这是 还与TRM7结合以在ACL中的tRNA位置34处形成保守的修饰。这些缺陷 由于人类TRM7（称为FTSJ1）中突变引起的修改会导致ID。此外，人类TRM732 （THADA）与2型糖尿病，肥胖和多囊卵巢综合征以及人类TRM734（WDR6）有关 还与人类健康有联系。因此，在提案的目标1中，学生将进行生化实验 确定TRM7在TRNA修饰中TRM732和TRM734的作用。在我们上一轮的资金中， 学生确定了缺乏TRNA修饰活动的TRM732和TRM734变体蛋白，这将是 了解每种蛋白质如何在tRNA修饰中起作用的宝贵工具。第二，虽然 以前已经研究了这些修饰在32和34时的累积作用，对此知之甚少 每次修改的个人角色本身。因此，在AIM 2中，我们建议确定每个修改的方式 单独影响酵母以及培养的人和果蝇细胞中的翻译。第三，我们建议 确定形成酵母中未发现的人tRNA ACL修饰所需的基因，并研究其在 翻译。在AIM 3中，我们将完成我们的工作，以确定为位置39添加修改的酶 某些人，动物和植物trnas。学生将继续测试候选基因进行修改 使用我们先前开发的方法来鉴定其他tRNA修饰基因的活性。一旦 鉴定了基因，我们将使用记者测定法来确定这种修饰在翻译中的作用。这 本文提出的研究符合NIH R15地区奖的标准，因为它给予了本科生 学生有机会充分参与研究，以增加对基础分子的了解 疾病的原因。学生将接受遗传，分子生物学和生化技术的培训， 帮助准备新一代的生物医学研究人员。