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 翻译是细胞将基因转化为蛋白质的整个过程中的关键步骤，而翻译中的缺陷 翻译与许多人类疾病有关。翻译的一个关键组成部分是转移RNA (tRNA)， 必须经过多种细胞酶的广泛化学修饰才能正常发挥作用。许多 这些酶及其形成的 tRNA 修饰在真核生物中是保守的，包括 将单细胞芽殖酵母转化为多细胞生物（例如人类），从而使酵母成为强大的模型 研究 tRNA 修饰作用的生物体。重要的是，tRNA 修饰缺陷会导致 智力障碍 (ID) 等多种神经系统疾病与许多其他疾病有关。这 NIH R15 AREA 提案旨在通过以下方式让本科生接触现代生物医学研究 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， 与甲基转移酶 Trm7 结合的蛋白质，在第 32 位形成高度保守的修饰 位于 tRNA 的关键反密码子环 (ACL) 区域。此外，我们将研究酵母Trm734，它 还与 Trm7 结合，在 ACL 中的 tRNA 位置 34 处形成保守修饰。这些方面的缺陷 人类 TRM7（称为 FTSJ1）突变引起的修饰会导致 ID。此外，人类TRM732 (THADA) 与 2 型糖尿病、肥胖和多囊卵巢综合征有关，而人类 TRM734 (WDR6) 也与人类健康有联系。因此，在提案的目标 1 中，学生将进行生化实验 确定 Trm732 和 Trm734 在 Trm7 修饰 tRNA 中的作用。在我们的上一轮融资中， 学生们鉴定出缺乏 tRNA 修饰活性的 Trm732 和 Trm734 变异蛋白，这将是 了解每种蛋白质在 tRNA 修饰中如何发挥作用的宝贵工具。其次，虽然 先前已研究过 32 和 34 处这些修饰的累积作用，但人们对这些修饰知之甚少。 每个修改本身的单独作用。因此，在目标 2 中，我们建议确定每个修改如何 单独影响酵母以及培养的人类和果蝇细胞中的翻译。第三，我们建议 鉴定形成酵母中未发现的人类 tRNA ACL 修饰所需的基因，并研究其在 翻译。在目标 3 中，我们将完成鉴定对 39 位进行修饰的酶的工作。 某些人类、动物和植物 tRNA。学生将继续测试候选基因的修饰 使用我们之前开发的方法来识别其他 tRNA 修饰基因的活性。一旦 基因被识别后，我们将使用报告基因检测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 AREA 奖的标准，因为它为本科生提供了 学生有机会充分参与研究，以增加对潜在分子的了解 疾病的原因。学生将接受遗传、分子生物学和生化技术方面的培训， 帮助培养新一代生物医学研究人员。