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

摘要 翻译是细胞将基因转化为蛋白质的整个过程中的关键一步，而基因缺陷 翻译与许多人类疾病有关。翻译的一个关键成分是转移RNA(TRNA)， 它必须被许多细胞酶进行广泛的化学修饰才能正常发挥作用。许多. 这些酶和它们形成的tRNA修饰在真核生物中是保守的，从 单细胞发芽酵母到多细胞生物体，如人类，从而使酵母成为一个强大的模型 研究tRNA修饰作用的有机体。重要的是，tRNA修饰中的缺陷会导致 各种神经疾病，如智力残疾(ID)，并与许多其他疾病有关。这 NIH R15区域提案旨在通过以下方式让本科生接触现代生物医学研究 从三个方面推进tRNA修饰研究领域。首先，我们建议研究酵母Trm732，一种 与甲基转移酶Trm7结合在第32位形成高度保守修饰的蛋白质 位于tRNA的关键反密码子环(ACL)区域。此外，我们还将研究酵母Trm734，它 还与Trm7结合，在ACL中的tRNA位置34形成保守的修饰。这些产品中的缺陷 人类TRM7(称为FTSJ1)的突变导致的修改会导致ID。此外，人类TRM732 (Thada)与2型糖尿病、肥胖和多囊卵巢综合征有关，人类TRM734(WDR6) 也与人类健康有关。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， 学生们发现了缺乏tRNA修饰活性的Trm732和Trm734变异蛋白，这将是 了解每种蛋白质如何在tRNA修饰中发挥作用的宝贵工具。第二，虽然 这些修饰在32和34处的累积作用以前已经被研究过，但对 每个修改本身的单独角色。因此，在目标2中，我们建议确定每项修改如何 分别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 鉴定酵母中未发现的形成人tRNA ACL修饰所需的基因，并研究其在 翻译。在目标3中，我们将完成我们的工作，以确定将修饰添加到第39位的酶 某些人类、动物和植物的tRNA。学生们将继续测试用于修饰的候选基因 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 基因被鉴定后，我们将使用记者分析来确定这种修饰在翻译中的作用。这个 这里提出的研究符合NIH R15区域奖的标准，因为它给本科生 学生有机会充分参与研究，以增加对潜在分子的知识 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。

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.