Regulation of telomerase activity and aging by 2'-O-methylation in telomerase RNA

端粒酶RNA中2-O-甲基化对端粒酶活性和衰老的调节

• 批准号: 8242209
• 负责人: YI-TAO YU
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8242209
• 关键词: Adopted; Affect; Aging; Binding; Binding Sites; Biological Assay; Boxing; Cells; Complementary DNA; Digestion; Disease; Enzymes; Eukaryotic Cell; Goals; Growth; Guide RNA; In Vitro; Intention; Investigation; Ku Protein; Lead; Libraries; Methylation; Modification; Nuclease P1; Nucleotides; Open Reading Frames; Phase; Phenotype; Process; Protein Binding; Proteins; RNA; RNA library; RNA-Directed DNA Polymerase; Regulation; Research; Screening procedure; Series; Site; Site-Directed Mutagenesis; Small Nuclear RNA; Structure; Techniques; Telomerase; Telomerase RNA Component; Testing; Thin Layer Chromatography; Work; Yeasts; base; cell growth; human disease; in vivo; novel; novel strategies; research study

DESCRIPTION (provided by applicant): In this application, we propose to develop a novel technique, namely, RNA-guided RNA 2'-O-methylation, for regulating telomerase activity in vivo. We also propose to understand how 2'-O-methylation, as a naturally occurring process, is adopted by cells to trigger the cessation of growth under certain conditions. Given the close relationship between telomerase activity and aging, we believe that our proposed work (under three specific aims, see below), once accomplished, will significantly advance our understanding of telomerase regulation, aging, and human diseases. Specific Aim 1--Targeting of nucleotides in the conserved pseudoknot region of TLC1 Using box C/D RNA-guided 2'-O-methylation, we have artificially targeted TLC1 at six sites within the conserved pseudoknot region, and identified at least three sites where modification led to altered telomerase activity (two led to enhancement and one to reduction). Building on these results, we plan to expand our modification targets to cover each and every site in this important pseudoknot region. Any sites at which 2'-O- methylation affects telomerase activity will be identified and selected (individully or in combination) for further telomerase activity analyses, telomerase RNP assembly and recruitment assays. Our studies will allow us to understand the basis of effects, thus offering a variety of choices to influence telomerase activity and aging. Specific Aim 2--Global screening of important 2'-OH groups in TLC1 via unbiased 2'-O-methylation Given that TLC1 is a long RNA molecule and has at least several important domains, we believe that some nucleotides (and their 2'-OH groups) outside of the pseudoknot region are important for function as well. To obtain a complete spectrum of the effects of 2'-O-methylation, we propose an unbiased full screen throughout the TLC1 sequence. Specifically, we will use TLC1 cDNA to construct an artificial box C/D guide RNA library, directing 2'-O-methylation to each and every nucleotide of TLC1. A growth phenotype screen assay will be carried out to identify important sites where 2'-O-methylation negatively impacts telomerase activity. Specific Aim 3--Identification of enzyme responsible for induced TLC1 2'-O-methylation Our preliminary results also showed that upon entry into the stationary phase, TLC1 was 2'-O-methylated (~50%) at several sites, including a site where modification reduces telomerase activity. This remarkable result prompted us to explore further the mechanism of this inducible 2'-O-methylation. Specifically, we propose to identify the enzyme responsible for this induced modification. To achieve this goal, we plan to conduct a series of experiments, including screening using yeast ORF libraries (if the activity is orchestrated by protein only) or a yeast box C/D guide RNA library (if a box C/D RNP is involved). Identification of the enzyme responsible for induced 2'-O-methylation will eventually lead to the elucidation of the mechanism of this modification, and will thus significantly advance our understanding of the regulation of telomerase activity, aging and diseases. PUBLIC HEALTH RELEVANCE: Relevance In this application, we propose to develop a novel approach, namely, RNA-guided RNA 2'-O-methylation, for regulating telomerase activity and aging, and to investigate naturally-occurring 2'-O-methylation that we have recently discovered in telomerase RNA. We believe that our proposed work will greatly enhance our understanding of how telomerase activity can be regulated in the cell, both artificially and naturally.

描述(由申请人提供)： 在这一应用中，我们建议开发一种新的技术，即RNA引导的RNA 2‘-O-甲基化，用于调节体内的端粒酶活性。我们还建议理解2‘-O-甲基化，作为一个自然发生的过程，是如何在特定条件下被细胞采用来触发生长停止的。鉴于端粒酶活性与衰老之间的密切关系，我们相信，我们提出的工作(在三个具体目标下，见下文)，一旦完成，将极大地促进我们对端粒酶调节、衰老和人类疾病的理解。具体目标1-利用box C/D RNA引导的2‘-O-甲基化靶向TLC1保守伪结区的核苷酸，我们人工将TLC1靶向保守伪结区内的六个位置，并确定了至少三个修饰导致端粒酶活性改变的位置(两个导致增强，一个导致降低)。在这些结果的基础上，我们计划扩大我们的修改目标，以涵盖这一重要伪结区的每一个地点。任何2‘-O-甲基化影响端粒酶活性的位点都将被识别和选择(单独或组合)，用于进一步的端粒酶活性分析、端粒酶RNP组装和招募分析。我们的研究将使我们了解影响端粒酶活性和衰老的基础，从而提供多种选择。特定目的2--通过无偏2‘-O-甲基化全面筛选TLC1中重要的2’-OH基团鉴于TLC1是一个长RNA分子，至少有几个重要的结构域，我们认为假结区域外的一些核苷酸(及其2‘-OH基团)对功能也很重要。为了获得2‘-O-甲基化影响的完整光谱，我们建议在整个TLC1序列中进行无偏全屏显示。具体地说，我们将使用TLC1cDNA构建一个人工框C/D引导RNA文库，将2‘-O-甲基化定向到TLC1的每个核苷酸上。将进行生长表型筛选试验，以确定2‘-O-甲基化对端粒酶活性产生负面影响的重要位置。特异性目标3--TLC1 2‘-O-甲基化酶的鉴定我们的初步结果还表明，进入固定相后，TLC1在几个位点发生了2’-O-甲基化(~50%)，包括一个修饰降低了端粒酶活性的位点。这一显著的结果促使我们进一步探索这种可诱导的2‘-O-甲基化的机制。具体地说，我们建议确定负责这种诱导修饰的酶。为了实现这一目标，我们计划进行一系列实验，包括使用酵母ORF文库(如果活性仅由蛋白质协调)或酵母盒C/D引导RNA文库(如果涉及盒C/D RNP)。鉴定诱导2‘-O-甲基化的酶最终将有助于阐明这种修饰的机制，从而极大地促进我们对端粒酶活性调节、衰老和疾病的理解。 公共卫生相关性： 在这一应用中，我们建议开发一种新的方法，即RNA引导的RNA2‘-O-甲基化，用于调节端粒酶活性和衰老，并研究我们最近在端粒酶RNA中发现的自然发生的2’-O-甲基化。我们相信，我们提出的工作将极大地增强我们对细胞中端粒酶活性如何调节的理解，无论是人工调节还是自然调节。