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

描述（由申请人提供）： 在本申请中，我们提出了一种新的技术，即RNA引导的RNA 2 '-O-甲基化，用于调节体内端粒酶活性。我们还建议了解2 '-O-甲基化作为一种自然发生的过程，是如何被细胞采用以在某些条件下触发生长停止的。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（根据三个具体目标，见下文），一旦完成，将显着推进我们对端粒酶调节，衰老和人类疾病的理解。具体目标1--靶向TLC 1保守假结区域的核苷酸利用盒C/D RNA指导的2 '-O-甲基化，我们人工靶向TLC 1保守假结区域内的六个位点，并鉴定了至少三个位点，其中修饰导致端粒酶活性改变（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，以覆盖这个重要的伪结区域的每个站点。将鉴定并选择（单独或组合地）2 '-O-甲基化影响端粒酶活性的任何位点用于进一步的端粒酶活性分析、端粒酶RNP组装和募集测定。我们的研究将使我们了解影响的基础，从而提供各种选择来影响端粒酶活性和衰老。具体目标2--通过无偏2 '-O-甲基化全面筛选TLC 1中重要的2'-OH基团鉴于TLC 1是一个长RNA分子，并且至少有几个重要的结构域，我们相信假结区域之外的一些核苷酸（及其2 '-OH基团）对功能也很重要。为了获得2 '-O-甲基化作用的完整谱，我们提出了在整个TLC 1序列中进行无偏全筛选。具体来说，我们将使用TLC 1 cDNA构建人工盒C/D引导RNA文库，将2 '-O-甲基化引导至TLC 1的每个核苷酸。将进行生长表型筛选试验，以鉴定2 '-O-甲基化对端粒酶活性产生负面影响的重要位点。我们的初步结果还表明，在进入稳定期时，TLC 1在几个位点被2 '-O-甲基化（~50%），包括修饰降低端粒酶活性的位点。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。具体来说，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指导RNA文库（如果涉及盒C/D RNP）进行筛选。鉴定负责诱导的2 '-O-甲基化的酶将最终导致这种修饰的机制的阐明，并因此将显着推进我们对端粒酶活性、衰老和疾病的调节的理解。

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.