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: 8516934
• 负责人: YI-TAO YU
• 金额: $ 21.9万
• 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 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8516934
• 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; 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; screening

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.

描述（由申请人提供）： 在此应用中，我们建议开发一种新型技术，即RNA引导的RNA 2'-O-甲基化，以调节体内端粒酶活性。我们还建议了解2'-O-甲基化是一种天然发生的过程，细胞如何在某些条件下触发生长的停止。鉴于端粒酶活动与衰老之间的密切关系，我们认为我们提出的工作（在三个特定目标下，见下文）一旦完成，将大大提高我们对端粒酶调节，衰老和人类疾病的理解。具体目标1-使用Box C/D RNA引导的2'-O-甲基化在保守的TLC1中的核苷酸靶向，我们在保守的Pseudoknot区域内的六个地点进行了人为靶向的TLC1，并至少确定了三个修改的位置，以更改远程望远镜的活性，以增强远程望远镜的活性，以增强远程触发的活性，以增强远程化的活性。在这些结果的基础上，我们计划扩展我们的修改目标，以涵盖这个重要的伪诺区中的每个站点。将确定并选择2'-O-甲基化影响端粒酶活性的任何位点（个体或组合），以进行进一步的端粒酶活性分析，端粒酶RNP组装和募集分析。我们的研究将使我们能够理解效果的基础，从而提供各种选择以影响端粒酶活性和衰老。特定的目标2 - 通过公正的2'-O-甲基化对TLC1中重要的2'-OH基团的全球筛选，因为TLC1是一个长的RNA分子，并且至少具有几个重要域，我们认为某些核苷酸（及其2'-oh组）在PSEUDOKNOT区域以外的PSEUDOKNOT区域很重要。为了获得2'-O-甲基化效应的完整谱，我们在整个TLC1序列中提出了一个无偏的全屏。具体而言，我们将使用TLC1 cDNA构建一个人造盒子C/D引导RNA库，将2'-O-甲基化引导到TLC1的每个核苷酸。将进行生长表型筛选分析，以确定2'-O-甲基化对端粒酶活性产生负面影响的重要部位。具体目的3-识别负责诱导TLC1 2'-O-甲基化的酶，我们的初步结果还表明，在进入固定相时，TLC1在几个地点被2'-O-甲基化（〜50％），包括修饰的位点减少端粒酶活性。这一出色的结果促使我们进一步探索了这种诱导2'-O-甲基化的机制。具体而言，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文库进行筛查（如果活动仅由蛋白质策划）或酵母盒C/D指南RNA库（如果涉及盒子C/D RNP）。鉴定负责诱导2'-O-甲基化的酶最终将导致这种修饰机理的阐明，因此将显着提高我们对端粒酶活性，衰老和疾病的调节的理解。

项目成果

Unusual base pairing during the decoding of a stop codon by the ribosome.

• DOI: 10.1038/nature12302
• 发表时间: 2013-08-01
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Fernandez, Israel S.;Chyan Leong Ng;Kelley, Ann C.;Wu, Guowei;Yu, Yi-Tao;Ramakrishnan, V.
• 通讯作者: Ramakrishnan, V.

Pseudouridine in mRNA: Incorporation, Detection, and Recoding.

• DOI: 10.1016/bs.mie.2015.03.009
• 发表时间: 2015
• 期刊: Methods in enzymology
• 作者: Wu G;Huang C;Yu YT
• 通讯作者: Yu YT

Inducing nonsense suppression by targeted pseudouridylation.

• DOI: 10.1038/nprot.2012.029
• 发表时间: 2012-03-29
• 期刊: Nature protocols
• 影响因子: 14.8

Insight into the mechanisms and functions of spliceosomal snRNA pseudouridylation.

• DOI: 10.4331/wjbc.v5.i4.398
• 发表时间: 2014-11-26
• 期刊: World journal of biological chemistry
• 作者: Adachi, Hironori;Yu, Yi-Tao
• 通讯作者: Yu, Yi-Tao

Structural insights into Gemin5-guided selection of pre-snRNAs for snRNP assembly.

Gemin5 指导选择用于 snRNP 组装的 pre-snRNA 的结构见解。

• DOI: 10.1101/gad.288340.116
• 发表时间: 2016-11-01
• 期刊: Genes & development
• 影响因子: 10.5
• 作者: Xu C;Ishikawa H;Izumikawa K;Li L;He H;Nobe Y;Yamauchi Y;Shahjee HM;Wu XH;Yu YT;Isobe T;Takahashi N;Min J
• 通讯作者: Min J