Telomere maintenance by the telomerase RNA-protein complex

端粒酶 RNA-蛋白质复合物维持端粒

• 批准号: 10090608
• 负责人: David Clifford Zappulla
• 金额: $ 32.9万
• 依托单位: LEHIGH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10090608
• 关键词: Acute; Address; Age; Aging; Animal Model; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Cell Proliferation; Cell Survival; Cell division; Cells; Cessation of life; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; Development; Disease; Distal; Enzymes; Genetic; Genome; Genomic Instability; Goals; Health; Homeostasis; Human; Human Chromosomes; In Vitro; Individual; Lead; Learning; Length; Link; Malignant Neoplasms; Mediating; Mission; Molecular; Multienzyme Complexes; Mutation; Nucleotides; Organ failure; Pathway interactions; Premature aging syndrome; Process; Proteins; RNA; RNA-Directed DNA Polymerase; Recruitment Activity; Regulation; Research; Resolution; Ribonucleoproteins; Role; Speed; Structure; System; Telomerase; Telomerase RNA Component; Telomere Maintenance; Testing; Two-Hybrid System Techniques; United States National Institutes of Health; Work; Yeast Model System; Yeasts; base; cell growth; deep sequencing; experimental study; human tissue; improved; insight; molecular targeted therapies; mutant; novel; prevent; protein complex; recruit; senescence; telomere; therapy development; yeast two hybrid system

PROJECT SUMMARY Without a dedicated mechanism to replicate their ends, human chromosomes shorten with each cell division, leading to senescence and death. A specialized RNA-protein enzyme complex, telomerase, counteracts this end-replication problem, lengthening telomeres to complete genome duplication. Telomerase becomes dormant during development of human tissues, leading to chromosome shortening with age. However, it reemerges in most human cancers, sustaining cell proliferation potential. Basic information is needed about how telomerase operates to maintain telomeres in order to develop therapies that control these process. Studies indicate that telomere length homeostasis is achieved by telomerase being preferentially recruited to the shortest telomeres, yet the system that controls this is not understood. In yeast, two pathways have been identified for telomerase recruitment, based on its Ku and Est1 subunits. This project seeks to identify how telomerase is selectively recruited to short telomeres to lengthen them. The research evaluates the hypotheses that (1) the Ku and Est1 telomerase-recruiting pathways operate in a concerted, stepwise manner and (2) that the telomeric Rif proteins regulate telomere length by antagonizing each recruitment step by specific independent mechanisms that weaken as telomere length shortens. Aim 1 will first use a new single-telomere system to define the length of a “short” telomere and each telomeric component functions to stimulate or suppress telomerase recruitment and action. The second goal of Aim 1 is to pinpoint where telomeric proteins and telomerase associate with DNA along the distal portions of chromosomes, using a deep-sequencing- based assay that has near single-nucleotide resolution. This will advance our understanding of telomere structure and telomerase regulation by revealing the arrangement of telomeric proteins along telomeric repeats and identifying where telomerase is recruited relative to chromosome ends. Aim 2 will determine how close to an individual chromosome end telomerase needs to be recruited to extend it and also test if the Ku pathway relies on Est1 to promote telomerase recruitment and activity. Finally, Aim 3 will investigate how telomerase, once recruited, accesses and extends a chromosome end by examining an essential, conserved telomerase RNA moiety recently found to be required after enzyme recruitment. The experiments will identify the proteins that bind telomerase RNA by an RNA-protein two-hybrid interaction assay as well as more conventional approaches. Overall, this project will provide critical mechanistic insights into how cells maintain telomere length in the major yeast model organism and establish the framework to understand telomere length regulation in humans. Given the critical function of telomere maintenance in sustaining cell growth potential in cancer and aging, learning how telomerase maintains telomeres will have a major impact on improving human health by revealing the best molecular targets for therapeutics.

项目概要 如果没有专门的机制来复制其末端，人类染色体会随着每次细胞分裂而缩短， 导致衰老和死亡。一种特殊的 RNA-蛋白酶复合物，即端粒酶，可以抵消这种现象 末端复制问题，延长端粒以完成基因组复制。端粒酶变成 在人体组织发育过程中处于休眠状态，导致染色体随着年龄的增长而缩短。然而，它 在大多数人类癌症中重新出现，维持细胞增殖潜力。需要有关的基本信息 端粒酶如何运作来维持端粒，以便开发控制这些过程的疗法。 研究表明，端粒长度稳态是通过优先招募端粒酶来实现的 最短的端粒，但控制端粒的系统尚不清楚。在酵母中，有两条途径 根据其 Ku 和 Est1 亚基确定用于端粒酶招募。该项目旨在确定如何 端粒酶被选择性地招募到短端粒上以延长它们。研究评估假设 (1) Ku 和 Est1 端粒酶招募途径以协调一致、逐步的方式运作，并且 (2) 端粒 Rif 蛋白通过特定的拮抗每个招募步骤来调节端粒长度 随着端粒长度缩短而减弱的独立机制。目标1将首先使用新的单端粒 系统定义“短”端粒的长度，每个端粒成分的功能是刺激或 抑制端粒酶的募集和活动。目标 1 的第二个目标是查明端粒蛋白在何处 端粒酶与染色体远端部分的DNA结合，使用深度测序- 基于具有接近单核苷酸分辨率的测定。这将增进我们对端粒的理解 通过揭示端粒蛋白沿端粒重复序列的排列来研究结构和端粒酶调控 并确定端粒酶相对于染色体末端的募集位置。目标 2 将确定距离有多近 需要招募单个染色体末端端粒酶来延长它，并测试 Ku 通路是否 依靠 Est1 来促进端粒酶的招募和活性。最后，目标 3 将研究端粒酶如何， 一旦招募，通过检查必需的、保守的端粒酶来访问并延伸染色体末端 最近发现酶招募后需要 RNA 部分。实验将鉴定蛋白质 通过 RNA-蛋白质双杂交相互作用测定以及更传统的方法结合端粒酶 RNA 接近。总体而言，该项目将为细胞如何维持端粒提供重要的机制见解 主要酵母模型生物体中的长度并建立理解端粒长度的框架 对人体的调节。鉴于端粒维持在维持细胞生长潜力中的关键功能 癌症和衰老，了解端粒酶如何维持端粒将对改善人类健康产生重大影响 通过揭示最佳的治疗分子靶点来促进健康。

项目成果

Structural Insights into Yeast Telomerase Recruitment to Telomeres.

酵母端粒酶招募到端粒的结构见解。

• DOI: 10.1016/j.cell.2017.12.008
• 发表时间: 2018-01-11
• 期刊: Cell
• 影响因子: 64.5
• 作者: Chen H;Xue J;Churikov D;Hass EP;Shi S;Lemon LD;Luciano P;Bertuch AA;Zappulla DC;Géli V;Wu J;Lei M
• 通讯作者: Lei M

A 4-Base-Pair Core-Enclosing Helix in Telomerase RNA Is Essential for Activity and for Binding to the Telomerase Reverse Transcriptase Catalytic Protein Subunit.

• DOI: 10.1128/mcb.00239-20
• 发表时间: 2020-11-20
• 期刊: Molecular and cellular biology
• 影响因子: 5.3
• 作者: Mefford MA;Hass EP;Zappulla DC
• 通讯作者: Zappulla DC