Telomere maintenance by the telomerase RNA-protein complex

项目摘要 如果没有专门的机制来复制它们的末端，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，端粒酶，抵消了这一点 末端复制问题，延长端粒以完成基因组复制。端粒酶成为 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。但 在大多数人类癌症中重新出现，维持细胞增殖潜力。基本信息需要关于 研究端粒酶如何维持端粒，以开发控制这些过程的疗法。 研究表明，端粒长度的稳态是通过端粒酶被优先募集到 最短的端粒，但控制这一点的系统尚不清楚。在酵母中， 基于其Ku和Est 1亚基鉴定为端粒酶募集。该项目旨在确定如何 端粒酶被选择性地募集来缩短端粒以延长它们。研究评估了假设 （1）Ku和Est 1端粒聚合酶募集途径以协调、逐步的方式运作，以及（2） 端粒Rif蛋白通过拮抗端粒长度的每个募集步骤， 独立的机制，随着端粒长度的缩短而减弱。AIM 1将首先使用一种新的单端粒 系统来定义“短”端粒的长度，并且每个端粒组分的功能是刺激或 抑制端粒酶募集和作用。Aim 1的第二个目标是确定端粒蛋白质 和端粒酶与染色体远端部分的DNA沿着结合，使用深度测序- 基于具有接近单核苷酸分辨率的测定。这将推进我们对端粒的理解 通过揭示端粒蛋白沿着端粒重复序列的排列， 以及鉴定端粒酶相对于染色体末端的募集位置。目标2将决定如何接近 一个单独的染色体末端端粒酶需要被招募来延长它， 依赖于Est 1促进端粒酶的募集和活性。最后，Aim 3将研究端粒酶， 一旦被招募，通过检测一种必需的、保守的端粒酶， 最近发现RNA部分在酶募集后是必需的。这些实验将鉴定出 其通过RNA-蛋白质双杂交相互作用测定结合端粒酶RNA， 接近。总的来说，这个项目将提供关键的机械见解细胞如何维持端粒 长度在主要酵母模式生物和建立框架，以了解端粒长度 人类的规则。考虑到端粒维持在维持细胞生长潜力中的关键作用， 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。 通过揭示治疗的最佳分子靶点来改善健康。

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.