Collaborative Research: Telomerase Regulation in Deep Branching Eukaryotes

合作研究：深分支真核生物中的端粒酶调控

• 批准号: 1616314
• 负责人: Kausik Chakrabarti
• 金额: $ 51.75万
• 依托单位: Carnegie-Mellon University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2017-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1616314&HistoricalAwards=false
• 关键词: Collaborative; Research; Telomerase; Regulation; Deep

This project focuses on telomerase, the enzyme that is responsible for making telomeres, structures that protect the ends of all eukaryotic chromosomes, reminiscent of the hard ends of shoelaces. The genetic mechanism of telomere replication by telomerase is better studied in yeast and mammalian cells; as far from each other as those two organisms are, they in fact represent relatively recent branches of eukaryotic phylogeny. In contrast, in this study an ancient, deep branching lineage of eukaryotes, kinetoplastid protists will be examined. The organism of choice, Trypanosoma brucei, has telomerase with unique structural and functional properties, and result should indicate how this enzyme works. The project will encourage full participation from women and underrepresented minorities in science. Apart from training graduate and postdoctoral trainees, this project will also implement a three-year undergraduate program (BIOKEYS) in both PIs' laboratories; the major goal of this program will be to teach undergraduate researchers to value interdisciplinary sciences early in their research careers. In addition to gaining experience from designing experiments and problem solving using high-end technologies, students will be teaching and learning from each other as part of a research group.Telomerase, a ribonucleoprotein enzyme, provides the major means for elongation of chromosome ends (telomeres), thus counteracting the loss of linear DNA ends in each cell cycle due to incomplete DNA replication by conventional DNA polymerases. Telomerase has two core components, the Telomerase Reverse Transcriptase (TERT) that catalyzes telomere elongation, and the telomerase RNA (TER), which provides a template for telomere DNA synthesis. The mechanisms of telomere elongation by telomerase are poorly understood in Trypanosoma brucei, a deep branching Kinetoplastid. Therefore, this project dissects structural, biochemical and genetic features of telomerase RNA in T. brucei to understand the mechanism of telomerase regulation in early eukaryotic species. The recent discovery of the T. brucei TER reveals novel features exclusive to deep branching eukaryotes, suggesting mechanistic differences in the process of telomere synthesis between T. brucei and higher eukaryotic organisms. Therefore, this project: (i) investigates telomerase RNA structure at a single nucleotide resolution using NMR and SHAPE chemistry, (ii) defines key TERT contact sites on TER that are essential for telomerase function in T. brucei using HITS-CLIP technology, and (iii) establishes the functional significance of TR domains by genetic manipulations and telomerase functional assays. Overall, this research will allow significant advances in understanding the mechanistic details of telomerase evolution in protists.

这个项目的重点是端粒酶，这种酶负责制造端粒，端粒是保护所有真核生物染色体末端的结构，让人想起鞋带的硬端。在酵母和哺乳动物细胞中，端粒酶复制端粒的遗传机制得到了更好的研究;尽管这两种生物彼此相距甚远，但它们实际上代表了真核生物发生学的相对较新的分支。相反，在这项研究中，一个古老的，深分支的真核生物谱系，动质体原生生物将进行检查。选择的生物体，布氏锥虫，具有独特的结构和功能特性的端粒酶，结果应该表明这种酶如何工作。该项目将鼓励妇女和代表性不足的少数群体充分参与科学。除了培训研究生和博士后学员外，该项目还将在两个PI的实验室实施为期三年的本科课程（BIOKEYS）;该课程的主要目标是教导本科研究人员在研究生涯的早期重视跨学科科学。除了通过设计实验和使用高端技术解决问题获得经验外，学生们还将作为研究小组的一部分进行教学和学习。端粒酶是一种核糖核蛋白酶，提供了延长染色体末端（端粒）的主要手段，从而抵消了由于常规DNA聚合酶的不完整DNA复制而导致的每个细胞周期中线性DNA末端的丢失。端粒酶有两个核心组分，催化端粒延伸的端粒酶逆转录酶（TERT）和为端粒DNA合成提供模板的端粒酶RNA（TER）。布氏锥虫是一种具有深分支的动质体，其端粒通过端粒酶延长的机制还不清楚。因此，本研究对T. brucei的研究，以了解早期真核生物中端粒酶的调控机制。最近发现的T.布氏杆菌TER揭示了深分支真核生物独有的新特征，表明T.布鲁氏菌和高等真核生物。因此，本项目：（i）使用NMR和SHAPE化学在单核苷酸分辨率下研究端粒酶RNA结构，（ii）定义TER上对于T.（iii）通过基因操作和端粒酶功能测定确定TR结构域的功能意义。总的来说，这项研究将使了解原生生物端粒酶进化的机制细节取得重大进展。