Collaborative Research: Telomerase Regulation in Deep Branching Eukaryotes

这个项目的重点是端粒酶，这是一种负责制造端粒的酶，端粒是保护所有真核细胞染色体末端的结构，让人想起鞋带的硬端。通过端粒酶复制端粒的遗传机制在酵母和哺乳动物细胞中得到了更好的研究；尽管这两种生物彼此相距遥远，但它们实际上代表了真核系统发育的相对较新的分支。相反，在这项研究中，将研究真核生物的一个古老的、深分支的谱系--动质原生生物。被选择的生物，布鲁氏锥虫，具有独特的结构和功能的端粒酶，结果应该表明这种酶是如何工作的。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生计划(BIOKEYS)；该计划的主要目标将是教会本科生研究人员在他们的研究生涯早期重视跨学科科学。除了从设计实验和使用高端技术解决问题中获得经验外，学生还将作为研究小组的一部分相互教授和学习。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，从而抵消了传统DNA聚合酶在每个细胞周期中因DNA复制不完全而造成的线形DNA末端的损失。端粒酶有两个核心成分，催化端粒延长的端粒酶逆转录酶(TERT)和为端粒DNA合成提供模板的端粒酶RNA(TER)。布鲁氏锥虫是一种深分枝动态体，端粒酶延长端粒的机制尚不清楚。因此，本课题对布氏毛滴虫端粒酶RNA的结构、生化和遗传特征进行了深入研究，以期了解早期真核生物中端粒酶的调控机制。最近布鲁氏毛滴虫的发现揭示了深分支真核生物所特有的新特征，表明布鲁氏毛滴虫和高等真核生物在端粒合成过程中存在机制上的差异。因此，本项目：(I)利用核磁共振和形状化学在单核苷酸分辨率下研究端粒酶RNA结构；(Ii)使用HITS-CLIP技术确定对布氏毛滴虫端粒酶功能至关重要的TERT上的关键接触位点；以及(Iii)通过遗传操作和端粒酶功能分析确定TR域的功能意义。总体而言，这项研究将在理解原生生物端粒酶进化的机制细节方面取得重大进展。

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.