CAREER: Molecular Evolution of Telomerase Ribonucleoprotein: A Phylogenetic Study of Telomerase RNA Structure and Function

端粒酶是一种独特的核糖核蛋白(RNP)，在纤毛虫、酵母菌和脊椎动物中其生化组成和生物发生途径存在很大差异。在不同的物种中，端粒酶RNA与不同的蛋白质结合，并与其他RNP酶共享不同的生物发生途径。端粒酶RNP进化的不寻常过程背后的分子机制尚不清楚。为了更好地理解端粒酶生物发生和调控的不同机制，从真核生物的所有主要代表中鉴定和鉴定端粒酶RNA及其相关蛋白是至关重要的。端粒酶RNP研究中最困难的挑战之一是鉴定端粒酶RNA同源物，这主要是由于物种之间缺乏序列相似性。本研究项目的目的是从无脊椎动物、植物、藻类和一些早期分枝的原生动物中鉴定端粒酶RNA基因，以便进行比较研究。为了实现这一具有挑战性的目标，设计了一种新的策略来克隆未知的端粒酶RNA基因。尽管端粒酶RNA具有特殊的序列和大小变化，但它在体外与端粒酶逆转录酶(TERT)蛋白形成稳定的复合体。利用这种特定的RNA-蛋白质相互作用，重组TERT蛋白将被用作诱饵，通过一种改进的体外选择方案来亲和纯化它们的端粒酶RNA。鉴定真核生物不同分支的端粒酶RNA之后，将对重组的RNP进行结构和功能表征。总之，该项目的结果将为端粒酶结构和功能的分子进化提供新的见解。端粒酶核糖核蛋白是一种独特的RNA-蛋白质酶，在真核细胞中维持基因组的稳定。端粒酶RNA不同寻常地采用不同物种特有的结构域，并与不同物种组的不同蛋白质组分相互作用。本项目旨在确定所有主要真核物种的端粒酶RNA的结构，并了解这些RNA的结构与功能的关系。从这项工作中获得的见解不仅将为端粒酶的分子进化提供全面的图景，而且还将为其他核糖核蛋白酶的研究提供基础知识。这项研究项目还包括对高中、本科生和研究生进行培训和指导。进行真实研究项目的实践经验将有助于激励和鼓励学生追求科学研究事业。

Telomerase is a unique ribonucleoprotein (RNP) that varies dramatically among ciliates, yeasts and vertebrates in its biochemical composition and biogenesis pathway. In different species, telomerase RNA associates with different proteins and shares distinct biogenesis pathways with other RNP enzymes. The molecular mechanism underlying the unusual course of telomerase RNP evolution remains unclear. To better understand the diverse mechanisms of telomerase biogenesis and regulation, it is essential to identify and characterize telomerase RNA and its associated proteins from all major representatives of eukaryotes. One of the most difficult challenges in studying the telomerase RNP is identifying telomerase RNA homologues, mainly due to the lack of sequence similarity between groups of species. The goal of this research project is to identify telomerase RNA genes from invertebrates, plants, algae and some early-branching protozoa for comparative studies. To achieve this challenging goal, a novel strategy is designed to clone unknown telomerase RNA genes. In spite of its unusual sequence and size variation, telomerase RNA forms a stable complex with telomerase reverse transcriptase (TERT) protein in vitro. Taking advantage of this specific RNA-protein interaction, recombinant TERT proteins will be used as bait to affinity-purify their telomerase RNA counterparts using a modified in vitro selection scheme. The identification of telomerase RNAs from different branches of eukaryotes will be followed by structural and functional characterization of the reconstituted RNP. Collectively, the results from this project will provide new insights into the molecular evolution of telomerase structure and function.Telomerase ribonucleoprotein is a unique RNA-protein enzyme that maintains genomic stability in eukaryotic cells. The telomerase RNA unusually adopts various species-specific structural domains and interacts with various protein components in different groups of species. This project aims to determine the structure of telomerase RNAs from all major groups of eukaryotic species and to understand the structure-function relationship of these RNAs. Insights gained from this work will not only offer a comprehensive picture of the molecular evolution of telomerase enzyme, but also provide fundamental knowledge applicable to the studies of other ribonucleoprotein enzymes. This research project also involves training and mentoring students at the high school, undergraduate and graduate levels. The hands-on experience in conducting real research projects will help motivate and encourage students to pursue research careers in science.