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.
The vast majority of enzymes in cells are composed entirely from protein. However, some enzymes function as a complex containing both RNA and protein components. The enzyme telomerase is one such RNA-protein enzyme that requires the cooperative efforts of the RNA and protein components. Telomerase is, furthermore, unique in it is responsible for cell immortality in higher eukaryotes and play an important role in ageing, cancer and various other human diseases. The telomerase protein and RNA components cooperatively catalyze DNA synthesis specifically to the ends of chromosomes, maintaining genome integrity and preventing disease. While the protein component is uniform and structure and sequence, the RNA component has massively diverged across groups of species. This divergence in the telomerase RNA structure and sequence is due to a fast evolution rate compared with the protein component. Telomerase has been identified and extensively studied in select groups of species: ciliates (simple single-celled water-born organisms), yeasts, vertebrates and recently in a single plant. However, very little is known of telomerase outside of these few groups of species, which would provide crucial insights into the function and evolution of the telomerase RNA. The difficult challenge for identifying and studying telomerase RNAs from additional groups of species is the massive divergence in structure and sequence, impeding identification by similarity. The central goal of this research program was to establish effective and universal methodologies and technologies for identifying the divergent telomerase RNAs from all major groups of species. The identification of these novel telomerase RNA will permit comparative analysis of the RNA structure and function. During the five year funding period for this project, we have developed and established numerous innovative strategies for cloning these then unknown telomerase RNAs. To validate our methodologies and technologies, we have successfully identified and characterized the telomerase RNA from numerous organisms, including species from filamentous fungi and invertebrates. Characterizing these newly identified species has served as a ‘missing links’ between vertebrate and yeast telomerase RNAs. These newly discovered telomerase RNAs reveal the intermediate structures, serving to fill in the gaps between these two seemingly divergent groups of species. Additionally, this research has contributed to our understanding of the RNA-protein interactions necessary for telomerase—and other RNA-protein enzymes—to function. We have initially mapped, and then determined, the specific structure of the RNA and protein where they come together to create a functional enzyme. Our work has also extended into understanding telomerase-related diseases, determining and validating the mutations which brought about the loss of enzyme function and damage to human health. In addition to our principle research, this lab has been actively involved in training students at the high school, undergraduate and graduate levels. These students (9 PhD students, 41 undergraduates and 10 high school students) have hands-on, direct contact with research and their work has also been published in research journals. Most of these students who have contributed to this project are pursuing careers in the science, technology and related disciplines. Moreover, we have created a web-based telomerase database (http://telomerase.asu.edu/) to provide easy access to telomerase-related information. This database has become an indispensable resource in the telomerase research community, and essential for basic science researchers, physicians and the general public.
