Identification and Characterization of Telomerase Components in Plants

Telomerase is a specialized ribonucleoprotein (RNP) complex responsible for telomere maintenance. Telomerase consists of three essential components: the catalytic subunit telomerase reverse transcriptase TERT, the telomerase RNA subunit TR, and telomerase accessory proteins. Studies of telomerase aim to define the components of telomerase RNP complexes and characterize their relations in terms of biogenesis, subunit interactions, and structures, which all provide significant implications for cellular aging and cancer. In the plant kingdom, TERT was defined 20 years ago in the model organism Arabidopsis thaliana. However, due to technological limitations, the precise RNA component and the complete list of accessory proteins are unknown. In this dissertation, I describe the identification and characterization of the bona fide telomerase RNA, termed AtTR, in A. thaliana, which lead to the discovery of 85 TR from three major clades of plants with a conserved secondary structure serving as an evolutionary bridge to connect divergent TRs in vertebrates and ciliates. In addition to TR, I characterized the previously identified telomerase-associated proteins, AtPOT1a and dyskerin. I found that AtPOT1a physically associates with TERT and telomeric DNA to promote telomerase recruitment on telomeres. I also detected a specific interaction between dyskerin and AtTR mediated by a unique three-way junction element supported by cryo-EM analysis. This study reinforced the conclusion that plant telomerase is an evolutionary bridge by presenting a chimeric RNP complex with ciliate-like AtTR and vertebrate-like telomerase-associated protein dyskerin and AtPOT1a. Furthermore, we re-evaluated a previously defined telomerase-associated RNA molecule AtTER2. We found that AtTER2 completely overlaps with the gene encoding tRNA Deaminase 3 (TAD3), which indirectly participates in telomere maintenance. Finally, we utilized quantitative mass spectrometry to define the associated proteins of telomerase core in A. thaliana. This study provided a framework to uncover telomerase-associated proteins and indications for telomerase RNP components in the plants. In summary, the studies presented in this dissertation reveal that Arabidopsis telomerase serves as an evolutionary bridge to unite vertebrate and ciliate telomerase RNP complexes by maintaining a chimeric complex, making it an exciting model system for study of telomere and telomerase biology