Architecture and Biogenesis of the Human Telomerase Holoenzyme

Telomerase adds simple sequence repeats to the ends of linear chromosomes to counteract the loss of end sequence inherent in conventional DNA replication. Telomerase is a ribonucleoprotein (RNP) whose catalytic activity arises from the cooperation of the telomerase reverse transcriptase protein (TERT) and the telomerase RNA (TER), which provides an integrated template for DNA repeat addition. TERs vary widely in sequence and structure but share a set of motifs required for TERT binding and catalytic activity. Species-specific TER motifs play essential roles in RNP biogenesis, stability, trafficking, and regulation. Human TER, or hTR, contains a motif shared with a large family of H/ACA RNAs that directs a pathway of RNP maturation. I have investigated the RNA and RNP architecture of the hTR H/ACA domain and defined the sequence and structural elements required for RNA accumulation and RNP assembly. I have shown that, like other eukaryotic H/ACA RNAs, hTR recruits two sets of H/ACA core proteins despite a noncanonical spacing of conserved elements in its 5' H/ACA hairpin (Chapter Two). I have found that an hTR-specific BIO box motif within the 3' H/ACA loop stimulates H/ACA core RNP assembly in a manner required for hTR accumulation in vivo (Chapter Three). Finally, I have generated stable cell lines expressing tagged hTR and TERT to enable RNA-based and protein-based affinity purification of telomerase complexes in order to identify hTR-interacting factors and investigate telomerase multimerization (Chapter Four)