Implications of the SHAPE of telomerase RNA

Telomerase is a specialized ribonucleoprotein complex that uses its RNA subunit to add six nucleotide G-rich repeats to the telomeric DNA at the end of our chromosomes, subsequently controlling cellular lifespan. Though telomerase is undetectable in somatic cells, it is up-regulated in approximately 90% of cancer cells. Crucial to enzyme activity is telomerase RNA (TR), whose diverse secondary structure is intrinsic for biological function. Due to the size of long RNAs and limitations of RNA structure determination techniques, high resolution structural data for full length TRs has been unavailable. Utilizing a novel chemical footprinting approach SHAPE, that gives high resolution structural data for every nucleotide in an RNA, we examined the structure of Tetrahymena thermophila (tTR) and human (hTR) TRs. The research presented here describes important structural aspects of these two telomerase RNAs. Previous studies have determined the solution structure of tTR via SHAPE. In the first part of this dissertation, we examined tTR via differential SHAPE to identify nucleotides that serve as molecular switches for RNA folding. We also analyzed two structural motifs from the stem IV helix, a portion of tTR that is essential for TERT binding and catalytic activity. Results from these studies indicate that local conformational flexibility impacts telomerase function. In part two, we determined the protein-free structure of human telomerase RNA. We initially interrogated the structure of the pseudoknot, CR4-CR5, and CR7 domains and mutants that give rise to pre-mature aging diseases showing that changes in structure instigate disease progression. We then determined the structure of the full length hTR, demonstrating that the pseudoknot forms an elongated hairpin, the template is base paired, and that the 5' RNA forms a G-quadraplex. We finally used the hTR structural data to identify the binding site of aminoglycosides that inhibit telomerase assemblage and activity by binding to hTR. As a principal component of telomerase, the contribution of TR structural information to RNA and telomerase biology is extremely important. With these data, comes the potential to develop rational approaches to modulate telomerase activity in aging and cancer drug platforms that take advantage of telomerase as a universal cancer target