Eukaryotic chromosomes are linear DNA strands that terminate with telomeres, protective regions of repetitive sequences that are coated in DNA-binding proteins. Telomeres gradually shorten with each cell division until they reach a critical length that induces a ‘telomere crisis’, resulting in programmed cell death or senescence. The telomerase enzyme prevents telomere crisis by using a reverse transcriptase (TERT) and integral telomerase RNA (TR) to synthesize new telomeric DNA and counteract DNA loss. While telomerase is beneficial for preserving self-renewing tissues, it is also a hallmark of most human cancers.
Despite decades of work since telomerase’s discovery in 1984, zero telomerase-targeting therapeutics are approved for clinical use today. A key obstacle in telomerase drug development was the lack of structural information available, but recent high-resolution cryogenic electron microscopy (cryo EM) models in combination with other structural studies have now eliminated this barrier. While a growing body of research is illuminating telomerase in higher molecular detail, much remains to be learned about the process of telomerase assembly; how the various protein and RNA components of this ribonucleoprotein (RNP) assemble to produce the active complex. The knowledge gained on this topic will accelerate therapeutic development and better enable us to understand diseases caused by telomerase deficiencies.
This thesis focuses on human telomerase biogenesis from an RNA structural perspective, highlighting the interplay between human TR (hTR) structure and protein-binding events that must be properly orchestrated to achieve the assembly of the active enzyme. I will focus first on in vitro studies that reveal hTR adopts heterogenous folds, and describe a discrete hTR conformational change that occurs upon TERT binding. Next, I will focus on how alternatively folded conformations of hTR persist in the cellular environment and their impact on the biogenesis of telomerase. In addition, I provide a detailed guide to designing, executing, and analyzing in-cell RNA chemical probing experiments from start to finish. Finally, I describe the future directions of my unfinished projects on the topics of hTR folding chaperones and disease-associated hTR mutations.
