Talk abstracts
Talk on Friday 04:15-04:30pm submitted by Kaitlin Klotz
High-Resolution Profiling of Telomerase RNA Structure Dynamics in the Eukaryotic Pathogen Trypanosoma brucei
Kaitlin E. Klotz (Department of Biological Sciences-University of North Carolina at Charlotte), Abhishek Dey, Anais Monroy-Eklund (Department of Biology-University of North Carolina at Chapel Hill), Justin Davis (Department of Biological Sciences-University of North Carolina at Charlotte), Arpita Saha, Bibo Li (Department of Biological, Geological and Environmental Sciences-Cleveland State University), Alain Laederach (Department of Biology-University of North Carolina at Chapel Hill), Kausik Chakrabarti (Department of Biological Sciences-University of North Carolina at Charlotte)
Abstract:
Telomerase is a ribonucleoprotein (RNP) comprised of a reverse transcriptase (TERT) and an RNA template (TR) for extending linear chromosomes to preserve genomic integrity and address the end replication problem within eukaryotic cells. The TR catalytic core, which is responsible for executing the reactions to add repeats to the ends of linear chromosomes, resides near the 5’ end of the telomerase RNP and it is functionally conserved amongst eukaryotes. Until recently, studying the conformation of the cellular telomerase RNP has been difficult due to its long, folded structure and low abundance in vivo. To address this problem and directly analyze the in-cell TbTR architecture, we employed a novel approach which coupled structure-specific in vivo chemical modification with mutational profiling, followed by next-generation sequencing to elucidate the conformation of the TbTR catalytic core. We investigated whether proper assembly of the catalytic core components of telomerase is a requirement for RNA folding and function through affinity purification of the telomerase RNP complex from T. brucei, then we probed the native RNA structure using mutational profiling. This immunoprecipitated telomerase demonstrated telomere repeat addition activity in a telomerase assay, suggesting that this RNA is a vital part of the active telomerase complex. Interestingly, we observed that T. brucei TR exists in two distinctly different folding states in discrete developmental stages within both the insect and mammalian host. Taken together, the described work provides the first detailed analysis of the in vivo folding architecture of telomerase RNA at nucleotide resolution. Because telomerase is the key mechanism employed by the parasite to preserve telomere integrity and thus, maintenance of the sub-telomeric virulence genes, we anticipate that developing a greater understanding of TR dynamics in telomerase function will provide crucial insights into T. brucei telomere synthesis, sub-telomeric virulence gene regulation and the ability of the parasite to survive.
Keywords: Telomerase RNA, Structure Dynamics, Trypanosoma brucei