Decoding RNA-Protein Interactions in Trypanosoma Telomerase

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Project Summary Telomerase is a unique ribonucleoprotein enzyme that processively adds telomeric repeats, copied from its integral RNA component, to the ends of linear chromosomes to prevent genome instability in eukaryotes. This proposal seeks to define RNA folding and RNA-protein interactions that are critically important for telomerase regulation in Trypanosoma brucei, an early divergent parasitic protist that proliferates through multiple morphologically distinct life cycle forms in humans and insects. In T. brucei, the telomere structure plays an important role in regulation of antigenic variation that enables the parasite to establish a long-term infection. Particularly, extremely short telomeres could jeopardize telomere integrity, stability of their (sub)telomeric virulence genes and parasite survival. Therefore, understanding the mechanism that controls telomere replication in T. brucei could provide important clues to control parasite proliferation. Telomerase is the major mechanism of telomere synthesis in T. brucei. Two highly conserved telomerase RNA structural domains, the RNA template and eCR4/5 independently bind the catalytic protein, telomerase reverse transcriptase (TERT) during telomere synthesis and are the only required RNA elements for in vitro reconstitution of catalytically active telomerase. However, T.brucei telomerase RNA has unusual sequence and structural composition in the above domains compared to ciliate, yeast and vertebrate telomerase RNAs, suggesting novel modes of regulation for telomere synthesis. Therefore, our hypothesis is that these unusual sequence and structural diversity of T. brucei telomerase RNA domains cause differences in RNA-protein interactions and conformational changes, resulting in species-specific telomerase assembly and activity. Our recent RNA structure probing data from two replicative stages of T. brucei suggests that RNA folding and telomerase activation could be developmentally regulated. To understand how stage -specific structural rearrangements and RNA-proteins interactions control telomerase regulation in T. brucei, in Aim 1 of the proposal we will determine molecular requirements of RNA-protein interactions in the above two domains in T. brucei telomerase. Aim 2 of this proposal will explore additional requirements for telomerase regulation by dissecting RNA-specific factors that are required for functional telomerase RNP assembly and activity. In summary, this research will lay the foundation for the PI's long-term goal to define core components of telomerase activation and interactions for telomere length homeostasis and genome integrity in a clinically important protist.