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.
