Administrative Supplement to Promote Diversity in DecodingRNA-Protein Interactions in Trypanosoma Telomerase

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. The
overall goal of this diversity research supplement proposal is to define the role of post-translational
modifications of the telomerase catalytic protein component, TERT in RNA-protein interactions and telomerase
regulation in Trypanosoma brucei. T. brucei is 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. Telomerase is the major regulator 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. Previous studies on higher eukaryotes revealed that
ubiquitination is one of the major post-translational modifications that affect the stability and activity of TERT
component in telomerase holoenzyme. However, the role of ubiquitination in regulating RNA-protein
interactions and telomerase activity remains unknown for any eukaryotic telomerase. Recently, we have
characterized the interactome of the bloodstream form T. brucei parasites as part of the proposed activities in
the parent proposal and found that T. brucei telomerase enzyme complex contains several ubiquitination -
specific enzymes and factors. Additionally, new experimental data revealed that TERT protein is differentially
ubiquitinated in the procyclic and bloodstream form developmental stages of T. brucei. Therefore, in this
supplement proposal, PI intends to expand the original proposal’s objectives to elucidate the mechanisms of
RNA-protein interactions of TERT and telomerase RNA during T.brucei development. Particularly, PI plans to
map the ubiquitination sites on the TERT protein and determine their roles in T. brucei telomerase function. 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.

Grant Number: 3R15AI166764-01A1S1
NIH Institute/Center: NIH
Principal Investigator: Kausik Chakrabarti