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

点击翻译按钮获取中文摘要

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.