RNA uridylation by terminal uridylyl transferases (TUTases) has recently emerged as the major transcriptome- shaping force in protists, fungi, plants and metazoan organisms. Pioneering work on mitochondrial enzymes from the kinetoplastid parasite Trypanosoma brucei introduced the first examples of TUTases? biological functions in RNA editing and small RNA biogenesis, and established their basic domain architecture and atomic details of UTP selectivity. African trypanosomes cycle between mammals and insects, and proceed through proliferating and non-proliferating stages in each host. Given the lack of transcriptional control for most protein- coding genes, the major contribution of post-transcriptional regulatory mechanisms to these transitions is well accepted. However, current efforts chiefly concentrate on RNA binding proteins that recognize sequence elements in 3? untranslated regions and function as positive or negative modulators of mRNA stability. This proposal focuses on cytosolic 3? uridylation and seeks to assess the transcriptome-wide occurrence of mRNA U-tailing upon developmental switches, and the potential coupling between mRNA uridylation and decay. We previously identified two cytosolic TUTases, TbTUT3 and TbTUT4, and now provide preliminary data supporting TbTUT3?s involvement in mRNA 3?-5? degradation.
Aim 1 will establish deep sequencing-based technology (Tail- Seq) and develop a bioinformatics pipeline for unbiased analysis of mRNA termini in laboratory-adapted monomorphic insect and bloodstream proliferative forms. The inquiry will be extended to a pleomorphic cell line capable of differentiating in vitro from the bloodstream proliferative slender form to the non-proliferative, transmission-competent stumpy form, and further into early insect form.
Aim 2 will link mRNA uridylation activity to a specific TUTase and test the hypothesis that uridylated mRNAs are targeted by the 3? exosome. This work will institute a powerful tool for studying trypanosomal gene expression in development and pathogenesis, and will yield a comprehensive view of mRNA 3? modifications. By investigating early diverged eukaryotes, it will also address the evolutionary conservation of uridylation-induced RNA decay and may validate TUTases as druggable targets.
The unicellular parasite Trypanosoma brucei causes human African trypanosomiasis (sleeping sickness), a neglected tropical disease that affects some of the most marginalized populations. The goal of this project is to understand the mechanisms by which uridylation may control the transcriptome of Trypanosoma brucei. Because uridylation affects a wide spectrum of RNA molecules and is required for the parasite?s survival, the proposed studies will significantly expand fundamental knowledge of essential processes in a medically and economically relevant organism.
