Developmental regulation of RNA editing in Trypanosoma brucei

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Project Summary Trypanosoma brucei is a parasitic protozoan and the causative agent of human African trypanosomiasis in 36 sub-Saharan African countries. The parasite progresses through several, required developmental stages throughout its life cycle as it transitions between its two hosts, the tsetse fly and mammals. These hosts offer very different nutritional environments which the parasite must be able to rapidly adapt to and exploit. To efficiently take advantage of both nutritional environments, T. brucei modulates its mitochondrial activity. However, while several respiratory subunits are encoded in the mitochondrial genome, most of these mitochondrial genes do not encode functional open reading frames. To generate translatable open reading frames, the parasite must post-transcriptionally modify the mRNAs by precise insertion and deletion of often hundreds of uridine residues in a process termed RNA editing. Concomitant with changing metabolic demands, editing of several mRNAs is differentially regulated between mammalian long slender bloodstream form (BSF) and insect vector procyclic form (PCF) parasites, the only stages in which RNA editing has been investigated in T. brucei. However, the mechanisms by which this regulation takes place and the precise points in the life cycle when it is triggered are unknown. Recent data call into question older studies regarding which transcripts undergo developmentally regulated editing, and use of different strains and growth conditions has likely also caused discrepancies. Our hypothesis is that editing of distinct mRNAs is differentially regulated throughout the T. brucei life cycle with regard to both timing and mechanism, and that post-translational modification of editing accessory factors contributes to this regulation. In Aim 1, we will establish a cell line that can transition through both proliferative and non-proliferative (transmissible) life cycle stages and use qRT-PCR and high throughput sequencing to define the editing profile of all 12 edited mRNAs in four life cycle stages. Developmentally regulated accumulation of edited apocytochrome b (CYb) mRNA is well established, and accessory factors RBP16 and MRP1/2 are required for this accumulation in PCF parasites. In Aim 2, we will test whether arginine methylation of RBP16 regulates CYb mRNA editing initiation and association of CYb mRNA with the editing holoenzyme. In Aim 3, we will determine whether the reported developmentally regulated phosphorylation of MRP1/2 accounts for the ability of this factor to support edited CYb mRNA levels specifically in PCF. This project builds upon the skills Dr. Smith acquired during his Ph.D. work on protein trafficking in T. brucei. The project will require him to develop new skills and knowledge, notably in RNA biology, protein-nucleic acid interactions, and bioinformatics. Dr. Smith will develop professional and academic skills with the help of mentors, and through courses offered at the University at Buffalo. He will present his data at regional and international meetings, and broaden his network of potential collaborators. The proposed project, with support from his mentoring committee, will expand Dr. Smith’s capabilities to a point where he will be comfortable starting an independent laboratory.