Comparative transcriptomics of blood stream and procyclic form of Trypanosoma brucei and investigation of its mRNA methyl cap structure and function (360G-Wellcome-093712_Z_10_Z)

In Human African trypanosomiasis (HAT), the causative parasite Trypanosoma brucei (T. brucei) undergoes a transition between the Tsetse fly vector (procyclic form) and mammalian host (blood stream form) (1). A better understanding of the disease transmission cycle is imperative for the development of new therapeutic targets because substantial changes in protein expression occur when the parasite transitions from one stage to the next. This proposed project's overarching question asks is the T. brucei proteome substantially controlled at the level of mRNA selection by ribosomes; and if so, what is the mechanism of this selection. Aim 1: Perform RNAseq (in collaboration with the Wellcome Trust Sanger Institute) of total polA+ and polyribosomal RNA purified from blood stream form (bsf) and procyclic form (pcf) T. brucei followed by bioinformatic analysis comparing the transcript profiles. Aim 2: Correlate transcriptome data with proteomic analysis, using the SILAC method, of global protein expression levels in bsf and pcf T. brucei. mRNA processing in eukaryotic cells involves the addition of the 7-methylguanosine cap (methyl cap) to the first nucleotide of the 5' end of the transcript. Since the methyl cap is essential for gene expression in all eukaryotes investigated we will investigate whether it is essential in trypanosomses and whether the enzymes that catalyze formation of the methyl cap are potential drug targets. In the vast majority of eukaryotes methyl cap formation is catalysed by a single guanylyltransferase and a single methyltransferase. Trypanosomes are highly unusual in that they have an additional guanylyltransferase and methyltransferase on a single polypeptide (2). Aim 3: Our approach is to knock out the single methyltransferase and the bifunctional methyltransfere/guanylyltransferase and determine the effect on cell viability. If either enzyme is required for cell viability, it will be pursued further as a drug target with the drug discovery unit. The effect of methyltransferase knock-out on transcript stability, splicing, and translation will also be investigated to determine the effect of the methyl cap in trypanosomes.