Characterisation of trans-acting factors involved in the regulation of life-cycle stage-specific mRNAs in Trypanosoma brucei

The vast majority of gene expression in African trypanosomes is regulated post-transcriptionally. The regulation of two model genes was investigated: GPI-PLC and ISG65 mRNAs are ~50-fold greater in bloodstream form trypanosomes than in insect procyclic forms. The GPI-PLC mRNA is selectively unstable in procyclic forms. The two model genes were used to identify trans-acting factors involved in the instability of the mRNAs in procyclic forms detected as causing an increase in GPI-PLC and/or ISG65 mRNA steady state levels. Two alternative approaches have been taken to achieve this: (1) RNAi ablation of homologues of factors known to have a role in mRNA stability in higher eukaryotic systems; and (2) a forward genetic screen using a reporter system to identify factors essential to the process of developmental regulation. Seven candidate genes were identified as putative orthologues of genes involved in mRNA maturation and turnover in other eukaryotes. Expression of each was ablated by RNAi in procyclic cells and ISG65 and GPI-PLC mRNA steady state levels measured. This approach identified two candidate genes: DRBD4 knockdown caused a substantial accumulation of the ISG65 mRNA and also a smaller but significant accumulation of both a full length and a truncated GPI-PLC mRNA species; DHH1 mRNA ablation caused accumulation of GPI-PLC mRNA. DRBD4 was initially selected as a putative orthologue of human pyrimidine tract biding protein. DRBD4 localised to discrete nuclear bodies and in vitro RNA-binding assays predicted G-rich RNA-binding specificity. The differential effects of DRBD4-depletion on ISG65 and GPI-PLC mRNA steady state levels suggested two alternative functions of DRBD4. The truncated GPI-PLC species resulted from an alternative pre-mRNA processing event intimating a role for DRBD4 as a processing site repressor. The impact on ISG65 mRNA steady state levels following DRBD4 knockdown was less straightforward. The half-life of ISG65 mRNA was unchanged despite substantial accumulation of both message and protein and, surprisingly, the results were instead consistent with either an alteration in the rate of transcription or processing. This suggested that ISG65 represents an unusual case in which differential expression is regulated at the level of mRNA production as opposed turnover. The role of DRBD4 in mediating this regulation remains unclear. DHH1 is a highly conserved RNA helicase with a role in promoting mRNA decapping in higher eukaryotes. DHH1 was present in small foci within the cytoplasm of the cells, similar to its location in yeast and mammalian cells.EThOS - Electronic Theses Online ServiceGBUnited Kingdo