Authors
A Trenaman1; R Wall1; D Horn1; 1 University of Dundee, UK Discussion
African Trypanosomes constitutively transcribe the majority of their genome in a polycistronic manner and have expanded their repertoire of RNA-binding proteins (RBPs), in order to modulate gene expression. Many of these RBPs are thought to act by binding 3' untranslated regions (UTRs) in the mRNA. Indeed, abundant stage-specific surface proteins are known to be subject to this form of regulation. Relatively few UTRs have been shown to play such a role, however, and the sequences responsible remain largely uncharacterised. We aim to identify these regulatory UTRs on a genome-wide scale and to improve our understanding of these trypanosome-specific adaptations. We assembled a reporter construct comprising a dual positive/negative selectable marker cassette, containing a blasticidin deaminase/thymidine kinase fusion gene. A small set of reference UTRs were used to validate the reporter system. We then assembled a Trypanosoma brucei genome-scale library by cloning genomic DNA fragments (1 - 3 kbp) immediately downstream of the reporter gene. The plasmid library has been used to assemble a T. brucei library and we have applied both positive (Blasticidin) and negative (Ganciclovir) selection to the library. These two screens have initially been performed in bloodstream form T. brucei with the aim of identifying 3’-UTRs associated with increased or reduced reporter gene expression, respectively. We are currently running a deep-sequencing step designed to quantify the relative contributions of several thousand UTRs. 
