Characterisation of the ESAG6 and ESAG7 3'UTRs involved in the iron starvation response in Trypanosoma brucei

Barnes, Chloe and Urbaniak, Mick and McKean, Paul (2021) Characterisation of the ESAG6 and ESAG7 3'UTRs involved in the iron starvation response in Trypanosoma brucei. Masters thesis, Lancaster University.

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Abstract

All organisms require iron for survival and the bloodstream form of the parasite Trypanosoma brucei has evolved a unique receptor that binds host transferrin to facilitate iron uptake. The TbTfR is encoded by two expression site associated genes, ESAG6 and ESAG7, and is able to bind transferrin with a variable affinity from a wide range of host organisms. Upon iron starvation, the parasite is able to rapidly upregulate the expression of the transferrin receptor via a post-transcriptional mechanism mediated by the ESAG6 3’UTR. Here the ESAG7 3’UTR is defined, and truncations were made of the ESAG6 and ESAG7 3’UTRs to identify motifs that are important for the upregulation of the receptor. The truncated sequences were ligated into a firefly luciferase reporter system and transfected into a tagged rRNA locus in a bloodstream form cell line. Luciferase assays were performed on the truncated cell lines to measure the upregulation of the reporter gene when iron starvation is induced. Normally, expression of the transferrin receptor is low under basal conditions and increases when the cells are incubated in media starved of iron. Under iron starvation conditions, it was observed that a number of the truncated cell lines were able to increase the expression of the reporter gene by a magnitude previously reported for the upregulation of the transferrin receptor. This response was only maintained when the 3’ end of the 3’UTR remained undisrupted. From the 3’UTR a putative motif has been identified that may be responsible for mediating the upregulation of the transferrin receptor under iron starvation conditions.