The transmission of Trypanosoma brucei, the causative agent of human African trypanosomiasis, depends upon the development in the bloodstream of 'stumpy forms' from non-transmissible 'slender forms'. In stumpy forms many mRNAs are downregulated and translation is generally repressed. However, a small subset of genes escape this repression and are upregulated, presumably as an adaptation for transmission. To understand the basic of this, regulatory sequences within the 3'UTR of a major stumpy-enriched transcript (an ESAG9 gene) have been characterised. This identified a signal responsible for gene silencing in slender forms and gene activation when cells develop to stumpy forms. An investigation was made of upstream open reading frames (uORFs) as a mechanism for the control of stumpy form gene expression. No evidence was found of uORF control, but one gene investigated was found to produce two transcripts through trans-splicing at different sites. These transcripts, which were found to exhibit some differential abundance between life-cycle stages, would generate a long and short form (from an internal ATG) of the encoded protein. Both are predicted to contain a UBA/TS-N (ubiquitin associated) domain, however, the longer form of the protein is also predicted to contain a transmembrane helix and cleavable signal peptide, suggesting a different localisation. However, ectopic expression of either protein form with a Ty epitope tag resulted in the same protein localisation. Additionally, the transcripts of two translational protein homologues, TbeIF4E4 and TbeIF6, were identified as upregulated in stumpy forms. Radiolabelled-methionine experiments and polysome analysis showed that overexpression or RNAi-mediated ablation of TbeIF6 resulted in a decrease in protein synthesis and decrease in translation. Unlike its archaeal homologue, TbeIF6 protein was not induced by coldshock treatment. Finally, to identify which transcripts escape translational repression in stumpy forms an analysis was made of polysome-associated transcripts by RNA-sequencing. This identified potentially interesting genes for further investigation, and showed that many procyclic-enriched transcripts were also enriched in stumpy form polysomeassociated RNA, confirming these cells as preadapted for transmission. Together, this work has characterised a 3’UTR regulatory element in a stumpy-enriched transcript, examined alternative trans-splicing of another transcript, investigated two translational protein homologues and identified transcripts that escape translational repression in the transmissible life-cycle stage of T. brucei.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:586308 |
| Date | January 2012 |
| Creators | Monk, Stephanie Lydia Spencer |
| Contributors | Matthews, Keith; Vazquez-Boland, Jose |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/8177 |
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