Trypanosomatid parasites such as Trypanosoma brucei have unusual mechanisms of gene expression including polycistronic transcription, mitochondrial RNA editing and trans-splicing. Additionally, these protists rely mainly on post-transcriptional regulation where RNA-binding proteins (RBP) have shown to play a major role. RBP6 and RBP10 are two examples of RBPs that play crucial roles in procyclic and bloodstream form parasites differentiation respectively, by post-transcriptional regulation. Over-expression of RBP6 is enough to promote differentiation into metacyclic trypomastigotes that are infective to mice. However, continuous expression is required, and this pattern does not reflect the natural expression in the tsetse fly or the influence of other RNA-binding proteins. RBP5 is a RBP with a single RNA-recognition motif similar to RBP6 and RBP10, whose expression is upregulated during the life stages within the salivary glands of tsetse flies. We hypothesize the RBP5 facilitates metacyclogenesis in the tsetse fly. To evaluate possible contributions to T. brucei differentiation, we will over-express RBP5 in procyclic cells alone and in combination with RBP6. Initial screening of cells over-expressing PTP-tagged RBP5 resulted in parasites with a moderate growing defect, and the scoring of nuclei and kinetoplasts in fixed cells showed a progressive accumulation of cells with 2 nuclei and 2 kinetoplasts (2N2K) and appearance of multinucleated cells. On the other hand, over-expression of non-tagged RBP5 generated a more severe growing defect, starting immediately after the first day of induction. The scoring of nuclei and kinetoplasts resulted in a drastic increase of 2N2K cells and a greater appearance of multinucleated cells, which suggests an irregular cell cycle progression. When developing the dual over-expression system, our cells over-expressing RBP6 were not able to differentiate into any stage, and when over-expressing RBP5 and RBP6 coordinately, no differentiation process was observed either. Together these data suggest that RBP5 might be a regulator of genes involved in the initiation of cytokinesis in T. brucei parasites, however a role in metacyclogenesis cannot be discarded since we were not able to obtain metacyclic parasites. This study helped us to get a better understanding of the post-transcriptional regulatory mechanisms that repress and regulate T. brucei cell cycle progression.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:masters_theses_2-1760 |
| Date | 25 October 2018 |
| Creators | Anaguano Pillajo, David |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses |
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