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Abstract

In African trypanosomes, the control of transcription initiation by RNA pol II is absent at the level of individual mRNAs. Nevertheless, gene expression changes dramatically during life cycle transitions in response to the changing host environments. In the bloodstream of the mammalian host, Trypanosoma brucei exist as proliferative long slender forms or as a non-dividing stumpy forms; the latter differentiate to procyclic forms in the midgut of the tsetse fly. Differential gene expression between life cycle stages is achieved through regulation of mRNA degradation and translation, and mainly relies on RNA binding proteins. The work focuses on the RNA binding protein RBP10. RBP10 is a bloodstream form specific cytoplasmic protein with a single RRM domain. Depletion of RBP10 in bloodstream forms or forced expression in the procyclic forms is lethal due to mis-regulation of developmentally expressed mRNAs. Bloodstream forms cells depleted of RBP10 differentiate to procyclic forms after transfer into procyclic growth media and incubation at 27oC; within three days, >80% of the cells express GPEET procyclin, and reposition their kinetoplast. Conversely, expression of RBP10 in procyclic cells for two days converts the cells to bloodstream forms. In such cells, eight VSG transcripts, including three with metacyclic promoters, were strongly up regulated, and ~16% of the cells had acquired a VSG surface coat. More importantly, a subset of the cells survived after transfer into bloodstream form growth media and incubation at 37oC, resulting in proliferating cells in about ten days. Tethering of RBP10 to a reporter mRNA inhibits translation and promotes mRNA degradation. RBP10 from bloodstream forms co-precipitated many procyclic specific mRNAs that are normally unstable in bloodstream forms. Indeed, 39% of the mRNAs up regulated after RBP10 depletion were bound by it; these included the transcript encoding procyclic surface coat EP procyclin, several enzymes needed for procyclic energy metabolism, regulatory proteins ZC3H21, ZC3H20, two kinases and a phosphatase. The UA(U)6 motif was found to be highly enriched in the 3' UTR of RBP10 mRNA targets. Binding of RBP10 to EP 3’ UTR was lost when the UA(U)6 motif was deleted from a reporter mRNA, and the motif was also necessary for its regulation by RBP10. In bloodstream forms RBP10 target mRNAs are likely to be blocked from translation hence degraded; this is important for survival. Perturbation of RBP10 expression therefore triggers a regulatory cascade that is sufficient to modulate T. brucei developmental capacity.