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PDF, English (PhD thesis) Download (21MB) | Lizenz: Regulating a post-transcriptional regulator of gene expression, the RNA-binding protein 10, in Trypanosoma brucei by Bishola Tshitenge, Tania underlies the terms of Creative Commons Attribution-NoDerivatives 4.0

Abstract

African trypanosomes are flagellated parasites that cause sleeping sickness disease in humans and nagana in cattle. In the bloodstream of the mammalian host, Trypanosoma brucei exist as proliferative long slender forms or as non-dividing stumpy forms; the latter differentiate to procyclic forms in the midgut of the tsetse fly. Since the majority of protein coding genes are arranged in polycistronic transcription units, trypanosomes rely almost exclusively on post-transcriptional mechanisms - mRNA processing, decay and translation - to regulate gene expression. The RNA-binding proteins assume the burden that is

carried by transcription factors in others eukaryotes and therefore they are critical at all stages. The RNA- binding protein 10 (RBP10), which is exclusively expressed in bloodstream forms, binds the motif UA(U)6

present in the 3’-untranslated regions of procyclic-specific mRNAs and targets them for destruction. This work focuses on understanding the mechanisms that regulate the differential expression of RBP10 in Trypanosoma brucei. Results of this study show that the developmental regulation of RBP10 expression is mediated by the long 3’-untranslated regions of its mRNA. Different regulatory sequences that enhance stability and translation of the RBP10 mRNA in bloodstream forms or destabilize and repress the mRNA in procyclics are scattered throughout the RBP10 3’-untranslated region. The only motif that was found to be involved in RBP10

mRNA translation in bloodstream forms is the (AU)10 element. RNAi-mediated depletion of the RNA- binding proteins, HNRNPFH, ZC3H28, ZC3H40 and ZC3H45 did not affect RBP10 mRNA levels in

bloodstream forms. Nevertheless, ZC3H28 associates with MKT1 complex, the translation initiation factor complex EIF4E4/EIF4G3 and several ribosomal proteins. The protein binds long mRNAs that have low ribosome occupancy and presumably might stabilize them. On the other hand, the depletion of the double RNA-binding protein 18 (DRBD18) in trypanosome bloodstream-forms affected cell growth and led to reduction of RBP10 protein levels. This was accompanied by accumulation of several shortened RBP10 mRNA isoforms, and loss of the longer mRNA species. The effect was seen in over 100 other mRNAs. DRBD18 associated with the proteins of the outer ring of the nuclear pore complex as well as those of the exon junction complex among others. Depletion of the nuclear export factor MEX67 did not impair RBP10 processing, suggesting that the effect of DRBD18 on the RBP10 mRNA was rather specific. After depletion of DRBD18, the longer RBP10 transcripts were trapped in the nucleus while the shorter ones were able to migrate to the cytosol. The proposed model implies that DRBD18 binds suboptimal processing signals in the 3’-UTRs of its target mRNAs, preventing their use and simultaneously allowing export of the bound mRNAs. In the absence of DRBD18, suboptimal splicing signals are bound by the splicing/polyadenylation machinery generating alternative polyadenylated mRNAs, which are exported in the cytosol.