In the protozoan parasite Trypanosoma brucei gene expression is mostly regulated on the post-‐transcriptional level. C3H zinc fingers have shown to play an important role in this process. So far, all described C3H zinc finger in Trypanosoma brucei have shown to stabilize their targets. This work focuses on the zinc finger protein ZC3H32. A large-‐scale isolation of free and membrane-‐bound polyribosomes from proyclic and bloodstream trypanosomes, which is also described in this thesis, suggested that this protein associates with polyribosomes. However, small-‐scale polyribosome isolations followed by Western blotting revealed that only a minor fraction of it is actually associated with polyribosomes. Immunofluorescence showed that ZC3H32 localizes to the cytoplasm. A yeast-‐two-‐ hybrid screen identified it as a putative binding partner of MKT1. This interaction was confirmed by co-‐immunoprecipitation. RNAi, as well as knock-‐out studies, showed that ZC3H32 is essential in the bloodstream form. Northern blotting, as well as a SILAC screen by Urbaniak et al. indicate that it is also enriched in this life-‐cycle stage as compared to procyclics. Artificial tethering of ZC3H32 to a reporter RNA lead to the RNA’s degradation, suggesting that this protein has a destabilizing effect. Tethering of ZC3H32 fragments revealed that both its N-‐terminal, as well as its C-‐terminal region are able to generate this destabilization effect, while the middle region, containing the zinc finger domains, can’t. RNA isolation from polyribosomal fractions showed that tethering of ZC3H32 also decreases the translation of the reporter RNA. High-‐throughput sequencing of poly-‐A+ RNA from a ZC3H32-‐RNAi cell line revealed 20 RNAs that were up-‐regulated upon ZC3H32 knock-‐down and thus might be putative targets. The upregulation was confirmed for three of these candidate RNAs. The majority of the putative ZC3H32 targets play a role in the trypanosome’s energy metabolism and 15 of them are up-‐regulated in procyclics. These results suggest that ZC3H32 might be involved in the stage-‐specific regulation of these RNAs in the bloodstream form.
|Supervisor:||Clayton, Prof. Dr. Christine|
|Date of thesis defense:||11 June 2014|
|Date Deposited:||20 Jun 2014 06:18|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||570 Life sciences|