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Abstract

The trypanosomatids are a group of eukaryotic parasites (Trypanosoma brucei, Trypanosoma cruzi and Leishmaniab sp.), which causes disease affecting humans and animals. Since most genes do not have individual promoters and regulating transcription factors, the mode of gene expression regulation is post-transcriptional: almost entirely determined by RNA binding proteins, stabilitzing complexes and the mRNA decay machinery. In my PhD, I worked on two projects in Trypanosoma brucei; a screen to identify the genome wide interactors of the CAF1-NOT complex and the role of RNA binding protein ZC3H30 in stress. The decay-promoting deadenylation machinery, the CAF1-NOT complex, degrades poly (A) tails, an event that marks mRNA for degradation. I did a genome-wide yeast-two-hybrid screen to discover novel proteins that might influence CAF1-NOT complex function in the parasite. I looked at the total pool of genes obtained after screening, and assessed the protein class and biological process of the candidates obtained. To assess interaction specificity I compared the yeast-two-hybrid screen data with those obtained from other trypanosome proteins. African trypanosomes undergo stress in their insect host in the form of heat shock or in their human host from the immune reponse, or during fever or intake of drugs. The parasites have developed differential gene expression to cope with stress using RNA binding proteins, The second part of the manuscript focuses on a zinc finger RNA binding protein ZC3H30, which is inessential for the growth of trypanosomes under normal conditions. However, conditions like heat shock, ER stress, oxidative stress and starvation stress, are lethal for the trypanosomes that lack ZC3H30. The absence of ZC3H30 has no effect on stress markers, except that major cytoplasmic HSP70 mRNA is more abundant. I have shown that ectopically expressed ZC3H30, when tethered to reporter mRNA, can reduce its transcript and encoded protein abundance. ZC3H30 is a bona fide component of stress granule; in presence of heat shock, ZC3H30 re-localises to stress granules from the cytoplasm. It pulls down another protein, which also migrates to stress granules upon heat shock and might also have a role in stress management for trypanosomes. By transcriptome profiling, procyclic cells lacking ZC3H30 showed no difference from wild type in transcript abundance, and specific targets were not found. However, procyclic form trypanosomes have a higher higher overall translation rate than wild type cells. Possibly ZC3H30 affects the mis-reading of mRNAs during translation, since the knockouts of ZC3H30 are less sensitive than wild type to aqminoglycoside translation inhibitors like hygromycin. Nevertheless, I have not been able to identify definitively the role of ZC3H30 in stress resistance.