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The suppressive role of 4EIP and PUF3 in gene expression during differentiation of Trypanosoma brucei

Marucha, Kevin

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Abstract

African trypanosomes are flagellated protozoan parasites that cause sleeping sickness in humans and nagana in cattle. During their life cycle, they change their morphology and metabolism through robust gene regulation processes. Trypanosomes have a unique polycistronic gene arrangement and have to rely almost entirely on post-transcriptional regulation mechanisms, which include mRNA processing, export, stability and translation, in order to regulate gene expression. RNA binding proteins and translation factors are involved. Trypanosomes have six eIF4Es of varying cap-binding affinities and five eIF4Gs, suggesting numerous possibilities for translation regulation. TbEIF4E1 does not interact with any of the eIF4Gs but instead interacts with 4E-interacting protein, 4EIP, which is the first focus of this thesis. Both TbEIF4E1 and Tb4EIP repress a reporter mRNA in a tethering assay, but suppression by TbEIF4E1 requires Tb4EIP. Bloodstream form Trypanosoma brucei lacking Tb4EIP have only a mild growth defect. At high parasitemia, bloodstream forms stop dividing, suppress translation and become stumpy forms, which are adapted to differentiate to procyclic forms when taken up by a tsetse fly. Interestingly, lack of Tb4EIP compromises stumpy formation, and the defect can be rescued by a truncated Tb4EIP that is unable to bind TbEIF4E1. Tb4EIP knockout stumpy forms have abnormally high protein synthesis rates indicating that Tb4EIP is required for translation suppression during differentiation to the stumpy form. RNA binding protein PUF3 is among 11 T. brucei pumilio domain containing proteins. Little was known about PUF3 save for its repression of a reporter mRNA and that it co-purifies with poly(A) mRNA. It is therefore the second focus of this thesis. PUF3-depleted monomorphic and pleomorphic bloodstream cells have a marginal growth defect but PUF3 knockout cells strangely lack this defect. Interestingly, when put to differentiate to stumpy and procyclic forms, PUF3-depleted pleomorphic bloodstream cells experience a delayed differentiation manifested by a low expression of the stumpy form marker PAD1 and procyclic surface coat proteins EP/GPEET. Nevertheless these cells eventually differentiated to viable procyclic forms. Surprisingly, pleomorphic bloodstream cells without PUF3 later seemed to adapt and lack this differentiation defect. The defect however persists in monomorphic cells, suggesting an adaptation mechanism in pleomorphic cells that equilibrates the cellular metabolism to life without PUF3. Using TRIBE (Targets of RNA binding proteins Identified by Editing) 295 putative targets of PUF3 were identified in stumpy-like cells. 79 of these targets are enriched in bloodstream forms while only 12 are enriched in procyclic forms, consistent with a role of PUF3 as a repressor during differentiation to procyclic forms. These targets include mRNAs encoding cytoskeleton proteins, protein kinases, RNA binding proteins, leucine rich repeat proteins, expression-site associated genes, chaperones and translation factors. The results here suggest that Tb4EIP and PUF3 fine-tune gene expression in readiness for differentiation.

Item Type: Dissertation
Supervisor: Clayton, Prof. Dr. Christine
Date of thesis defense: 7 December 2018
Date Deposited: 17 Jan 2019 09:38
Date: 2019
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
610 Medical sciences Medicine
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