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Purification of specific mRNP via the nascent polypeptide The RNA Binding Proteins ZC3H22 and ZC3H38

Inchaustegui Gil, Diana Patricia

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Abstract

The main determinants of the cytoplasmic fate of an mRNA are the interactions between RNA-binding proteins (RBPs) and cis-regulatory motifs, present in the untranslated regions (UTRs) of the mRNAs. It is expected that translating mRNAs associate with many RBPs and other proteins that are recruited but do not necessarily interact directly with the mRNA, thus forming the messenger ribonucleo-protein particles (mRNPs). In the present dissertation, a method to detect key factors involved in the regulation of gene expression in trypanosomes was tested. The aim was to affinity purify specific ribosome-associated mRNPs and detect their protein components. The purification relies on three streptavidin binding peptides (3SBPs) at the N-terminus of the nascent polypeptide. These 3SBPs connected to the actively translating mRNAs on polyribosomes will bind to the streptavidin matrix. The average yield of the reporter mRNA was 16% relative to the input polysomal fraction. The reporter was also eight-fold enriched compared to the housekeeping gene. The method was validated using a known RNA-protein interaction in trypanosomes. A zinc finger protein, ZC3H11, binds to an AU-rich element present in the HSP70 3’-UTR and stabilizes this mRNA upon heat shock. Two independent purifications were made, one using a reporter containing the complete HSP70 3’-UTR and another without the AU-rich element, as a negative control. Indeed, ZC3H11 was detected in the purification when the AU-rich element was present in the HSP70 3’-UTR, and was absent from the control purification. The limitation of the method was the detection by mass spectrometry. Furthermore, two RBPs were studied, zinc finger proteins ZC3H22 and ZC3H38. ZC3H22 was found by quantitative mass spectrometry when purifying the EP 3’-UTR reporter using the affinity purification method described above. This protein seems to be present in the polyribosomes but down regulation of the gene by RNAi was only achieved to 30%, the protein was still produced and no changes in the growth phenotype observed. Only a single conditional knockout was obtained but not the double knockout. The role of this protein in procyclic trypanosomes is still not discovered. The protein ZC3H38 was previously found in our laboratory by Dr. Erben to increase target mRNA expression. My Tethering assay showed indeed, that the protein stabilized the reporter mRNA approximately 2-fold and increased 1.5-folds the amount of reporter protein produced. Analysing different parts of the protein by tethering assay also indicates that a region containing the HNPY domain might be responsible for the reporter mRNA stabilization. ZC3H38 protein is mainly localized in the cytoplasm. RNAi against ZC3H38 mRNA presents a change in the growth phenotype after 24 hours of tetracycline induction in bloodstream trypanosomes. Currently, more experiments are being carried out in order to further characterize this protein.

Item Type: Dissertation
Supervisor: Clayton, Prof. Dr Christine
Date of thesis defense: 26 June 2015
Date Deposited: 13 Jul 2015 07:22
Date: 2015
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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