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Structural studies of proteins involved in mitochondrial RNA processing in Trypanosome brucei

Defense Thèse de doctorat : Univ. Genève, 2015 - Sc. 4828 - 2015/09/25
Abstract The mitochondrion is an essential membrane-bound organelle that functions as an energy-producing factory. For this thesis, we focused our attention on critical proteins involved in mitoRNAs biogenesis in the model organism Trypanosoma brucei belonging to the genus kinetoplastids. In the first project, we carried out structural and functional studies of an essential protein named RNA Editing TUTase 1 (RET1) that participates in the maturation process of guide RNAs by adding a poly (U) tails at their 3'-end. Furthermore, the RET1 protein also forms an integral part of the polyadenylation/uridylation complex, which renders mRNA translationally competent. We have determined the atomic structure of RET1 to a resolution of up to 1.6 Å in its apo form, bound to its substrate UMPNPP, a non-hydrolysable form of UTP to a resolution of 1.8 Å and a catalytically inactive RET1 protein (D473A) bound to UTP to a resolution of 2.2 Å. This study provides insights into the overall architecture of the enzyme, the UTP binding specificity and highlights important residues for RNA binding and catalytic activity. In the second project, we purified and crystallized a heterodimeric complex that belongs to the family of Pentatricopeptide Repeat (PPR) family called Kinetoplast polyadenylation/uridylation factors (KPAFs) 1&2. These two proteins form an integral part of the polyadenylation/uridylation complex and help coordinate the addition of long A/U tails that are essential for producing translationally competent mRNAs. We have successfully expressed, purified, crystallized the KPAF complex and collected diffraction data up to 4.8 Å. Taken together, the work in this thesis has provided insights into UTP specificity, RNA recognition and domain architecture of RET1. The thesis also provides an advanced framework for the purification and crystallization of the KPAF complex for future researchers in the field.
Keywords MitochondriaProcessingTrypanosomeBruceiRNAPolyuridylationEditingStructureAtomicModel
URN: urn:nbn:ch:unige-760285
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Research group Groupe Schalch
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RAJAPPA, Lional. Structural studies of proteins involved in mitochondrial RNA processing in Trypanosome brucei. Université de Genève. Thèse, 2015. doi: 10.13097/archive-ouverte/unige:76028 https://archive-ouverte.unige.ch/unige:76028

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Deposited on : 2015-10-13

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