Caractérisation structurale et fonctionnelle de la protéine Bcd1, impliquée dans la biogenèse des snoRNP à boîtes C/D chez la levure Saccharomyces cerevisiae

Abstract : The protein Bcd1 is a nuclear factor essential for the cellular viability of the yeast Saccharomyces cerevisiae. It is described as required to ensure box C/D snoRNA stability. These small non-coding RNAs associate with an invariable set of 4 proteins to form the box C/D snoRNPs that are crucial players in ribosome biogenesis. Indeed, some of these particles participate in mechanisms for the maturation of the ribosomal RNA precursor (prerRNA) and the vast majority of the other particles are catalysts of 2’-O-methylation of riboses. Bcd1p is not present in mature particles, but is one of the assembly factors in addition to the Rsa1p:Hit1p and R2TP (Rvb1p:Rvb2p:Tah1p:Pih1p) sub-complexes. Our analysis of the different Bcd1p fragments has firstly shown that the essential function of Bcd1p relies on its N-terminal region (residues 1 to 96). It comprises a double zinc finger domain from the zf-HIT family, also present in another box C/D snoRNP assembly factor, the protein Hit1. We solved the 3D solution structure of these two zinc fingers and showed that these are modules for the interaction of Bcd1p with the Rvb1/2 proteins. Secondly, we identified the C-terminal region (residues 120 to 303) of Bcd1p as being sufficient to interact with the histone chaperone Rtt106p. The 3D solution structure of this domain of Bcd1p was determined by NMR. Different approaches of hydrogen/deuterium kinetic exchange and cross-link experiments followed by mass spectrometry analysis, NMR titration, and SAXS allowed us to obtain information about the interaction surfaces on each of the two proteins. A fragment defined from NMR data on the free Bcd1p allowed us to obtain crystals of the Bcd1p:Rtt106p complex, opening the perspective to solve its 3D structure by X-ray diffraction. Furthermore, functional studies started in order to determine the importance of this complex formation in box C/D snoRNP biogenesis and the impact of Bcd1p on the interaction of Rtt106p with nucleosomes
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Benoît Bragantini. Caractérisation structurale et fonctionnelle de la protéine Bcd1, impliquée dans la biogenèse des snoRNP à boîtes C/D chez la levure Saccharomyces cerevisiae. Biochimie [q-bio.BM]. Université de Lorraine, 2016. Français. ⟨NNT : 2016LORR0295⟩. ⟨tel-01752418⟩

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