Etudes structurales de Mob1, une protéine impliquée dans la sortie de mitose

Les protéines de la famille Mob1-Phocéine sont retrouvées dans toutes les cellules eucaryotes. Chez la levure, ces protéines sont des sous-unités régulatrices des protéines kinases NDR (Nuclear Dbf2 Related) impliquées dans le contrôle du cycle cellulaire. Leur fonction biologique est actuellement peu décrite. Dans ce travail, nous présentons la structure en solution de la protéine Mob1 de Xenopus laevis résolue par RMN hétéronucléaire multidimensionelle. La structure présente un repliement constitué d'un faisceau de quatre hélices central lévogyre, d'une hélice de liaison, de deux petites hélices flanquantes et d'une longue boucle flexible. La structure et des études par spéctrométrie de flamme ont permis de mettre en évidence un site de liaison au zinc. L'observation des changements des déplacements chimiques après addition d'un peptide représentant la région basique du domaine régulateur N-terminal de la kinase NDR ont permis d'identifier et de localiser une interaction spécifique entre la protéine Mob1 et la kinase. Nos données suggèrent que la kinase NDR est l'homologue fonctionnel de Dbf2 dans les cellules animales et contribue à la compréhension de la fonction moléculaire des protéines Mob1.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

FRET Analysis of RNA –Protein Interactions Using Spinach Aptamers

The method development to analyze direct RNA–protein interaction is of high importance as not many homogeneous assay formats are available. The discovery of fluorescent light-up aptamers (FLAPs), short RNA aptamers that switch the fluorescence of small, cell-permeable, and nontoxic organic chromophores on, paved the road for their utilization in direct RNA–protein interactions. The combination with fluorescent proteins as biological fluorophores enabled the development of homogeneous assays that are in principle even encodable on genomic level. Here the rules and methods to design a homogeneous in vitro assay for the detection and quantification of a direct RNA–protein interaction will be described. The design and application of a homogeneous assay to observe and quantify the interaction of the Pseudomonas aeruginosa bacteriophage coat protein 7 (PP7) with its binding RNA sequence (pp7-RNA) will be shown. For this, the Spinach-DFHBI aptamer as RNA fusion and the red fluorescent mCherry as protein fusion was used. The methods presented here do not require any chemical modification of proteins or RNAs which make them relatively easy to use and to adopt on other systems. As all fluorophores are fusion tags to the according biomolecules, standard cloning strategies and molecular biology technologies are sufficient and make this method available for a broad community of researchers

Backbone resonance assignment of the human uracil DNA glycosylase-2

International audienceThe HIV-1 viral protein R (Vpr) is incorporated into virus particle during budding suggesting that its presence in the mature virion is required in the early steps of the virus life cycle in newly infected cells. Vpr is released into the host cell cytoplasm to participate to the translocation of the preintegration complex (PIC) into the nucleus for integration of the viral DNA into the host genome. Actually, Vpr plays a key role in the activation of the transcription of the HIV-1 long terminal repeat (LTR), mediates cell cycle arrest in G2 to M transition, facilitates apoptosis and controls the fidelity of reverse transcription. Moreover, Vpr drives the repair enzyme uracil DNA glycosylase (UNG2) towards degradation. UNG2 has a major role in "Base excision repair" (BER) whose main function is to maintain genome integrity by controlling DNA uracilation. The interaction of Vpr with the cellular protein UNG2 is a key event in various stages of retroviral replication and its role remains to be defined. We have performed the structural study of UNG2 by NMR and we report its (1HN, 15N, 13Cα, 13Cβ and 13C') chemical shift backbone assignment and its secondary structure in solution as predicted by TALOS-N. We aim to determine with accuracy by NMR, the residues of UNG2 interacting with Vpr, characterize their interaction and use the local structure of UNG2 and its interface with Vpr to propose potential ligands disturbing this interaction

Letter to the Editor: Resonance Assignments and Topology of the 15 N, 13 C Labelled 23 kDa Core Domain of Xenopus Mob1

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A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold

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Selective RNase H Cleavage of Target RNAs from a tRNA Scaffold

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NMR Solution Structure of Mob1, a Mitotic Exit Network Protein and its Interaction with an NDR Kinase Peptide

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Design of cross-linked RNA/protein complexes for structural studies

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