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Accueil > Départements > Biologie des Génomes > Olivier NAMY : Génomique, Structure et Traduction

Publications de l’équipe

2017


  • L. Bidou, O. Bugaud, V. Belakhov, T. Baasov, et O. Namy, « Characterization of new-generation aminoglycoside promoting premature termination codon readthrough in cancer cells », RNA biology, p. 1-11, 2017.
    Résumé : Nonsense mutations, generating premature termination codons (PTCs), account for 10% to 30% of the mutations in tumor suppressor genes. Nonsense translational suppression, induced by small molecules including gentamicin and G418, has been suggested as a potential therapy to counteract the deleterious effects of nonsense mutations in several genetic diseases and cancers. We describe here that NB124, a synthetic aminoglycoside derivative recently developed especially for PTC suppression, strongly induces apoptosis in human tumor cells by promoting high level of PTC readthrough. Using a reporter system, we showed that NB124 suppressed several of the PTCs encountered in tumor suppressor genes, such as the p53 and APC genes. We also showed that NB124 counteracted p53 mRNA degradation by nonsense-mediated decay (NMD). Both PTC suppression and mRNA stabilization contributed to the production of a full-length p53 protein capable of activating p53-dependent genes, thereby specifically promoting high levels of apoptosis. This new-generation aminoglycoside thus outperforms the only clinically available readthrough inducer (gentamicin). These results have important implications for the development of personalised treatments of PTC-dependent diseases and for the development of new drugs modifying translation fidelity.
    Mots-clés : Aminoglycoside, Apoptosis, cancer, DBG, GST, p53, stop codon readthrough.

  • O. Bugaud, N. Barbier, H. Chommy, N. Fiszman, A. Le Gall, D. Dullin, M. Saguy, N. Westbrook, K. Perronet, et O. Namy, « Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single molecule fluorescence microscopy », RNA (New York, N.Y.), 2017.
    Résumé : Protein synthesis is a complex multi-step process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labelling of the ribosomes are much more complicated. Here we use a non-canonical translation initiation based on internal ribosome entry sites (IRES) and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA. We explore initiation by two types of IRES, the intergenic IRES of Cricket Paralysis virus (CrPV) and the hepatitis C (HCV) IRES, and show that they both strongly limit the rate of the first elongation steps compared to the following ones suggesting that those first elongation cycles do not correspond to a canonical elongation. This new system opens the possibility to study both IRES-mediated initiation and elongation kinetics of eukaryotic translation and will undoubtedly be a valuable tool to investigate the role of translation machinery modifications in human diseases.
    Mots-clés : DBG, Eukaryotic translation, GST, IRES, RNA, Single molecule.

2016


  • A. Baudin-Baillieu, I. Hatin, R. Legendre, et O. Namy, « Translation Analysis at the Genome Scale by Ribosome Profiling », Methods in Molecular Biology (Clifton, N.J.), vol. 1361, p. 105-124, 2016.
    Résumé : Ribosome profiling is an emerging approach using deep sequencing of the mRNA part protected by the ribosome to study protein synthesis at the genome scale. This approach provides new insights into gene regulation at the translational level. In this review we describe the protocol to prepare polysomes and extract ribosome protected fragments before to deep sequence them.
    Mots-clés : DBG, Genome, GST, High-Throughput Nucleotide Sequencing, Polyribosomes, Protein Biosynthesis, Recoding, Ribo-seq, ribosome profiling, Ribosomes, RNA, Messenger, Translation regulation.

  • I. Sermet-Gaudelus et O. Namy, « New Pharmacological Approaches to Treat Patients with Cystic Fibrosis with Nonsense Mutations », American Journal of Respiratory and Critical Care Medicine, vol. 194, nᵒ 9, p. 1042-1044, 2016.

  • P. C. Thiaville, R. Legendre, D. Rojas-Benítez, A. Baudin-Baillieu, I. Hatin, G. Chalancon, A. Glavic, O. Namy, et V. de Crécy-Lagard, « Global translational impacts of the loss of the tRNA modification t(6)A in yeast », Microbial Cell (Graz, Austria), vol. 3, nᵒ 1, p. 29-45, 2016.
    Résumé : The universal tRNA modification t(6)A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t(6)A37 leads to severe growth defects in baker's yeast, phenotypes similar to those caused by defects in mcm(5)s(2)U34 synthesis. Mutants in mcm(5)s(2)U34 can be suppressed by overexpression of tRNA(Lys)UUU, but we show t(6)A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t(6)A and mcm(5)s(2)U are not determinants for each other's formation. Our results suggest that t(6)A deficiency, like mcm(5)s(2)U deficiency, leads to protein folding defects, and show that the absence of t(6)A led to stress sensitivities (heat, ethanol, salt) and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t(6)A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs) were increased in the mutants. The global consequences on translation caused by t(6)A absence were examined by ribosome profiling. Interestingly, the absence of t(6)A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t(6)A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I34:C3 pairs while increasing the elongation rate of rare tRNAs and G34:U3 pairs. This work reveals that the consequences of t(6)A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes.
    Mots-clés : DBG, GST, modified nucleosides, ribosome profiling, t6A, translation, tRNA.

2015


  • S. Blanchet, M. Rowe, T. Von der Haar, C. Fabret, S. Demais, M. J. Howard, et O. Namy, « New insights into stop codon recognition by eRF1 », Nucleic Acids Research, vol. 43, nᵒ 6, p. 3298-3308, 2015.
    Résumé : In eukaryotes, translation termination is performed by eRF1, which recognizes stop codons via its N-terminal domain. Many previous studies based on point mutagenesis, cross-linking experiments or eRF1 chimeras have investigated the mechanism by which the stop signal is decoded by eRF1. Conserved motifs, such as GTS and YxCxxxF, were found to be important for termination efficiency, but the recognition mechanism remains unclear. We characterized a region of the eRF1 N-terminal domain, the P1 pocket, that we had previously shown to be involved in termination efficiency. We performed alanine scanning mutagenesis of this region, and we quantified in vivo readthrough efficiency for each alanine mutant. We identified two residues, arginine 65 and lysine 109, as critical for recognition of the three stop codons. We also demonstrated a role for the serine 33 and serine 70 residues in UGA decoding in vivo. NMR analysis of the alanine mutants revealed that the correct conformation of this region was controlled by the YxCxxxF motif. By combining our genetic data with a structural analysis of eRF1 mutants, we were able to formulate a new model in which the stop codon interacts with eRF1 through the P1 pocket.
    Mots-clés : Codon, Terminator, DBG, GST, Models, Molecular, Mutagenesis, Nuclear Magnetic Resonance, Biomolecular, Peptide Termination Factors, Protein Conformation, Protein Structure, Tertiary, Recombinant Proteins, Saccharomyces cerevisiae Proteins.

  • R. Legendre, A. Baudin-Baillieu, I. Hatin, et O. Namy, « RiboTools: a Galaxy toolbox for qualitative ribosome profiling analysis », Bioinformatics (Oxford, England), vol. 31, nᵒ 15, p. 2586-2588, 2015.
    Résumé : MOTIVATION: Ribosome profiling provides genome-wide information about translational regulation. However, there is currently no standard tool for the qualitative analysis of Ribo-seq data. We present here RiboTools, a Galaxy toolbox for the analysis of ribosome profiling (Ribo-seq) data. It can be used to detect translational ambiguities, stop codon readthrough events and codon occupancy. It provides a large number of plots for the visualisation of these events.
    Mots-clés : Codon, Terminator, Computational Biology, Databases, Genetic, DBG, Gene Expression Profiling, Gene Expression Regulation, Genome, Fungal, GST, High-Throughput Nucleotide Sequencing, Humans, Prions, Protein Biosynthesis, Ribosomes, Saccharomyces cerevisiae, Software.
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Publications majeures avant 2015

1.New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae.
Blanchet S, Cornu D, Argentini M, Namy O.
Nucleic Acids Res. 2014 ;42(15):10061-72.

2. Genome-wide translational changes induced by the prion [PSI+].
Baudin-Baillieu A, Legendre R, Kuchly C, Hatin I, Demais S, Mestdagh C, Gautheret D, Namy O.
Cell Rep. 2014 Jul 24 ;8(2):439-48.

3. Sense from nonsense : therapies for premature stop codon diseases.
Bidou L, Allamand V, Rousset JP, Namy O.
Trends Mol Med. 2012 Nov ;18(11):679-88.

4. Rescue of non-sense mutated p53 tumor suppressor gene by aminoglycosides.
Floquet C, Deforges J, Rousset JP, Bidou L.
Nucleic Acids Res. 2011 Apr ;39(8):3350-62.

5. Nucleotide modifications in three functionally important regions of the Saccharomyces cerevisiae ribosome affect translation accuracy.
Baudin-Baillieu A, Fabret C, Liang XH, Piekna-Przybylska D, Fournier MJ, Rousset JP.
Nucleic Acids Res. 2009 Dec ;37(22):7665-77.

6. Molecular dissection of translation termination mechanism identifies two new critical regions in eRF1.
Hatin I, Fabret C, Rousset JP, Namy O.
Nucleic Acids Res. 2009 Apr ;37(6):1789-98.

7. Epigenetic control of polyamines by the prion [PSI+].
Namy O, Galopier A, Martini C, Matsufuji S, Fabret C, Rousset JP.
Nat Cell Biol. 2008 Sep ;10(9):1069-75.

8. A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting.
Namy O, Moran SJ, Stuart DI, Gilbert RJ, Brierley I.
Nature. 2006 May 11 ;441(7090):244-7.

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