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

Publications de l’équipe

2019


  • A. Aliouat, I. Hatin, P. Bertin, P. François, V. Stierlé, O. Namy, S. Salhi, et O. Jean-Jean, « Divergent effects of translation termination factor eRF3A and nonsense-mediated mRNA decay factor UPF1 on the expression of uORF carrying mRNAs and ribosome protein genes », RNA biology, p. 1-13, oct. 2019.
    Résumé : In addition to its role in translation termination, eRF3A has been implicated in the nonsense-mediated mRNA decay (NMD) pathway through its interaction with UPF1. NMD is a RNA quality control mechanism, which detects and degrades aberrant mRNAs as well as some normal transcripts including those that harbour upstream open reading frames in their 5' leader sequence. In this study, we used RNA-sequencing and ribosome profiling to perform a genome wide analysis of the effect of either eRF3A or UPF1 depletion in human cells. Our bioinformatics analyses allow to delineate the features of the transcripts controlled by eRF3A and UPF1 and to compare the effect of each of these factors on gene expression. We find that eRF3A and UPF1 have very different impacts on the human transcriptome, less than 250 transcripts being targeted by both factors. We show that eRF3A depletion globally derepresses the expression of mRNAs containing translated uORFs while UPF1 knockdown derepresses only the mRNAs harbouring uORFs with an AUG codon in an optimal context for translation initiation. Finally, we also find that eRF3A and UPF1 have opposite effects on ribosome protein gene expression. Together, our results provide important elements for understanding the impact of translation termination and NMD on the human transcriptome and reveal novel determinants of ribosome biogenesis regulation.
    Mots-clés : DBG, eRF3, GSPT1, GST, nonsense-mediated mRNA decay, ribosome protein genes, translation termination, uORF, UPF1.

  • E. M. S. Brito, V. M. Romero-Núñez, C. A. Caretta, P. Bertin, J. C. Valerdi-Negreros, R. Guyoneaud, et M. Goñi-Urriza, « The bacterial diversity on steam vents from Paricutín and Sapichu volcanoes », Extremophiles: Life Under Extreme Conditions, févr. 2019.
    Résumé : Vapor steam vents are prevailing structures on geothermal sites in which local geochemical conditions allow the development of extremophilic microorganisms. We describe the structure of the prokaryotic community able to grow on the walls and rocks of such microecosystems in two terrestrial Mexican volcanoes: Paricutín (PI and PII samples) and its satellite Sapichu (S sample). The investigated samples showed similar diversity indices, with few dominant OTUs (abundance > 1%): 21, 16 and 23, respectively for PI, PII and S. However, each steam vent showed a particular community profile: PI was dominated by photosynthetic bacteria (Cyanobacteria and Chloroflexia class), PII by Actinobacteria and Proteobacteria, and S by Ktedonobacteria class, Acidobacteria and Cyanobacteria phyla. Concerning the predicted metabolic potential, we found a dominance of cellular pathways, especially the ones for energy generation with metabolisms for sulfur respiration, nitrogen fixation, methanogenesis, carbon fixation, photosynthesis, and metals, among others. We suggest a different maturity stage for the three studied fumaroles, from the youngest (PI) to the oldest (S and PII), also influenced by the temperature and other geochemical parameters. Furthermore, four anaerobic strains were isolated, belonging to Clostridia class (Clostridium sphenoides, C. swellfunanium and Anaerocolumna cellulosilytica) and to Bacilli class (Paenibacillus azoreducens).
    Mots-clés : Anaerobic bacteria, DBG, Extreme environment, GST, Microbial biodiversity, Predictive metagenomics profiling, Volcanic fumaroles.

  • E. Durand, I. Gagnon-Arsenault, J. Hallin, I. Hatin, A. K. Dube, L. Nielly-Thibault, O. Namy, et C. R. Landry, « Turnover of ribosome-associated transcripts from de novo ORFs produces gene-like characteristics available for de novo gene emergence in wild yeast populations », Genome Research, vol. 29, nᵒ 6, p. 932-943, juin 2019.
    Résumé : Little is known about the rate of emergence of de novo genes, what their initial properties are, and how they spread in populations. We examined wild yeast populations (Saccharomyces paradoxus) to characterize the diversity and turnover of intergenic ORFs over short evolutionary timescales. We find that hundreds of intergenic ORFs show translation signatures similar to canonical genes, and we experimentally confirmed the translation of many of these ORFs in laboratory conditions using a reporter assay. Compared with canonical genes, intergenic ORFs have lower translation efficiency, which could imply a lack of optimization for translation or a mechanism to reduce their production cost. Translated intergenic ORFs also tend to have sequence properties that are generally close to those of random intergenic sequences. However, some of the very recent translated intergenic ORFs, which appeared <110 kya, already show gene-like characteristics, suggesting that the raw material for functional innovations could appear over short evolutionary timescales.
    Mots-clés : DBG, drosophila-yakuba, evolution, genome, GST, in-vivo, map, origin, sequences, translation.

2018



  • S. Blanchet, D. Cornu, I. Hatin, H. Grosjean, P. Bertin, et O. Namy, « Deciphering the reading of the genetic code by near-cognate tRNA », Proceedings of the National Academy of Sciences, vol. 115, nᵒ 12, p. 3018-3023, mars 2018.

  • S. Bouffard, E. Dambroise, A. Brombin, S. Lempereur, I. Hatin, M. Simion, R. Corre, F. Bourrat, J. - S. Joly, et F. Jamen, « Fibrillarin is essential for S-phase progression and neuronal differentiation in zebrafish dorsal midbrain and retina », Developmental Biology, févr. 2018.
    Résumé : Fibrillarin (Fbl) is a highly conserved protein that plays an essential role in ribosome biogenesis and more particularly in the methylation of ribosomal RNAs and rDNA histones. In cellular models, FBL was shown to play an important role in tumorigenesis and stem cell differentiation. We used the zebrafish as an in vivo model to study Fbl function during embryonic development. We show here that the optic tectum and the eye are severely affected by Fbl depletion whereas ventral regions of the brain are less impacted. The morphogenesis defects are associated with impaired neural differentiation and massive apoptosis. Polysome gradient experiments show that fbl mutant larvae display defects in ribosome biogenesis and activity. Strikingly, flow cytometry analyses revealed different S-phase profiles between wild-type and mutant cells, suggesting a defect in S-phase progression.
    Mots-clés : Cell cycle regulation, Danio rerio, DBG, Differentiation, GST, Neural progenitors, Optic tectum, Ribosome biogenesis.


  • W. Briand, O. Dao, G. Garnier, R. Guegan, B. Marta, C. Maupu, J. Miesch, K. Papadopoulo, A. Radoux, J. Rojahn, Y. Zhu, C. Aubry, P. Bouloc, S. Bury-Moné, A. Ferré, S. Lautru, O. Namy, et M. Sabeti-Azad, « Dégradation d’un anticancéreux dans les eaux usées - Une médaille d’or pour l’équipe GO Paris-Saclay », Medecine Sciences: M/S, vol. 34, nᵒ 12, p. 1111-1114, déc. 2018.
    Résumé : iGEM (pour <i>international genetically engineered machine)<i/> est un concours international autour de la biologie synthétique réunissant des étudiants de toutes disciplines (mathématiques, physique, biologie, arts, etc.). « L’objectif est de construire un système biologique fonctionnel complexe, en assemblant des composants individuels moléculaires simples et standardisés (fragments d’ADN), appelés « briques biologiques » (biobriques), sorte de « legos » moléculaires, entreposés au MIT (<i>Massachusetts Institute of Technology<i/>) (le <i>registry of standard biological parts<i/> contient environ 20 000 biobriques). C’est une démarche proche de celle de l’ingénieur qui assemble des circuits électroniques ». En 2004, lors de sa création par le MIT (<b>→<b/>), la compétition iGEM regroupait une quarantaine de projets ; 14 ans plus tard, elle accueille 350 équipes (6 000 étudiants, avec leurs instructeurs) issues des universités du monde entier. Elle culmine en un <i>Giant Jamboree<i/> de quatre jours à Boston en novembre, au cours duquel les équipes présentent leur projet. Le « wiki » de la compétition (www.igem.org) présente l’ensemble des projets ainsi que le palmarès. Cette année, ont été décernées 114 médailles d’or, 68 d’argent et 107 de bronze. Neuf équipes françaises étaient engagées.<b>(→) Voir l’article de J. Peccoud et L. Coulombel, dont certains passages sont repris dans ce « chapo », m/s n° 5, mai 2007, page 551<b/>
    Mots-clés : ACTINO, Animals, Antineoplastic Agents, Biodegradation, Environmental, Bioreactors, CHERDIR, DBG, Escherichia coli, Escherichia coli Proteins, gamma-Glutamyl Hydrolase, GST, Humans, I2BC, Metabolic Engineering, Methotrexate, MICROBIO, Multienzyme Complexes, Organisms, Genetically Modified, Peptide Synthases, RNASTR, SRRB, Waste Water, Water Pollutants, Chemical, Water Purification.
    Pièce jointe Full Text PDF 1.5 Mo (source)
    Pièce jointe Full Text PDF 1.5 Mo (source)

  • N. Planchard, P. Bertin, M. Quadrado, C. Dargel-Graffin, I. Hatin, O. Namy, et H. Mireau, « The translational landscape of Arabidopsis mitochondria », Nucleic Acids Research, vol. 46, nᵒ 12, p. 6218-6228, juill. 2018.
    Résumé : Messenger RNA translation is a complex process that is still poorly understood in eukaryotic organelles like mitochondria. Growing evidence indicates though that mitochondrial translation differs from its bacterial counterpart in many key aspects. In this analysis, we have used ribosome profiling technology to generate a genome-wide snapshot view of mitochondrial translation in Arabidopsis. We show that, unlike in humans, most Arabidopsis mitochondrial ribosome footprints measure 27 and 28 bases. We also reveal that respiratory subunits encoding mRNAs show much higher ribosome association than other mitochondrial mRNAs, implying that they are translated at higher levels. Homogenous ribosome densities were generally detected within each respiratory complex except for complex V, where higher ribosome coverage corroborated with higher requirements for specific subunits. In complex I respiratory mutants, a reorganization of mitochondrial mRNAs ribosome association was detected involving increased ribosome densities for certain ribosomal protein encoding transcripts and a reduction in translation of a few complex V mRNAs. Taken together, our observations reveal that plant mitochondrial translation is a dynamic process and that translational control is important for gene expression in plant mitochondria. This study paves the way for future advances in the understanding translation in higher plant mitochondria.
    Mots-clés : DBG, GST.

  • I. Pranke, L. Bidou, N. Martin, S. Blanchet, A. Hatton, S. Karri, D. Cornu, B. Costes, B. Chevalier, D. Tondelier, E. Girodon, M. Coupet, A. Edelman, P. Fanen, O. Namy, I. Sermet-Gaudelus, et A. Hinzpeter, « Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons », ERJ open research, vol. 4, nᵒ 1, janv. 2018.
    Résumé : Premature termination codons (PTCs) are generally associated with severe forms of genetic diseases. Readthrough of in-frame PTCs using small molecules is a promising therapeutic approach. Nonetheless, the outcome of preclinical studies has been low and variable. Treatment efficacy depends on: 1) the level of drug-induced readthrough, 2) the amount of target transcripts, and 3) the activity of the recoded protein. The aim of the present study was to identify, in the cystic fibrosis transmembrane conductance regulator (CFTR) model, recoded channels from readthrough therapy that may be enhanced using CFTR modulators. First, drug-induced readthrough of 15 PTCs was measured using a dual reporter system under basal conditions and in response to gentamicin and negamycin. Secondly, exon skipping associated with these PTCs was evaluated with a minigene system. Finally, incorporated amino acids were identified by mass spectrometry and the function of the predicted recoded CFTR channels corresponding to these 15 PTCs was measured. Nonfunctional channels were subjected to CFTR-directed ivacaftor-lumacaftor treatments. The results demonstrated that CFTR modulators increased activity of recoded channels, which could also be confirmed in cells derived from a patient. In conclusion, this work will provide a framework to adapt treatments to the patient's genotype by identifying the most efficient molecule for each PTC and the recoded channels needing co-therapies to rescue channel function.
    Mots-clés : DBG, GST.

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, févr. 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.), août 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.

  • J. Erales, V. Marchand, B. Panthu, S. Gillot, S. Belin, S. E. Ghayad, M. Garcia, F. Laforêts, V. Marcel, A. Baudin-Baillieu, P. Bertin, Y. Couté, A. Adrait, M. Meyer, G. Therizols, M. Yusupov, O. Namy, T. Ohlmann, Y. Motorin, F. Catez, et J. - J. Diaz, « Evidence for rRNA 2'-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes », Proceedings of the National Academy of Sciences of the United States of America, nov. 2017.
    Résumé : Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2'-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2'-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.
    Mots-clés : 2′-O-methylation, DBG, fibrillarin, GST, ribosomal RNA, RNA epigenetics, translational control.

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, nov. 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, janv. 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, mars 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, août 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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