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Accueil > Départements > Biologie des Génomes > Dominique FOURMY : Structure et dynamique des ARN

Publications de l’équipe

2017


  • M. Nakashima, R. Yamagami, C. Tomikawa, Y. Ochi, T. Moriya, H. Asahara, D. Fourmy, S. Yoshizawa, T. Oshima, et H. Hori, « Long and branched polyamines are required for maintenance of the ribosome, tRNA(His) and tRNA(Tyr) in Thermus thermophilus cells at high temperatures », Genes to Cells: Devoted to Molecular & Cellular Mechanisms, 2017.
    Résumé : Thermus thermophilus is an extremely thermophilic eubacterium that produces various polyamines. Aminopropylagmatine ureohydrolase (SpeB) and SAM decarboxylase-like protein 1 (SpeD1) are involved in the biosynthesis of spermidine from arginine. Because long and branched polyamines in T. thermophilus are synthesized from spermidine, the speB and speD1 gene-deleted strains (ΔspeB and ΔspeD1, respectively) cannot synthesize long and branched polyamines. Although neither strain grew at high temperatures (>75°C) in minimal medium, both strains survived at 80°C when they were cultured at 70°C until the mid-log phase and then shifted to 80°C. We therefore prepared the ΔspeB and ΔspeD1 cells using this culture method. Microscopic analysis showed that both strains can survive for 10 h after the temperature shift. Although the modification levels of 2'-O-methylguanosine at position 18, N(7) -methylguanosine at position 46, 5-methyluridine at position 54 and N(1) -methyladenosine at position 58 in the class I tRNA from both strains were normal, amounts of tRNA(Tyr) , tRNA(His) , rRNAs and 70S ribosomes were decreased after the temperature shift. Furthermore, in vivo protein synthesis in both strains was completely lost 10 h after the temperature shift. Thus, long and branched polyamines are required for at least the maintenance of 70S ribosome and some tRNA species at high temperatures.
    Mots-clés : DBG, RNASTR.

  • T. Plénat, S. Yoshizawa, et D. Fourmy, « DNA-Guided Delivery of Single Molecules into Zero-Mode Waveguides », ACS applied materials & interfaces, 2017.
    Résumé : Zero-mode waveguides (ZMWs) are powerful analytical tools corresponding to optical nanostructures fabricated in a thin metallic film capable of confining an excitation volume to the range of attoliters. This small volume of confinement allows single-molecule fluorescence experiments to be performed at physiologically relevant concentrations of fluorescently labelled biomolecules. Exactly one molecule to be studied must be attached at the floor of the ZMW for signal detection and analysis; however, the massive parallelism of these nanoarrays suffers from a Poissonian-limited distribution of these biomolecules. To date, there is no method available that provides full single molecule occupancy of massively arrayed ZMWs. Here we report the performance of a DNA-guided method that uses steric exclusion properties of large DNA molecules to bias the Poissonian-limited delivery of single molecules. Non-Poissonian statistics were obtained with DNA molecules that contain a free-biotinylated extremity for efficient binding to the floor of the ZMW, which resulted in a decrease of accessibility for a second molecule. Both random coil and condensed DNA conformations drove non-Poissonian single-molecule delivery into ZMWs arrays. The results suggest that an optimal balance between rigidity and flexibility of the macromolecule is critical for favourable accessibility and single occupancy. The optimized method provides means for full exploitation of these massively parallelized analytical tools.
    Mots-clés : DBG, RNASTR.

2016


  • M. Costa, H. Walbott, D. Monachello, E. Westhof, et F. Michel, « Crystal structures of a group II intron lariat primed for reverse splicing », Science (New York, N.Y.), vol. 354, nᵒ 6316, 2016.
    Résumé : The 2'-5' branch of nuclear premessenger introns is believed to have been inherited from self-splicing group II introns, which are retrotransposons of bacterial origin. Our crystal structures at 3.4 and 3.5 angstrom of an excised group II intron in branched ("lariat") form show that the 2'-5' branch organizes a network of active-site tertiary interactions that position the intron terminal 3'-hydroxyl group into a configuration poised to initiate reverse splicing, the first step in retrotransposition. Moreover, the branchpoint and flanking helices must undergo a base-pairing switch after branch formation. A group II-based model of the active site of the nuclear splicing machinery (the spliceosome) is proposed. The crucial role of the lariat conformation in active-site assembly and catalysis explains its prevalence in modern splicing.
    Mots-clés : DBG, RIBOZYMO, RNASTR.

  • H. Grosjean et E. Westhof, « An integrated, structure- and energy-based view of the genetic code », Nucleic Acids Research, vol. 44, nᵒ 17, p. 8020-8040, 2016.
    Résumé : The principles of mRNA decoding are conserved among all extant life forms. We present an integrative view of all the interaction networks between mRNA, tRNA and rRNA: the intrinsic stability of codon-anticodon duplex, the conformation of the anticodon hairpin, the presence of modified nucleotides, the occurrence of non-Watson-Crick pairs in the codon-anticodon helix and the interactions with bases of rRNA at the A-site decoding site. We derive a more information-rich, alternative representation of the genetic code, that is circular with an unsymmetrical distribution of codons leading to a clear segregation between GC-rich 4-codon boxes and AU-rich 2:2-codon and 3:1-codon boxes. All tRNA sequence variations can be visualized, within an internal structural and energy framework, for each organism, and each anticodon of the sense codons. The multiplicity and complexity of nucleotide modifications at positions 34 and 37 of the anticodon loop segregate meaningfully, and correlate well with the necessity to stabilize AU-rich codon-anticodon pairs and to avoid miscoding in split codon boxes. The evolution and expansion of the genetic code is viewed as being originally based on GC content with progressive introduction of A/U together with tRNA modifications. The representation we present should help the engineering of the genetic code to include non-natural amino acids.
    Mots-clés : DBG, RNASTR.

  • D. Hamdane, H. Grosjean, et M. Fontecave, « Flavin-Dependent Methylation of RNAs: Complex Chemistry for a Simple Modification », Journal of Molecular Biology, vol. 428, nᵒ 24 Pt B, p. 4867-4881, 2016.
    Résumé : RNA methylation is the most abundant and evolutionarily conserved chemical modification of bases or ribose in noncoding and coding RNAs. This rather simple modification has nevertheless major consequences on the function of maturated RNA molecules and ultimately on their cellular fates. The methyl group employed in the methylation is almost universally derived from S-adenosyl-L-methionine via a simple SN2 displacement reaction. However, in some rare cases, the carbon originates from N5,N10-methylenetetrahydrofolate (CH2=THF). Here, a methylene group is transferred first and requires a subsequent reduction step (2e(-)+H(+)) via the flavin adenine dinucleotide hydroquinone (FADH(-)) to form the final methylated derivative. This FAD/folate-dependent mode of chemical reaction, called reductive methylation, is thus far more complex than the usual simple S-adenosyl-L-methionine-dependent one. This reaction is catalyzed by flavoenzymes, now named TrmFO and RlmFO, which respectively modify transfer and ribosomal RNAs. In this review, we briefly recount how these new RNA methyltransferases were discovered and describe a novel aspect of the chemistry of flavins, wherein this versatile biological cofactor is not just a simple redox catalyst but is also a new methyl transfer agent acting via a critical CH2=(N5)FAD iminium intermediate. The enigmatic structural reorganization of these enzymes that needs to take place during catalysis in order to build their active center is also discussed. Finally, recent findings demonstrated that this flavin-dependent mechanism is also employed by enzymatic systems involved in DNA synthesis, suggesting that the use of this cofactor as a methylating agent of biomolecules could be far more usual than initially anticipated.
    Mots-clés : 5-methyluridine, DBG, flavoenzyme, methylation mechanism, methyltransferase, RNA, RNASTR.

  • M. Okuda, D. Fourmy, et S. Yoshizawa, « Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs », Nucleic Acids Research, 2016.
    Résumé : Fluorogenic RNA aptamers provide a powerful tool for study of RNA analogous to green fluorescent protein for the study of proteins. Spinach and Broccoli are RNAs selected in vitro or in vivo respectively to bind to an exogenous chromophore. They can be genetically inserted into an RNA of interest for live-cell imaging. Spinach aptamer has been altered to increase thermal stability and stabilize the desired folding. How well these fluorogenic RNA aptamers behave when inserted into structured cellular RNAs and how aptamer properties might be affected remains poorly characterized. Here, we report a study of the performance of distinct RNA Spinach and Broccoli aptamer sequences in isolation or inserted into the small subunit of the bacterial ribosome. We found that the ribosomal context helped maintaining the yield of the folded Baby Spinach aptamer; other versions of Spinach did not perform well in the context of ribosomes. In vivo, two aptamers clearly stood out. Baby Spinach and Broccoli aptamers yielded fluorescence levels markedly superior to all previous Spinach sequences including the super-folder tRNA scaffolded tSpinach2. Overall, the results suggest the use of Broccoli and Baby Spinach aptamers for live cell imaging of structured RNAs.
    Mots-clés : DBG, RNASTR.

  • R. Yamagami, C. Tomikawa, N. Shigi, A. Kazayama, S. - I. Asai, H. Takuma, A. Hirata, D. Fourmy, H. Asahara, K. Watanabe, S. Yoshizawa, et H. Hori, « Folate-/FAD-dependent tRNA methyltransferase from Thermus thermophilus regulates other modifications in tRNA at low temperatures », Genes to Cells: Devoted to Molecular & Cellular Mechanisms, vol. 21, nᵒ 7, p. 740-754, 2016.
    Résumé : TrmFO is a N(5) , N(10) -methylenetetrahydrofolate (CH2 THF)-/FAD-dependent tRNA methyltransferase, which synthesizes 5-methyluridine at position 54 (m(5) U54) in tRNA. Thermus thermophilus is an extreme-thermophilic eubacterium, which grows in a wide range of temperatures (50-83 °C). In T. thermophilus, modified nucleosides in tRNA and modification enzymes form a network, in which one modification regulates the degrees of other modifications and controls the flexibility of tRNA. To clarify the role of m(5) U54 and TrmFO in the network, we constructed the trmFO gene disruptant (∆trmFO) strain of T. thermophilus. Although this strain did not show any growth retardation at 70 °C, it showed a slow-growth phenotype at 50 °C. Nucleoside analysis showed increase in 2'-O-methylguanosine at position 18 and decrease in N(1) -methyladenosine at position 58 in the tRNA mixture from the ∆trmFO strain at 50 °C. These in vivo results were reproduced by in vitro experiments with purified enzymes. Thus, we concluded that the m(5) U54 modification have effects on the other modifications in tRNA through the network at 50 °C. (35) S incorporations into proteins showed that the protein synthesis activity of ∆trmFO strain was inferior to the wild-type strain at 50 °C, suggesting that the growth delay at 50 °C was caused by the inferior protein synthesis activity.
    Mots-clés : DBG, Flavin-Adenine Dinucleotide, Folic Acid, Guanosine, Mutation, RNA, Transfer, RNASTR, Temperature, Thermus thermophilus, tRNA Methyltransferases, Uridine.

  • S. Yoshizawa, « Preface », Progress in Molecular Biology and Translational Science, vol. 139, p. xi, 2016.
    Mots-clés : DBG, Nanoparticles, Nanotechnology, RNA, RNASTR.

2015

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Principales publications avant 2015

- Grosjean H., Breton M., Sirand-Pugnet P., Tardy F., Thiaucourt F., Citti C., Barré A., Yoshizawa S., Fourmy D., Crécy-Lagard V. and Blanchard A. (2014). Predicting the minimal translation apparatus : lessons from the reductive evolution of Mollicutes. PloS Genet, 10(5):e1004363.

- Kim, S.H., Yoshizawa, S., Takeuchi, S., Fujii, T., and Fourmy, D. (2013). Ultra-high density protein spots achieved by on chip digitalized protein synthesis. Analyst, DOI : 10.1039/C3AN00976A.

- Fourmy, D., and Yoshizawa, S. (2012). Protein-RNA footprinting : an evolving tool. Wiley Interdiscip Rev RNA 3, 557-566.

- Valérie de Crécy-Lagard1, C.M., and Henri Grosjean (2012). Decoding in Candidatus Riesia pediculicola, close to a minimal tRNA modification set ? Trends in Cell Biology vol 7 11-23.

- Yoshizawa, S. (2012). Micro and nanotechnological tools for study of RNA. Biochimie 94, 1588-1594.

- Kawano, R., Osaki, T., Sasaki, H., Takinoue, M., Yoshizawa, S., and Takeuchi, S. (2011) Rapid Detection of a Cocaine-Binding Aptamer Using Biological Nanopores on a Chip. J Am Chem Soc, 133 (22) 8474-7.

- White, K. H., Orzechowski, M., Fourmy, D., and Visscher, K.(2011) Mechanical unfolding of the Beet Western Yellow Virus -1 frameshift signal. J Am Chem Soc, 133 (25) 9775-82.

- Auxilien, S., Rasmussen, A., Rose, S., Brochier-Armanet, C., Husson, C., Fourmy, D., Grosjean, H. and Douthwaite, S. (2011) Specificity shifts in the rRNA and tRNA nucleotide targets of archaeal and bacterial m5U methyltransferases. RNA, 17, 45-53.

- Yoshizawa S, and Böck A. (2009) The many levels of control on bacteial selenoprotein synthesis. BBA, 1790, 1404-1414.

- Ota S, Yoshizawa S, and Takeuchi S. (2009) Microfluidic formation of monodisperse, cell-sized and unilamellar vesicles. Angewandte Chemie (International ed.), 48, 6533-6537.

- Mazauric M.H., Seol.Y, Yoshizawa S., Visscher K. and Fourmy D. (2009) Interaction of the HIV-1 frameshift signal with the ribosome. Nucl Ac Res, 37, 7654-7664.

- Mazauric M.H., Leroy J.L., Visscher K., Yoshizawa S. and Fourmy D. (2009) Footprinting analysis of BWYV pseudoknot-ribosome complexes. RNA, 15, 1775-1786.

- Soler N., Fourmy D. and Yoshizawa S. (2007) Structural basis for a molecular switch in winged-helix domains of elongation factor selB. J Mol Biol, 370, 728-741.

- Yoshizawa S. Rasubala L., Ose T., Kohda D., Fourmy, D., and Maenaka K. (2005) Structural basis for messenger RNA recognition by elongation factor SelB. Nat StrucMol Biol, 12, 198-203.

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