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Publications Département Biologie des Génomes


  • T. Candelli, D. Challal, J. - B. Briand, J. Boulay, O. Porrua, J. Colin, et D. Libri, « High-resolution transcription maps reveal the widespread impact of roadblock termination in yeast », The EMBO journal, janv. 2018.
    Résumé : Transcription termination delimits transcription units but also plays important roles in limiting pervasive transcription. We have previously shown that transcription termination occurs when elongating RNA polymerase II (RNAPII) collides with the DNA-bound general transcription factor Reb1. We demonstrate here that many different DNA-binding proteins can induce termination by a similar roadblock (RB) mechanism. We generated high-resolution transcription maps by the direct detection of RNAPII upon nuclear depletion of two essential RB factors or when the canonical termination pathways for coding and non-coding RNAs are defective. We show that RB termination occurs genomewide and functions independently of (and redundantly with) the main transcription termination pathways. We provide evidence that transcriptional readthrough at canonical terminators is a significant source of pervasive transcription, which is controlled to a large extent by RB termination. Finally, we demonstrate the occurrence of RB termination around centromeres and tRNA genes, which we suggest shields these regions from RNAPII to preserve their functional integrity.
    Mots-clés : BDG, DBG, pervasive transcription, Rap1, roadblock termination, TENOR, transcription readthrough, transcription termination mechanism.

  • C. Dard-Dascot, D. Naquin, Y. d'Aubenton-Carafa, K. Alix, C. Thermes, et E. van Dijk, « Systematic comparison of small RNA library preparation protocols for next-generation sequencing », BMC genomics, vol. 19, nᵒ 1, p. 118, 2018.
    Résumé : BACKGROUND: Next-generation sequencing technologies have revolutionized the study of small RNAs (sRNAs) on a genome-wide scale. However, classical sRNA library preparation methods introduce serious bias, mainly during adapter ligation steps. Several types of sRNA including plant microRNAs (miRNA), piwi-interacting RNAs (piRNA) in insects, nematodes and mammals, and small interfering RNAs (siRNA) in insects and plants contain a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This inhibits 3' adapter ligation and makes library preparation particularly challenging. To reduce bias, the NEBNext kit (New England Biolabs) uses polyethylene glycol (PEG), the NEXTflex V2 kit (BIOO Scientific) uses both randomised adapters and PEG, and the novel SMARTer (Clontech) and CATS (Diagenode) kits avoid ligation altogether. Here we compared these methods with Illumina's classical TruSeq protocol regarding the detection of normal and 2' OMe RNAs. In addition, we modified the TruSeq and NEXTflex protocols to identify conditions that improve performance. RESULTS: Among the five kits tested with their respective standard protocols, the SMARTer and CATS kits had the lowest levels of bias but also had a strong formation of side products, and as a result performed relatively poorly with biological samples; NEXTflex detected the largest numbers of different miRNAs. The use of a novel type of randomised adapters called MidRand-Like (MRL) adapters and PEG improved the detection of 2' OMe RNAs both in the TruSeq as well as in the NEXTflex protocol. CONCLUSIONS: While it is commonly accepted that biases in sRNA library preparation protocols are mainly due to adapter ligation steps, the ligation-free protocols were not the best performing methods. Our modified versions of the TruSeq and NEXTflex protocols provide an improved tool for the study of 2' OMe RNAs.
    Mots-clés : 2’-O-methyl RNA, ANGE, Bias, CHERDIR, DBG, Library preparation, Next-generation sequencing, NGS, PF, Small RNA.

  • D. Drubay, D. Gautheret, et S. Michiels, « A benchmark study of scoring methods for non-coding mutations », Bioinformatics (Oxford, England), janv. 2018.
    Résumé : Motivation: Detailed knowledge of coding sequences has led to different candidate models for pathogenic variant prioritization. Several deleteriousness scores have been proposed for the non-coding part of the genome, but no large-scale comparison has been realized to date to assess their performance. Results: We compared the leading scoring tools (CADD, FATHMM-MKL, Funseq2 and GWAVA) and some recent competitors (DANN, SNP and SOM scores) for their ability to discriminate assumed pathogenic variants from assumed benign variants (using the ClinVar, COSMIC and 1000 genomes project databases). Using the ClinVar benchmark, CADD was the best tool for detecting the pathogenic variants that are mainly located in protein coding gene regions. Using the COSMIC benchmark, FATHMM-MKL, GWAVA and SOMliver outperformed the other tools for pathogenic variants that are typically located in lincRNAs, pseudogenes, and other parts of the non-coding genome. However, all tools had low precision, which could potentially be improved by future non-coding genome feature discoveries. These results may have been influenced by the presence of potential benign variants in the COSMIC database. The development of a gold standard as consistent as ClinVar for these regions will be necessary to confirm our tool ranking. Availability and Implementation: The Snakemake, C ++ and R codes are freely available from and supported on Linux. Contact: Supplementary information: Supplementary results are available at Bioinformatics online.
    Mots-clés : DBG, SSFA.

  • L. Laboureur, V. Guérineau, S. Auxilien, S. Yoshizawa, et D. Touboul, « Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry », Journal of Chromatography. A, vol. 1537, p. 118-127, févr. 2018.
    Résumé : A method based on supercritical fluid chromatography coupled to high resolution mass spectrometry for the profiling of canonical and modified nucleosides was optimized, and compared to classical reverse-phase liquid chromatography in terms of separation, number of detected modified nucleosides and sensitivity. Limits of detection and quantification were measured using statistical method and quantifications of twelve nucleosides of a tRNA digest from E. coli are in good agreement with previously reported data. Results highlight the complementarity of both separation techniques to cover the largest view of nucleoside modifications for forthcoming epigenetic studies.
    Mots-clés : DBG, Epigenetic, High resolution mass spectrometry, Nucleoside, RNA, RNASTR, Supercritical fluid chromatography.

  • N. Morellet, X. Li, S. A. Wieninger, J. L. Taylor, J. Bischerour, S. Moriau, E. Lescop, B. Bardiaux, N. Mathy, N. Assrir, M. Bétermier, M. Nilges, A. B. Hickman, F. Dyda, N. L. Craig, et E. Guittet, « Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase », Nucleic Acids Research, janv. 2018.
    Résumé : The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.
    Mots-clés : DBG, MICMAC.

  • B. Negrutskii, D. Vlasenko, M. Mirande, P. Futernyk, et A. El'skaya, « mRNA-Independent way to regulate translation elongation rate in eukaryotic cells », IUBMB life, févr. 2018.
    Résumé : The question of what governs the translation elongation rate in eukaryotes has not yet been completely answered. Earlier, different availability of different tRNAs was considered as a main factor involved, however, recent data revealed that the elongation rate does not always depend on tRNA availability. Here, we offer another, codon-independent approach to explain specific tRNA-dependence of the elongation rate in eukaryotes. We hypothesize that the exit rate of eukaryotic translation elongation factor 1A (eEF1A)*GDP from the 80S ribosome depends on the protein affinity to specific aminoacyl-tRNA remaining on the ribosome after GTP hydrolysis. Subsequently, a slower dissociation of eEF1A*GDP from certain aminoacyl-tRNAs in the ribosome can negatively influence the ribosomal elongation rate in a tRNA-dependent and mRNA-independent way. The specific tRNA-dependent departure rate of eEF1A*GDP from the ribosome is suggested to be a novel factor contributing to the overall translation elongation control in eukaryotic cells. © 2018 IUBMB Life, 2018.
    Mots-clés : BDG, DBG, eukaryotic gene expression, MARS, protein synthesis, Ribosome.

  • M. Poidevin, M. Sato, I. Altinoglu, M. Delaplace, C. Sato, et Y. Yamaichi, « Mutation in ESBL Plasmid fromEscherichia coliO104:H4 Leads Autoagglutination and Enhanced Plasmid Dissemination. », Front Microbiol, vol. 9, p. 130, 2018.
    Résumé : Conjugative plasmids are one of the main driving force of wide-spreading of multidrug resistance (MDR) bacteria. They are self-transmittable via conjugation as carrying the required set of genes andcis-acting DNA locus for direct cell-to-cell transfer. IncI incompatibility plasmids are nowadays often associated with extended-spectrum beta-lactamases producing Enterobacteria in clinic and environment. pESBL-EA11 was isolated fromEscherichia coliO104:H4 outbreak strain in Germany in 2011. During the previous study identifying transfer genes of pESBL-EA11, it was shown that transposon insertion at certain DNA region of the plasmid, referred to as Hft, resulted in great enhancement of transfer ability. This suggested that genetic modifications can enhance dissemination of MDR plasmids. Such 'superspreader' mutations have attracted little attention so far despite their high potential to worsen MDR spreading. Present study aimed to gain our understanding on regulatory elements that involved pESBL transfer. While previous studies of IncI plasmids indicated that immediate downstream gene of Hft,traA, is not essential for conjugative transfer, here we showed that overexpression of TraA in host cell elevated transfer rate of pESBL-EA11. Transposon insertion or certain nucleotide substitutions in Hft led strong TraA overexpression which resulted in activation of essential regulator TraB and likely overexpression of conjugative pili. Atmospheric Scanning Electron Microscopy observation suggested that IncI pili are distinct from other types of conjugative pili (such as long filamentous F-type pili) and rather expressed throughout the cell surface. High transfer efficiency in the mutant pESBL-EA11 was involved with hyperpiliation which facilitates cell-to-cell adhesion, including autoagglutination. The capability of plasmids to evolve to highly transmissible mutant is alarming, particularly it might also have adverse effect on host pathogenicity
    Mots-clés : DBG, EQYY.

  • L. Shi, F. Koll, O. Arnaiz, et J. Cohen, « The Ciliary Protein IFT57 in the Macronucleus of Paramecium », The Journal of Eukaryotic Microbiology, vol. 65, nᵒ 1, p. 12-27, janv. 2018.
    Résumé : The intraflagellar transport IFT57 protein is essential for ciliary growth and maintenance. Also known as HIPPI, human IFT57 can be translocated to the nucleus via a molecular partner of the Huntingtin, Hip1, inducing gene expression changes. In Paramecium tetraurelia, we identified four IFT57 genes forming two subfamilies IFT57A/B and IFT57C/D arising from whole genome duplications. The depletion of proteins of the two subfamilies induced ciliary defects and IFT57A and IFT57C localized in basal bodies and cilia. We observed that IFT57A, but not IFT57C, is also present in the macronucleus and able to traffic toward the developing anlage during autogamy. Analysis of chimeric IFT57A-IFT57C-GFP-tagged proteins allowed us to identify a region of IFT57A necessary for nuclear localization. We studied the localization of the unique IFT57 protein of Paramecium caudatum, a species, which diverged from P. tetraurelia before the whole genome duplications. The P. caudatumIFT57C protein was excluded from the nucleus. We also analyzed whether the overexpression of IFT57A in Paramecium could affect gene transcription as the human protein does in HeLa cells. The expression of some genes was indeed affected by overexpression of IFT57A, but the set of affected genes poorly overlaps the set of genes affected in human cells.
    Mots-clés : ANGE, BIOCELL, BIOCIL, cilia, DBG, HIPPI, IFT57 /HIPPI, intraflagellar transport, intraflagellar transport (IFT), Macronucleus, MICMAC, Paramecium.

  • C. Souaid, S. Bloyer, et D. Noordermeer, « Promoter–Enhancer Looping and Regulatory Neighborhoods », in Nuclear Architecture and Dynamics, Elsevier, 2018, p. 435-456.

  • J. Soutourina, « Transcription regulation by the Mediator complex », Nature Reviews. Molecular Cell Biology, vol. 19, nᵒ 4, p. 262-274, avr. 2018.
    Résumé : Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.
    Mots-clés : DBG, GTR.

  • C. Tomikawa, S. Auxilien, V. Guérineau, Y. Yoshioka, K. Miyoshi, H. Hori, D. Fourmy, K. Takai, et S. Yoshizawa, « Characterization of redundant tRNAIles with CAU and UAU anticodons in Lactobacillus plantarum », Journal of Biochemistry, vol. 163, nᵒ 3, p. 233-241, mars 2018.
    Résumé : In most eubacteria, the minor AUA isoleucine codon is decoded by tRNAIle2, which has a lysidine (L) in the anticodon loop. The lysidine is introduced by tRNAIle-lysidine synthetase (TilS) through post-transcriptional modification of cytidine to yield an LAU anticodon. Some bacteria, Lactobacillus plantarum for example, possess two tRNAIle2(UAU) genes in addition to, two tRNAIle2(CAU) genes and the tilS gene. tRNA expression from all these genes would generate redundancy in a tRNA that decodes a rare AUA codon. In this study, we investigated the tRNA expression from these genes in L. plantarum and characterized the corresponding tRNAs. The tRNAIle2(CAU) gene products are modified by TilS to produce tRNAIle2(LAU), while tRNAIle2(UAU) lacks modification especially in the anticodon sequence. We found that tRNAIle2(LAU) is charged with isoleucine but tRNAIle2(UAU) is not. Our results suggest that the tRNAIle2 redundancy may be related to different roles of these tRNAs in the cell.
    Mots-clés : aminoacylation, BDG, DBG, Mass Spectrometry, modification, rare codon, RNASTR, translation, tRNA.


  • A. F. Amorim, D. Pinto, L. Kuras, et L. Fernandes, « Absence of Gim proteins, but not GimC complex, alter stress-induced transcription », Biochimica Et Biophysica Acta, avr. 2017.
    Résumé : Saccharomyces cerevisiae GimC (mammalian Prefoldin) is a hexameric (Gim1-6) cytoplasmic complex involved in the folding pathway of actin/tubulin. In contrast to a shared role in GimC complex, we show that absence of individual Gim proteins results in distinct stress responses. No concomitant alteration in F-actin integrity was observed. Transcription of stress responsive genes is altered in gim2Δ, gim3Δ and gim6Δ mutants: TRX2 gene is induced in these mutants but with a profile diverging from type cells, whereas CTT1 and HSP26 fail to be induced. Remaining gimΔ mutants display stress transcript abundance comparable to wild type cells. No alteration in the nuclear localization of the transcriptional activators for TRX2 (Yap1) and CTT1/HSP26 (Msn2) was observed in gim2Δ. In accordance with TRX2 induction, RNA polymerase II occupancy at TRX2 discriminates the wild type from gim2Δ and gim6Δ. In contrast, RNA polymerase II occupancy at CTT1 is similar in wild type and gim2Δ, but higher in gim6Δ. The absence of active RNA polymerase II at CTT1 in gim2Δ, but not in wild type and gim1Δ, explains the respective CTT1 transcript outputs. Altogether our results put forward the need of Gim2, Gim3 and Gim6 in oxidative and osmotic stress activated transcription; others Gim proteins are dispensable. Consequently, the participation of Gim proteins in activated-transcription is independent from the GimC complex.
    Mots-clés : DBG, Gim proteins, PEPS, stress, Transcription regulation.

  • O. Arnaiz, E. Van Dijk, M. Bétermier, M. Lhuillier-Akakpo, A. de Vanssay, S. Duharcourt, E. Sallet, J. Gouzy, et L. Sperling, « Improved methods and resources for paramecium genomics: transcription units, gene annotation and gene expression », BMC genomics, vol. 18, nᵒ 1, p. 483, juin 2017.
    Résumé : BACKGROUND: The 15 sibling species of the Paramecium aurelia cryptic species complex emerged after a whole genome duplication that occurred tens of millions of years ago. Given extensive knowledge of the genetics and epigenetics of Paramecium acquired over the last century, this species complex offers a uniquely powerful system to investigate the consequences of whole genome duplication in a unicellular eukaryote as well as the genetic and epigenetic mechanisms that drive speciation. High quality Paramecium gene models are important for research using this system. The major aim of the work reported here was to build an improved gene annotation pipeline for the Paramecium lineage. RESULTS: We generated oriented RNA-Seq transcriptome data across the sexual process of autogamy for the model species Paramecium tetraurelia. We determined, for the first time in a ciliate, candidate P. tetraurelia transcription start sites using an adapted Cap-Seq protocol. We developed TrUC, multi-threaded Perl software that in conjunction with TopHat mapping of RNA-Seq data to a reference genome, predicts transcription units for the annotation pipeline. We used EuGene software to combine annotation evidence. The high quality gene structural annotations obtained for P. tetraurelia were used as evidence to improve published annotations for 3 other Paramecium species. The RNA-Seq data were also used for differential gene expression analysis, providing a gene expression atlas that is more sensitive than the previously established microarray resource. CONCLUSIONS: We have developed a gene annotation pipeline tailored for the compact genomes and tiny introns of Paramecium species. A novel component of this pipeline, TrUC, predicts transcription units using Cap-Seq and oriented RNA-Seq data. TrUC could prove useful beyond Paramecium, especially in the case of high gene density. Accurate predictions of 3' and 5' UTR will be particularly valuable for studies of gene expression (e.g. nucleosome positioning, identification of cis regulatory motifs). The P. tetraurelia improved transcriptome resource, gene annotations for P. tetraurelia, P. biaurelia, P. sexaurelia and P. caudatum, and Paramecium-trained EuGene configuration are available through ParameciumDB ( ). TrUC software is freely distributed under a GNU GPL v3 licence ( ).
    Mots-clés : ANGE, Autogamy, Cap-Seq, Ciliate, DBG, Differential gene expression, Gene annotation, MICMAC, RNA-Seq, TSS.

  • J. Audoux, N. Philippe, R. Chikhi, M. Salson, M. Gallopin, M. Gabriel, J. Le Coz, E. Drouineau, T. Commes, et D. Gautheret, « DE-kupl: exhaustive capture of biological variation in RNA-seq data through k-mer decomposition », Genome Biology, vol. 18, nᵒ 1, p. 243, déc. 2017.
    Résumé : We introduce a k-mer-based computational protocol, DE-kupl, for capturing local RNA variation in a set of RNA-seq libraries, independently of a reference genome or transcriptome. DE-kupl extracts all k-mers with differential abundance directly from the raw data files. This enables the retrieval of virtually all variation present in an RNA-seq data set. This variation is subsequently assigned to biological events or entities such as differential long non-coding RNAs, splice and polyadenylation variants, introns, repeats, editing or mutation events, and exogenous RNA. Applying DE-kupl to human RNA-seq data sets identified multiple types of novel events, reproducibly across independent RNA-seq experiments.
    Mots-clés : BIM, DBG, SSFA.

  • S. Barral, Y. Morozumi, H. Tanaka, E. Montellier, J. Govin, M. de Dieuleveult, G. Charbonnier, Y. Couté, D. Puthier, T. Buchou, F. Boussouar, T. Urahama, F. Fenaille, S. Curtet, P. Héry, N. Fernandez-Nunez, H. Shiota, M. Gérard, S. Rousseaux, H. Kurumizaka, et S. Khochbin, « Histone Variant H2A.L.2 Guides Transition Protein-Dependent Protamine Assembly in Male Germ Cells », Molecular Cell, vol. 66, nᵒ 1, p. 89-101.e8, 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.

  • W. V. Bienvenut, J. - P. Scarpelli, J. Dumestier, T. Meinnel, et C. Giglione, « EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples », BMC Bioinformatics, vol. 18, nᵒ 1, 2017.
    Mots-clés : DBG, DIR, PROMTI, SICS.

  • M. Boudard, D. Barth, J. Bernauer, A. Denise, et J. Cohen, « GARN2: coarse-grained prediction of 3D structure of large RNA molecules by regret minimization », Bioinformatics (Oxford, England), avr. 2017.
    Résumé : Motivation: Predicting the 3D structure of RNA molecules is a key feature towards predicting their functions. Methods which work at atomic or nucleotide level are not suitable for large molecules. In these cases, coarse-grained prediction methods aim to predict a shape which could be refined later by using more precise methods on smaller parts of the molecule. Results: We developed a complete method for sampling 3D RNA structure at a coarse-grained model, taking a secondary structure as input. One of the novelties of our method is that a second step extracts two best possible structures close to the native, from a set of possible structures. Although our method benefits from the first version of GARN, some of the main features on GARN2 are very different. GARN2 is much faster than the previous version and than the well-known methods of the state-of-art. Our experiments show that GARN2 can also provide better structures than the other state-of-the-art methods. Availability and implementations: GARN2 is written in Java. It is freely distributed and available at: . Contacts: , Supplementary information: Supplementary data are available at Bioinformatics online.
    Mots-clés : BIM, DBG.

  • C. Bouthier de la Tour, M. Mathieu, L. Meyer, P. Dupaigne, F. Passot, P. Servant, S. Sommer, E. Le Cam, et F. Confalonieri, « In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium », PloS One, vol. 12, nᵒ 5, p. e0177751, 2017.
    Résumé : The bacterium Deinococcus radiodurans possesses a set of Deinococcus-specific genes highly induced after DNA damage. Among them, ddrC (dr0003) was recently re-annotated, found to be in the inverse orientation and called A2G07_00380. Here, we report the first in vivo and in vitro characterization of the corrected DdrC protein to better understand its function in irradiated cells. In vivo, the ΔddrC null mutant is sensitive to high doses of UV radiation and the ddrC deletion significantly increases UV-sensitivity of ΔuvrA or ΔuvsE mutant strains. We show that the expression of the DdrC protein is induced after γ-irradiation and is under the control of the regulators, DdrO and IrrE. DdrC is rapidly recruited into the nucleoid of the irradiated cells. In vitro, we show that DdrC is able to bind single- and double-stranded DNA with a preference for the single-stranded DNA but without sequence or shape specificity and protects DNA from various nuclease attacks. DdrC also condenses DNA and promotes circularization of linear DNA. Finally, we show that the purified protein exhibits a DNA strand annealing activity. Altogether, our results suggest that DdrC is a new DNA binding protein with pleiotropic activities. It might maintain the damaged DNA fragments end to end, thus limiting their dispersion and extensive degradation after exposure to ionizing radiation. DdrC might also be an accessory protein that participates in a single strand annealing pathway whose importance in DNA repair becomes apparent when DNA is heavily damaged.
    Mots-clés : DBG, RBA.

  • P. Brézellec, M. - A. Petit, S. Pasek, I. Vallet-Gely, C. Possoz, et J. - L. Ferat, « Domestication of Lambda Phage Genes into a Putative Third Type of Replicative Helicase Matchmaker », Genome Biology and Evolution, vol. 9, nᵒ 6, p. 1561-1566, juin 2017.
    Résumé : At the onset of the initiation of chromosome replication, bacterial replicative helicases are recruited and loaded on the DnaA-oriC nucleoprotein platform, assisted by proteins like DnaC/DnaI or DciA. Two orders of bacteria appear, however, to lack either of these factors, raising the question of the essentiality of these factors in bacteria. Through a phylogenomic approach, we identified a pair of genes that could have substituted for dciA. The two domesticated genes are specific of the dnaC/dnaI- and dciA-lacking organisms and apparently domesticated from lambdoid phage genes. They derive from λO and λP and were renamed dopC and dopE, respectively. DopE is expected to bring the replicative helicase to the bacterial origin of replication, while DopC might assist DopE in this function. The confirmation of the implication of DopCE in the handling of the replicative helicase at the onset of replication in these organisms would generalize to all bacteria and therefore to all living organisms the need for specific factors dedicated to this function.
    Mots-clés : DBG, dciA, dnaC, EMC2, lambda phage, OCB, replication initiation, replicative helicase, viral gene domestication.

  • 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.

  • S. Calderari, M. Ria, C. Gérard, T. C. Nogueira, O. Villate, S. C. Collins, H. Neil, N. Gervasi, C. Hue, N. Suarez-Zamorano, C. Prado, M. Cnop, M. - T. Bihoreau, P. J. Kaisaki, J. - B. Cazier, C. Julier, M. Lathrop, M. Werner, D. L. Eizirik, et D. Gauguier, « Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons », Genomics, sept. 2017.
    Résumé : The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q<0.05; enrichment range 1.40-9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting β-cells and neurons and underline the existence of trans‑nosology pathways in diabetes and its co-morbidities.
    Mots-clés : Alzheimer's disease, ChIP sequencing, DBG, Diabetes mellitus, Goto-Kakizaki rat, GTR, Quantitative trait locus, Single nucleotide polymorphism.

  • K. Contrepois, C. Coudereau, B. A. Benayoun, N. Schuler, P. - F. Roux, O. Bischof, R. Courbeyrette, C. Carvalho, J. - Y. Thuret, Z. Ma, C. Derbois, M. - C. Nevers, H. Volland, C. E. Redon, W. M. Bonner, J. - F. Deleuze, C. Wiel, D. Bernard, M. P. Snyder, C. E. Rübe, R. Olaso, F. Fenaille, et C. Mann, « Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression », Nature Communications, vol. 8, p. 14995, mai 2017.
    Résumé : The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases.
    Mots-clés : DBG, SEN.

  • T. N. Dalia, S. H. Yoon, E. Galli, F. - X. Barre, C. M. Waters, et A. B. Dalia, « Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases », Nucleic Acids Research, mai 2017.
    Résumé : Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ∼50% without selection in cis. Consequently, editing constructs are generated in a single polymerase chain reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.
    Mots-clés : DBG, EMC2.

  • J. - F. Dallery, N. Lapalu, A. Zampounis, S. Pigné, I. Luyten, J. Amselem, A. H. J. Wittenberg, S. Zhou, M. V. de Queiroz, G. P. Robin, A. Auger, M. Hainaut, B. Henrissat, K. - T. Kim, Y. - H. Lee, O. Lespinet, D. C. Schwartz, M. R. Thon, et R. J. O'Connell, « Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters », BMC genomics, vol. 18, nᵒ 1, p. 667, août 2017.
    Résumé : BACKGROUND: The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. RESULTS: Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications. CONCLUSION: The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.
    Mots-clés : accessory chromosomes, BIM, Colletotrichum higginsianum, DBG, Fungal genome, optical map, secondary metabolism genes, segmental duplication, SMRT sequencing, subtelomeres, transposable elements.

  • A. Dostálová, S. Rommelaere, M. Poidevin, et B. Lemaitre, « Thioester-containing proteins regulate the Toll pathway and play a role in Drosophila defence against microbial pathogens and parasitoid wasps », BMC biology, vol. 15, nᵒ 1, p. 79, sept. 2017.
    Résumé : BACKGROUND: Members of the thioester-containing protein (TEP) family contribute to host defence in both insects and mammals. However, their role in the immune response of Drosophila is elusive. In this study, we address the role of TEPs in Drosophila immunity by generating a mutant fly line, referred to as TEPq (Δ) , lacking the four immune-inducible TEPs, TEP1, 2, 3 and 4. RESULTS: Survival analyses with TEPq (Δ) flies reveal the importance of these proteins in defence against entomopathogenic fungi, Gram-positive bacteria and parasitoid wasps. Our results confirm that TEPs are required for efficient phagocytosis of bacteria, notably for the two Gram-positive species tested, Staphylococcus aureus and Enterococcus faecalis. Furthermore, we show that TEPq (Δ) flies have reduced Toll pathway activation upon microbial infection, resulting in lower expression of antimicrobial peptide genes. Epistatic analyses suggest that TEPs function upstream or independently of the serine protease ModSP at an initial stage of Toll pathway activation. CONCLUSIONS: Collectively, our study brings new insights into the role of TEPs in insect immunity. It reveals that TEPs participate in both humoral and cellular arms of immune response in Drosophila. In particular, it shows the importance of TEPs in defence against Gram-positive bacteria and entomopathogenic fungi, notably by promoting Toll pathway activation.
    Mots-clés : Beauveria, Complement, DBG, Drosophila, Entomopathogenic fungus, EQYY, innate immunity, Insect, Parasitoid wasp, Phagocytosis.

  • E. Dubois, N. Mathy, V. Régnier, J. Bischerour, C. Baudry, R. Trouslard, et M. Bétermier, « Multimerization properties of PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements », Nucleic Acids Research, janv. 2017.
    Résumé : During sexual processes, the ciliate Paramecium eliminates 25-30% of germline DNA from its somatic genome. DNA elimination includes excision of ∼45 000 short, single-copy internal eliminated sequences (IESs) and depends upon PiggyMac (Pgm), a domesticated piggyBac transposase that is essential for DNA cleavage at IES ends. Pgm carries a core transposase region with a putative catalytic domain containing three conserved aspartic acids, and a downstream cysteine-rich (CR) domain. A C-terminal extension of unknown function is predicted to adopt a coiled-coil (CC) structure. To address the role of the three domains, we designed an in vivo complementation assay by expressing wild-type or mutant Pgm-GFP fusions in cells depleted for their endogenous Pgm. The DDD triad and the CR domain are essential for Pgm activity and mutations in either domain have a dominant-negative effect in wild-type cells. A mutant lacking the CC domain is partially active in the presence of limiting Pgm amounts, but inactive when Pgm is completely absent, suggesting that presence of the mutant protein increases the overall number of active complexes. We conclude that IES excision involves multiple Pgm subunits, of which at least a fraction must contain the CC domain.
    Mots-clés : DBG, DSMC, MICMAC.

  • S. Duigou et F. Boccard, « Long range chromosome organization in Escherichia coli: The position of the replication origin defines the non-structured regions and the Right and Left macrodomains », PLoS genetics, vol. 13, nᵒ 5, p. e1006758, mai 2017.
    Résumé : The Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell.
    Mots-clés : DBG, OCB.

  • 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.

  • C. Esnault, D. Leiber, C. Toffano-Nioche, Z. Tanfin, et M. - J. Virolle, « Another example of enzymatic promiscuity: the polyphosphate kinase of Streptomyces lividans is endowed with phospholipase D activity », Applied Microbiology and Biotechnology, vol. 101, nᵒ 1, p. 139-145, janv. 2017.
    Résumé : Polyphosphate kinases (PPK) from different bacteria, including that of Streptomyces lividans, were shown to contain the typical HKD motif present in phospholipase D (PLD) and showed structural similarities to the latter. This observation prompted us to investigate the PLD activity of PPK of S. lividans, in vitro. The ability of PPK to catalyze the hydrolysis of phosphatidylcholine (PC), the PLD substrate, was assessed by the quantification of [(3)H]phosphatidic acid (PA) released from [(3)H]PC-labeled ELT3 cell membranes. Basal cell membrane PLD activity as well as GTPγS-activated PLD activity was higher in the presence than in absence of PPK. After abolition of the basal PLD activity of the membranes by heat or tryptic treatment, the addition of PPK to cell membranes was still accompanied by an increased production of PA demonstrating that PPK also bears a PLD activity. PLD activity of PPK was also assessed by the production of choline from hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of the Amplex Red reagent and compared to two commercial PLD enzymes. These data demonstrated that PPK is endowed with a weak but clearly detectable PLD activity. The question of the biological signification, if any, of this enzymatic promiscuity is discussed.
    Mots-clés : Amino Acid Motifs, Cell Membrane, Choline, DBG, eBio, Hydrolysis, Lipid droplets, MESMIC, MICROBIO, PF, Phosphatidic Acids, Phosphatidylcholines, Phospholipase D, Phosphotransferases (Phosphate Group Acceptor), Polyphosphate kinase, Promiscuous enzyme, Protein Conformation, SSFA, Streptomyces lividans.

  • T. Eychenne, M. Werner, et J. Soutourina, « Toward understanding of the mechanisms of Mediator function in vivo: Focus on the preinitiation complex assembly », Transcription, vol. 8, nᵒ 5, p. 328-342, 2017.
    Résumé : Mediator is a multisubunit complex conserved in eukaryotes that plays an essential coregulator role in RNA polymerase (Pol) II transcription. Despite intensive studies of the Mediator complex, the molecular mechanisms of its function in vivo remain to be fully defined. In this review, we will discuss the different aspects of Mediator function starting with its interactions with specific transcription factors, its recruitment to chromatin and how, as a coregulator, it contributes to the assembly of transcription machinery components within the preinitiation complex (PIC) in vivo and beyond the PIC formation.
    Mots-clés : coactivator, DBG, eukaryotic transcription, functional genomics, Genetics, GTR, human, Mediator, preinitiation complex, RNA Polymerase II, Structural Biology, yeast Saccharomyces cerevisiae.

  • P. J. Fabre, M. Leleu, B. H. Mormann, L. Lopez-Delisle, D. Noordermeer, L. Beccari, et D. Duboule, « Large scale genomic reorganization of topological domains at the HoxD locus », Genome Biology, vol. 18, nᵒ 1, p. 149, août 2017.
    Résumé : BACKGROUND: The transcriptional activation of HoxD genes during mammalian limb development involves dynamic interactions with two topologically associating domains (TADs) flanking the HoxD cluster. In particular, the activation of the most posterior HoxD genes in developing digits is controlled by regulatory elements located in the centromeric TAD (C-DOM) through long-range contacts. RESULTS: To assess the structure-function relationships underlying such interactions, we measured compaction levels and TAD discreteness using a combination of chromosome conformation capture (4C-seq) and DNA FISH. We assessed the robustness of the TAD architecture by using a series of genomic deletions and inversions that impact the integrity of this chromatin domain and that remodel long-range contacts. We report multi-partite associations between HoxD genes and up to three enhancers. We find that the loss of native chromatin topology leads to the remodeling of TAD structure following distinct parameters. CONCLUSIONS: Our results reveal that the recomposition of TAD architectures after large genomic re-arrangements is dependent on a boundary-selection mechanism in which CTCF mediates the gating of long-range contacts in combination with genomic distance and sequence specificity. Accordingly, the building of a recomposed TAD at this locus depends on distinct functional and constitutive parameters.
    Mots-clés : CHRODY, Chromatin organization, CTCF, DBG, Enhancer, Gene regulation, Hox, Limb development, Regulatory landscape, TAD, Topologically associating domains.

  • B. Felden et P. Bouloc, « Regulatory RNAs in bacteria: From identification to function », Methods, vol. 117, p. 1-2, 2017.

  • S. Fieulaine, R. Alves de Sousa, L. Maigre, K. Hamiche, M. Alimi, J. - M. Bolla, A. Taleb, A. Denis, J. - M. Pagès, I. Artaud, T. Meinnel, et C. Giglione, « Corrigendum: A unique peptide deformylase platform to rationally design and challenge novel active compounds », Scientific Reports, vol. 7, p. 39365, janv. 2017.

  • K. Forslund, C. Pereira, S. Capella-Gutierrez, A. Sousa da Silva, A. Altenhoff, J. Huerta-Cepas, M. Muffato, M. Patricio, K. Vandepoele, I. Ebersberger, J. Blake, J. T. Fernández Breis, Quest for Orthologs Consortium, B. Boeckmann, T. Gabaldón, E. Sonnhammer, C. Dessimoz, et S. Lewis, « Gearing up to handle the mosaic nature of life in the quest for orthologs », Bioinformatics (Oxford, England), août 2017.
    Résumé : The Quest for Orthologs (QfO) is an open collaboration framework for experts in comparative phylogenomics and related research areas who have an interest in highly accurate orthology predictions and their applications. We here report highlights and discussion points from the QfO meeting 2015 held in Barcelona. Achievements in recent years have established a basis to support developments for improved orthology prediction and to explore new approaches. Central to the QfO effort is proper benchmarking of methods and services, as well as design of standardized datasets and standardized formats to allow sharing and comparison of results. Simultaneously, analysis pipelines have been improved, evaluated, and adapted to handle large datasets. All this would not have occurred without the long-term collaboration of Consortium members. Meeting regularly to review and coordinate complementary activities from a broad spectrum of innovative researchers clearly benefits the community. Highlights of the meeting include addressing sources of and legitimacy of disagreements between orthology calls, the context dependency of orthology definitions, special challenges encountered when analyzing very anciently rooted orthologies, orthology in the light of whole-genome duplications, and the concept of orthologous versus paralogous relationships at different levels, including domain-level orthology. Furthermore, particular needs for different applications (e.g. plant genomics, ancient gene families, and others) and the infrastructure for making orthology inferences available (e.g. interfaces with model organism databases) were discussed, with several ongoing efforts that are expected to be reported on during the upcoming 2017 QfO meeting.
    Mots-clés : BIM, DBG.

  • E. Galli, C. Midonet, E. Paly, et F. - X. Barre, « Fast growth conditions uncouple the final stages of chromosome segregation and cell division in Escherichia coli », PLOS Genetics, vol. 13, nᵒ 3, p. e1006702, mars 2017.

  • E. Galli, E. Paly, et F. - X. Barre, « Late assembly of the Vibrio cholerae cell division machinery postpones septation to the last 10% of the cell cycle », Scientific Reports, vol. 7, p. 44505, mars 2017.

  • A. Glatigny, P. Gambette, A. Bourand-Plantefol, G. Dujardin, et M. - H. Mucchielli-Giorgi, « Development of an in silico method for the identification of subcomplexes involved in the biogenesis of multiprotein complexes in Saccharomyces cerevisiae », BMC systems biology, vol. 11, nᵒ 1, p. 67, juill. 2017.
    Résumé : BACKGROUND: Large sets of protein-protein interaction data coming either from biological experiments or predictive methods are available and can be combined to construct networks from which information about various cell processes can be extracted. We have developed an in silico approach based on these information to model the biogenesis of multiprotein complexes in the yeast Saccharomyces cerevisiae. RESULTS: Firstly, we have built three protein interaction networks by collecting the protein-protein interactions, which involved the subunits of three complexes, from different databases. The protein-protein interactions come from different kinds of biological experiments or are predicted. We have chosen the elongator and the mediator head complexes that are soluble and exhibit an architecture with subcomplexes that could be functional modules, and the mitochondrial bc 1 complex, which is an integral membrane complex and for which a late assembly subcomplex has been described. Secondly, by applying a clustering strategy to these networks, we were able to identify subcomplexes involved in the biogenesis of the complexes as well as the proteins interacting with each subcomplex. Thirdly, in order to validate our in silico results for the cytochrome bc1 complex we have analysed the physical interactions existing between three subunits by performing immunoprecipitation experiments in several genetic context. CONCLUSIONS: For the two soluble complexes (the elongator and mediator head), our model shows a strong clustering of subunits that belong to a known subcomplex or module. For the membrane bc 1 complex, our approach has suggested new interactions between subunits in the early steps of the assembly pathway that were experimentally confirmed. Scripts can be downloaded from the site: .
    Mots-clés : BIM, BIOCELL, BIOMIT, Complex assembly, DBG, Graph clustering, PPI network, Protein complex, Protein-protein interactions, Subcomplex.

  • J. Gruchota, C. Denby Wilkes, O. Arnaiz, L. Sperling, et J. K. Nowak, « A meiosis-specific Spt5 homolog involved in non-coding transcription », Nucleic Acids Research, janv. 2017.
    Résumé : Spt5 is a conserved and essential transcriptional regulator that binds directly to RNA polymerase and is involved in transcription elongation, polymerase pausing and various co-transcriptional processes. To investigate the role of Spt5 in non-coding transcription, we used the unicellular model Paramecium tetraurelia In this ciliate, development is controlled by epigenetic mechanisms that use different classes of non-coding RNAs to target DNA elimination. We identified two SPT5 genes. One (STP5v) is involved in vegetative growth, while the other (SPT5m) is essential for sexual reproduction. We focused our study on SPT5m, expressed at meiosis and associated with germline nuclei during sexual processes. Upon Spt5m depletion, we observed absence of scnRNAs, piRNA-like 25 nt small RNAs produced at meiosis. The scnRNAs are a temporal copy of the germline genome and play a key role in programming DNA elimination. Moreover, Spt5m depletion abolishes elimination of all germline-limited sequences, including sequences whose excision was previously shown to be scnRNA-independent. This suggests that in addition to scnRNA production, Spt5 is involved in setting some as yet uncharacterized epigenetic information at meiosis. Our study establishes that Spt5m is crucial for developmental genome rearrangements and necessary for scnRNA production.
    Mots-clés : ANGE, DBG.

  • R. Grzela, J. Nusbaum, S. Fieulaine, F. Lavecchia, W. V. Bienvenut, C. Dian, T. Meinnel, et C. Giglione, « The C-terminal residue of phage Vp16 PDF, the smallest peptide deformylase, acts as an offset element locking the active conformation », Scientific Reports, vol. 7, nᵒ 1, p. 11041, sept. 2017.
    Résumé : Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this mechanism. Recent metagenomics studies revealed new idiosyncratic PDF forms as the most abundant family of viral sequences. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity. We provide here the first evidence that viral PDFs, including the shortest PDF identified to date, Vp16 PDF, display deformylase activity in vivo, despite the absence of the key ribosome-interacting C-terminal region. Moreover, characterization of phage Vp16 PDF underscores unexpected structural and molecular features with the C-terminal Isoleucine residue significantly contributing to deformylase activity both in vitro and in vivo. This residue fully compensates for the absence of the usual long C-domain. Taken together, these data elucidate an unexpected mechanism of enzyme natural evolution and adaptation within viral sequences.
    Mots-clés : B3S, DBG, IMAPP, PROMTI.

  • R. Grzela, J. Nusbaum, S. Fieulaine, F. Lavecchia, M. Desmadril, N. Nhiri, A. Van Dorsselaer, S. Cianferani, E. Jacquet, T. Meinnel, et C. Giglione, « Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts », Biochimica Et Biophysica Acta, oct. 2017.
    Résumé : Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genomes. While various hypotheses dealing with the occurrence of these intriguing sequences have been made, no further characterization and functional studies have been described thus far. In this study, we characterize the bacteriophage Vp16 PDF enzyme, as representative member of the newly identified C-terminally truncated viral PDFs. We show here that conditions classically used for bacterial PDFs lead to an enzyme exhibiting weak activity. Nonetheless, our integrated biophysical and biochemical approaches reveal specific effects of pH and metals on Vp16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp16 specific activity to values similar to those of bacterial PDFs. We next show that Vp16PDF is as sensitive to the natural inhibitor compound of PDFs, actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp16 and E. coli PDFs bound to actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness.
    Mots-clés : AMIG, B3S, BDG, DBG, Enzyme mechanism, IMAPP, MIP, N-terminal methionine excision, Peptide deformylase, PF, PIM, PROMTI, Structure, Virus.

  • F. Guérin, O. Arnaiz, N. Boggetto, C. Denby Wilkes, E. Meyer, L. Sperling, et S. Duharcourt, « Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements », BMC genomics, vol. 18, nᵒ 1, p. 327, avr. 2017.
    Résumé : BACKGROUND: DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. RESULTS: We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. CONCLUSIONS: We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.
    Mots-clés : ANGE, DBG, Flow Cytometry, High throughput sequencing, ITm DNA transposons, Non-LTR retrotransposons, Programmed DNA elimination.

  • E. Krzywinska, C. Kantari-Mimoun, Y. Kerdiles, M. Sobecki, T. Isagawa, D. Gotthardt, M. Castells, J. Haubold, C. Millien, T. Viel, B. Tavitian, N. Takeda, J. Fandrey, E. Vivier, V. Sexl, et C. Stockmann, « Loss of HIF-1α in natural killer cells inhibits tumour growth by stimulating non-productive angiogenesis », Nature Communications, vol. 8, nᵒ 1, p. 1597, nov. 2017.
    Résumé : Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1α in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1α in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels.
    Mots-clés : DBG, TENOR.

  • H. Lalucque, F. Malagnac, K. Green, V. Gautier, P. Grognet, L. Chan Ho Tong, B. Scott, et P. Silar, « IDC2 and IDC3, two genes involved in cell non-autonomous signaling of fruiting body development in the model fungus Podospora anserina », Developmental Biology, vol. 421, nᵒ 2, p. 126-138, janv. 2017.
    Résumé : Filamentous ascomycetes produce complex multicellular structures during sexual reproduction. Little is known about the genetic pathways enabling the construction of such structures. Here, with a combination of classical and reverse genetic methods, as well as genetic mosaic and graft analyses, we identify and provide evidence for key roles for two genes during the formation of perithecia, the sexual fruiting bodies, of the filamentous fungus Podospora anserina. Data indicate that the proteins coded by these two genes function cell-non-autonomously and that their activity depends upon conserved cysteines, making them good candidate for being involved in the transmission of a reactive oxygen species (ROS) signal generated by the PaNox1 NADPH oxidase inside the maturing fruiting body towards the PaMpk1 MAP kinase, which is located inside the underlying mycelium, in which nutrients are stored. These data provide important new insights to our understanding of how fungi build multicellular structures.
    Mots-clés : DBG, Developmental mutants, DSMC, Fungal development, Multicellular fruiting bodies, Perithecium, Podospora anserina.

  • T. N. Le Lam, C. Morvan, W. Liu, C. Bohn, Y. Jaszczyszyn, et P. Bouloc, « Finding sRNA-associated phenotypes by competition assays: An example with Staphylococcus aureus », Methods, vol. 117, p. 21-27, 2017.
    Mots-clés : DBG, NGS, PF, SRRB.

  • J. Lehmann, « Induced fit of the peptidyl-transferase center of the ribosome and conformational freedom of the esterified amino acids », RNA (New York, N.Y.), vol. 23, nᵒ 2, p. 229-239, févr. 2017.
    Résumé : The catalytic site of most enzymes can efficiently handle only one substrate. In contrast, the ribosome is capable of polymerizing at a similar rate at least 20 different kinds of amino acids from aminoacyl-tRNA carriers while using just one catalytic site, the peptidyl-transferase center (PTC). An induced-fit mechanism has been uncovered in the PTC, but a possible connection between this mechanism and the uniform handling of the substrates has not been investigated. We present an analysis of published ribosome structures supporting the hypothesis that the induced fit eliminates unreactive rotamers predominantly populated for some A-site aminoacyl esters before induction. We show that this hypothesis is fully consistent with the wealth of kinetic data obtained with these substrates. Our analysis reveals that induction constrains the amino acids into a reactive conformation in a side-chain independent manner. It allows us to highlight the rationale of the PTC structural organization, which confers to the ribosome the very unusual ability to handle large as well as small substrates.
    Mots-clés : aminoacyl-tRNA, DBG, EF-P, induced fit, peptidyl-transferase center, Ribosome, SSFA.

  • S. Li, E. Porcel, H. Remita, S. Marco, M. Réfrégiers, M. Dutertre, F. Confalonieri, et S. Lacombe, « Platinum nanoparticles: an exquisite tool to overcome radioresistance », Cancer Nanotechnology, vol. 8, nᵒ 1, p. 4, 2017.
    Résumé : BACKGROUD: Small metallic nanoparticles are proposed as potential nanodrugs to optimize the performances of radiotherapy. This strategy, based on the enrichment of tumours with nanoparticles to amplify radiation effects in the tumour, aims at increasing the cytopathic effect in tumours while healthy tissue is preserved, an important challenge in radiotherapy. Another major cause of radiotherapy failure is the radioresistance of certain cancers. Surprisingly, the use of nanoparticles to overcome radioresistance has not, to the best of our knowledge, been extensively investigated. The mechanisms of radioresistance have been extensively studied using Deinococcus radiodurans, the most radioresistant organism ever reported, as a model. METHODS: In this work, we investigated the impact of ultra-small platinum nanoparticles (1.7 nm) on this organism, including uptake, toxicity, and effects on radiation responses. RESULTS: We showed that the nanoparticles penetrate D. radiodurans cells, despite the 150 nm cell wall thickness with a minimal inhibition concentration on the order of 4.8 mg L(-1). We also found that the nanoparticles amplify gamma ray radiation effects by >40%. CONCLUSIONS: Finally, this study demonstrates the capacity of metallic nanoparticles to amplify radiation in radioresistant organisms, thus opening the perspective to use nanoparticles not only to improve tumour targeting but also to overcome radioresistance.
    Mots-clés : DBG, Deinococcus radiodurans, Metallic nanoparticles, Radio-enhancement, Radioresistance, Radiosensitization, RBA.

  • B. Michel et S. J. Sandler, « Replication Restart in Bacteria », Journal of Bacteriology, p. JB.00102-17, mars 2017.

  • B. Michel et A. K. Sinha, « The inactivation of rfaP, rarA or sspA gene improves the viability of the Escherichia coli DNA polymerase III holD mutant », Molecular Microbiology, mars 2017.
    Résumé : The Escherichia coli holD mutant is poorly viable because the stability of holoenzyme polymerase III (Pol III HE) on DNA is compromised. Consequently, the SOS response is induced and the SOS polymerases DinB and Pol II further hinder replication. Mutations that restore the holD mutant viability belong to two classes, those that stabilize Pol III on DNA and those that prevent the deleterious effects of DinB over-production. We identified a dnaX mutation and the inactivation of rfaP and sspA genes as belonging to the first class of holD mutant suppressors. dnaX encodes a Pol III clamp loader subunit that interacts with HolD. rfaP encodes a lipopolysaccharide kinase that acts in outer membrane biogenesis. Its inactivation improves the holD mutant growth in part by affecting potassium import, previously proposed to stabilize Pol III HE on DNA by increasing electrostatic interactions. sspA encodes a global transcriptional regulator and growth of the holD mutant in its absence suggests that SspA controls genes that affect protein-DNA interactions. The inactivation of rarA belongs to the second class of suppressor mutations. rarA inactivation has a weak effect but is additive with other suppressor mutations. Our results suggest that RarA facilitates DinB binding to abandoned forks.
    Mots-clés : DBG, STABAC.

  • M. Mirande, « The Aminoacyl-tRNA Synthetase Complex », Sub-Cellular Biochemistry, vol. 83, p. 505-522, 2017.
    Résumé : Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that specifically aminoacylate one tRNA molecule by the cognate amino acid. They are a family of twenty enzymes, one for each amino acid. By coupling an amino acid to a specific RNA triplet, the anticodon, they are responsible for interpretation of the genetic code. In addition to this translational, canonical role, several aminoacyl-tRNA synthetases also fulfill nontranslational, moonlighting functions. In mammals, nine synthetases, those specific for amino acids Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met and Pro, associate into a multi-aminoacyl-tRNA synthetase complex, an association which is believed to play a key role in the cellular organization of translation, but also in the regulation of the translational and nontranslational functions of these enzymes. Because the balance between their alternative functions rests on the assembly and disassembly of this supramolecular entity, it is essential to get precise insight into the structural organization of this complex. The high-resolution 3D-structure of the native particle, with a molecular weight of about 1.5 MDa, is not yet known. Low-resolution structures of the multi-aminoacyl-tRNA synthetase complex, as determined by cryo-EM or SAXS, have been reported. High-resolution data have been reported for individual enzymes of the complex, or for small subcomplexes. This review aims to present a critical view of our present knowledge of the aminoacyl-tRNA synthetase complex in 3D. These preliminary data shed some light on the mechanisms responsible for the balance between the translational and nontranslational functions of some of its components.
    Mots-clés : Aminoacyl-tRNA synthetase (AARS), Core synthetases, Crystal Structure, DBG, MARS, MSC assembly, Multi-aminacyl-tRNA synthetase complex (MSC).

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