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Publications Département Biologie des Génomes


  • 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, 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, 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.

  • 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.

  • S. Berlivet, I. Hmitou, H. Picaud, et M. Gérard, « Efficient Depletion of Essential Gene Products for Loss-of-Function Studies in Embryonic Stem Cells », Methods in Molecular Biology (Clifton, N.J.), vol. 1622, p. 91-100, 2017.
    Résumé : The development of the CRISPR/Cas9 technology has provided powerful methods to target genetic alterations. However, investigating the function of genes essential for cell survival remains problematic, because genetic ablation of these genes results in cell death. As a consequence, cells recombined at the targeted gene and fully depleted of the gene product cannot be obtained. RNA interference is well suited for the study of essential genes, but this approach often results in a partial depletion of the targeted gene product, which can lead to misinterpretations. We previously developed the pHYPER shRNA vector, a high efficiency RNA interference vector, which is based on a 2.5-kb mouse genomic fragment encompassing the H1 gene. We provide here a pHYPER-based protocol optimized to study the function of essential gene products in mouse embryonic stem cells.
    Mots-clés : DBG, Electroporation, Embryonic stem cell, Essential genes, pHYPER, Puromycin selection, REMOD, RNA Interference, shRNA.

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

  • 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), 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.

  • 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, 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, 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.

  • 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, 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, 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.

  • 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.

  • 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, 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, 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, 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.

  • 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, 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.

  • 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, 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.

  • 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, 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.

  • 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, 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).

  • 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.

  • R. I. Ponce-Toledo, P. Deschamps, P. López-García, Y. Zivanovic, K. Benzerara, et D. Moreira, « An Early-Branching Freshwater Cyanobacterium at the Origin of Plastids », Current biology: CB, vol. 27, nᵒ 3, p. 386-391, 2017.
    Résumé : Photosynthesis evolved in eukaryotes by the endosymbiosis of a cyanobacterium, the future plastid, within a heterotrophic host. This primary endosymbiosis occurred in the ancestor of Archaeplastida, a eukaryotic supergroup that includes glaucophytes, red algae, green algae, and land plants [1-4]. However, although the endosymbiotic origin of plastids from a single cyanobacterial ancestor is firmly established, the nature of that ancestor remains controversial: plastids have been proposed to derive from either early- or late-branching cyanobacterial lineages [5-11]. To solve this issue, we carried out phylogenomic and supernetwork analyses of the most comprehensive dataset analyzed so far including plastid-encoded proteins and nucleus-encoded proteins of plastid origin resulting from endosymbiotic gene transfer (EGT) of primary photosynthetic eukaryotes, as well as wide-ranging genome data from cyanobacteria, including novel lineages. Our analyses strongly support that plastids evolved from deep-branching cyanobacteria and that the present-day closest cultured relative of primary plastids is Gloeomargarita lithophora. This species belongs to a recently discovered cyanobacterial lineage widespread in freshwater microbialites and microbial mats [12, 13]. The ecological distribution of this lineage sheds new light on the environmental conditions where the emergence of photosynthetic eukaryotes occurred, most likely in a terrestrial-freshwater setting. The fact that glaucophytes, the first archaeplastid lineage to diverge, are exclusively found in freshwater ecosystems reinforces this hypothesis. Therefore, not only did plastids emerge early within cyanobacteria, but the first photosynthetic eukaryotes most likely evolved in terrestrial-freshwater settings, not in oceans as commonly thought.
    Mots-clés : chloroplasts, Cyanobacteria, DBG, evolution, molecular phylogeny, phylogenomics, Plastids, RBA.

  • W. V. Bienvenut, C. Giglione, et T. Meinnel, « SILProNAQ: A Convenient Approach for Proteome-Wide Analysis of Protein N-Termini and N-Terminal Acetylation Quantitation », in Protein Terminal Profiling, vol. 1574, O. Schilling, Éd. New York, NY: Springer New York, 2017, p. 17-34.

  • L. Shi, K. France, O. Arnaiz, et J. Cohen, « The Ciliary Protein IFT57 in the Macronucleus of Paramecium », The Journal of Eukaryotic Microbiology, 2017.
    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 Paramecium tetraurelia before the whole genome duplications. The Paramecium caudatum IFT57C 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. This article is protected by copyright. All rights reserved.
    Mots-clés : ANGE, BIOCELL, BIOCIL, cilia, DBG, IFT57 /HIPPI, intraflagellar transport (IFT), Macronucleus, Paramecium.

  • A. Thiébaut, T. Delaveau, M. Benchouaia, J. Boeri, M. Garcia, G. Lelandais, et F. Devaux, « The CCAAT-Binding Complex Controls Respiratory Gene Expression and Iron Homeostasis in Candida Glabrata », Scientific Reports, vol. 7, nᵒ 1, p. 3531, 2017.
    Résumé : The CCAAT-binding complex (CBC) is a heterotrimeric transcription factor which is widely conserved in eukaryotes. In the model yeast S. cerevisiae, CBC positively controls the expression of respiratory pathway genes. This role involves interactions with the regulatory subunit Hap4. In many pathogenic fungi, CBC interacts with the HapX regulatory subunit to control iron homeostasis. HapX is a bZIP protein which only shares with Hap4 the Hap4Like domain (Hap4L) required for its interaction with CBC. Here, we show that CBC has a dual role in the pathogenic yeast C. glabrata. It is required, along with Hap4, for the constitutive expression of respiratory genes and it is also essential for the iron stress response, which is mediated by the Yap5 bZIP transcription factor. Interestingly, Yap5 contains a vestigial Hap4L domain. The mutagenesis of this domain severely reduced Yap5 binding to its targets and compromised its interaction with Hap5. Hence, Yap5, like HapX in other species, acts as a CBC regulatory subunit in the regulation of iron stress response. This work reveals new aspects of iron homeostasis in C. glabrata and of the evolution of the role of CBC and Hap4L-bZIP proteins in this process.
    Mots-clés : BIM, DBG.

  • C. Voisset, M. Blondel, G. W. Jones, G. Friocourt, G. Stahl, S. Chédin, V. Beringue, et R. Gillet, « The double life of the ribosome: when its protein folding activity supports prion propagation », Prion, p. 00-00, mars 2017.

  • S. Wang, T. Hassold, P. Hunt, M. A. White, D. Zickler, N. Kleckner, et L. Zhang, « Inefficient Crossover Maturation Underlies Elevated Aneuploidy in Human Female Meiosis », Cell, vol. 168, nᵒ 6, p. 977-989.e17, 2017.
    Résumé : Meiosis is the cellular program that underlies gamete formation. For this program, crossovers between homologous chromosomes play an essential mechanical role to ensure regular segregation. We present a detailed study of crossover formation in human male and female meiosis, enabled by modeling analysis. Results suggest that recombination in the two sexes proceeds analogously and efficiently through most stages. However, specifically in female (but not male), ∼25% of the intermediates that should mature into crossover products actually fail to do so. Further, this "female-specific crossover maturation inefficiency" is inferred to make major contributions to the high level of chromosome mis-segregation and resultant aneuploidy that uniquely afflicts human female oocytes (e.g., giving Down syndrome). Additionally, crossover levels on different chromosomes in the same nucleus tend to co-vary, an effect attributable to global per-nucleus modulation of chromatin loop size. Maturation inefficiency could potentially reflect an evolutionary advantage of increased aneuploidy for human females.
    Mots-clés : DBG, DSMC.

  • N. Xie, G. Ruprich-Robert, F. Chapeland-Leclerc, E. Coppin, H. Lalucque, S. Brun, R. Debuchy, et P. Silar, « Inositol-phosphate signaling as mediator for growth and sexual reproduction in Podospora anserina », Developmental Biology, 2017.
    Résumé : The molecular pathways involved in the development of multicellular fruiting bodies in fungi are still not well known. Especially, the interplay between the mycelium, the female tissues and the zygotic tissues of the fruiting bodies is poorly documented. Here, we describe PM154, a new strain of the model ascomycetes Podospora anserina able to mate with itself and that enabled the easy recovery of new mutants affected in fruiting body development. By complete genome sequencing of spod1, one of the new mutants, we identified an inositol phosphate polykinase gene as essential, especially for fruiting body development. A factor present in the wild type and diffusible in mutant hyphae was able to induce the development of the maternal tissues of the fruiting body in spod1, but failed to promote complete development of the zygotic ones. Addition of myo-inositol in the growth medium was able to increase the number of developing fruiting bodies in the wild type, but not in spod1. Overall, the data indicated that inositol and inositol polyphosphates were involved in promoting fruiting body maturation, but also in regulating the number of fruiting bodies that developed after fertilization. The same effect of inositol was seen in two other fungi, Sordaria macrospora and Chaetomium globosum. Key role of the inositol polyphosphate pathway during fruiting body maturation appears thus conserved during the evolution of Sordariales fungi.
    Mots-clés : DBG, Developmental mutants, DSMC, Fungal development, Inositol, Inositol kinase, Multicellular fruiting bodies, Perithecium, Podospora anserina.


  • E. Allemand, M. P. Myers, J. Garcia-Bernardo, A. Harel-Bellan, A. R. Krainer, et C. Muchardt, « A Broad Set of Chromatin Factors Influences Splicing », PLoS genetics, vol. 12, nᵒ 9, p. e1006318, 2016.
    Résumé : Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing.
    Mots-clés : DBG, LEC.

  • S. Bakari, M. Lembrouk, L. Sourd, F. Ousalem, F. André, S. Orlowski, M. Delaforge, et A. Frelet-Barrand, « Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1 », Molecular Biotechnology, vol. 58, nᵒ 4, p. 299-310, 2016.
    Mots-clés : B3S, DBG, LSOD, PEPS.

  • R. Balbontín, N. Villagra, M. Pardos de la Gándara, G. Mora, N. Figueroa-Bossi, et L. Bossi, « Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues », Molecular Microbiology, vol. 100, nᵒ 1, p. 139-155, 2016.
    Résumé : The iroN gene of Salmonella enterica and uropathogenic Escherichia coli encodes the outer membrane receptor of Fe(3+) -bound salmochelin, a siderophore tailored to evade capture by the host's immune system. The iroN gene is under negative control of the Fur repressor and transcribed under iron limiting conditions. We show here that transcriptional de-repression is not sufficient to allow iroN expression, as this also requires activation by either of two partially homologous small RNAs (sRNAs), RyhB1 and RyhB2. The two sRNAs target the same sequence segment approximately in the middle of the 94-nucleotide 5' untranslated region (UTR) of iroN mRNA. Several lines of evidence suggest that base pair interaction stimulates iroN mRNA translation. Activation does not result from the disruption of a secondary structure masking the ribosome binding site; rather it involves sequences at the 5' end of iroN 5' UTR. In vitro 'toeprint' assays revealed that this upstream site binds the 30S ribosomal subunit provided that RyhB1 is paired with the mRNA. Altogether, our data suggest that RyhB1, and to lesser extent RyhB2, activate iroN mRNA translation by promoting entry of the ribosome at an upstream 'standby' site. These findings add yet an additional nuance to the polychromatic landscape of sRNA-mediated regulation.
    Mots-clés : 5' Untranslated Regions, Bacterial Outer Membrane Proteins, Bacterial Proteins, Base Sequence, Binding Sites, Codon, Initiator, Conserved Sequence, DBG, Gene Expression Regulation, Bacterial, Nucleic Acid Conformation, Nucleotide Motifs, Protein Binding, Receptors, Cell Surface, RGSP, Ribosomes, RNA Stability, RNA, Bacterial, RNA, Messenger, RNA-Binding Proteins.

  • E. Barbier, A. Lagorce, A. Hachemi, M. Dutertre, A. Gorlas, L. Morand, C. Saint-Pierre, J. - L. Ravanat, T. Douki, J. Armengaud, D. Gasparutto, F. Confalonieri, et J. Breton, « Oxidative DNA Damage and Repair in the Radioresistant Archaeon Thermococcus gammatolerans », Chemical Research in Toxicology, vol. 29, nᵒ 11, p. 1796-1809, 2016.
    Résumé : The hyperthermophilic archaeon Thermococcus gammatolerans can resist huge doses of γ-irradiation, up to 5.0 kGy, without loss of viability. The potential to withstand such harsh conditions is probably due to complementary passive and active mechanisms, including repair of damaged chromosomes. In this work, we documented the formation and repair of oxidative DNA lesions in T. gammatolerans. The basal level of the oxidized nucleoside, 8-oxo-2'-deoxyguanosine (8-oxo-dGuo), was established at 9.2 (± 0.9) 8-oxo-dGuo per 10(6) nucleosides, a higher level than those usually measured in eukaryotic cells or bacteria. A significant increase in oxidative damage, i.e., up to 24.2 (± 8.0) 8-oxo-dGuo/10(6) nucleosides, was measured for T. gammatolerans exposed to a 5.0 kGy dose of γ-rays. Surprisingly, the yield of radiation-induced modifications was lower than those previously observed for human cells exposed to doses corresponding to a few grays. One hour after irradiation, 8-oxo-dGuo levels were significantly reduced, indicating an efficient repair. Two putative base excision repair (BER) enzymes, TGAM_1277 and TGAM_1653, were demonstrated both by proteomics and transcriptomics to be present in the cells without exposure to ionizing radiation. Their transcripts were moderately upregulated after gamma irradiation. After heterologous production and purification of these enzymes, biochemical assays based on electrophoresis and MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry indicated that both have a β-elimination cleavage activity. TGAM_1653 repairs 8-oxo-dGuo, whereas TGAM_1277 is also able to remove lesions affecting pyrimidines (1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd)). This work showed that in normal growth conditions or in the presence of a strong oxidative stress, T. gammatolerans has the potential to rapidly reduce the extent of DNA oxidation, with at least these two BER enzymes as bodyguards with distinct substrate ranges.
    Mots-clés : DBG, RBA.

  • 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.

  • M. Blondel, F. Soubigou, J. Evrard, P. H. Nguyen, N. Hasin, S. Chédin, R. Gillet, M. - A. Contesse, G. Friocourt, G. Stahl, G. W. Jones, et C. Voisset, « Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation », Scientific Reports, vol. 6, p. 32117, 2016.
    Résumé : 6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI(+)] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI(+)]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI(+)] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases.
    Mots-clés : DBG, PEPS.

  • K. Bomblies, G. Jones, C. Franklin, D. Zickler, et N. Kleckner, « The challenge of evolving stable polyploidy: could an increase in "crossover interference distance" play a central role? », Chromosoma, vol. 125, nᵒ 2, p. 287-300, 2016.
    Résumé : Whole genome duplication is a prominent feature of many highly evolved organisms, especially plants. When duplications occur within species, they yield genomes comprising multiple identical or very similar copies of each chromosome ("autopolyploids"). Such genomes face special challenges during meiosis, the specialized cellular program that underlies gamete formation for sexual reproduction. Comparisons between newly formed (neo)-autotetraploids and fully evolved autotetraploids suggest that these challenges are solved by specific restrictions on the positions of crossover recombination events and, thus, the positions of chiasmata, which govern the segregation of homologs at the first meiotic division. We propose that a critical feature in the evolution of these more effective chiasma patterns is an increase in the effective distance of meiotic crossover interference, which plays a central role in crossover positioning. We discuss the findings in several organisms, including the recent identification of relevant genes in Arabidopsis arenosa, that support this hypothesis.
    Mots-clés : Chiasmata, Chromosomes, Plant, Crossing Over, Genetic, Crossover interference, DBG, DSMC, Evolution, Molecular, Homologous chromosomes, Meiosis, Plants, Polyploidy, Recombination.

  • L. Bossi et N. Figueroa-Bossi, « Competing endogenous RNAs: a target-centric view of small RNA regulation in bacteria », Nature Reviews. Microbiology, vol. 14, nᵒ 12, p. 775-784, 2016.
    Résumé : Many bacterial regulatory small RNAs (sRNAs) have several mRNA targets, which places them at the centre of regulatory networks that help bacteria to adapt to environmental changes. However, different mRNA targets of any given sRNA compete with each other for binding to the sRNA; thus, depending on relative abundances and sRNA affinity, competition for regulatory sRNAs can mediate cross-regulation between bacterial mRNAs. This 'target-centric' perspective of sRNA regulation is reminiscent of the competing endogenous RNA (ceRNA) hypothesis, which posits that competition for a limited pool of microRNAs (miRNAs) in higher eukaryotes mediates cross-regulation of mRNAs. In this Opinion article, we discuss evidence that a similar network of RNA crosstalk operates in bacteria, and that this network also includes crosstalk between sRNAs and competition for RNA-binding proteins.
    Mots-clés : DBG, RGSP.

  • P. Bouloc et F. Repoila, « Fresh layers of RNA-mediated regulation in Gram-positive bacteria », Current Opinion in Microbiology, vol. 30, p. 30-35, 2016.
    Résumé : Bacterial regulatory RNAs have been defined as diverse classes of cis and trans elements that may intervene at each step of gene expression, from RNA and protein synthesis to degradation. Here, we report on a few examples from Gram-positive bacteria that extend the definition of regulatory RNAs to include 5' and 3' UTRs that also act as cis and trans regulators. New examples unveil the existence of cis and trans acting regulatory RNAs on a single molecule. Also, we highlight data showing that a key RNA chaperone in Enterobacteriaceae, Hfq, does not fulfill the same role in Gram-positive Firmicutes.
    Mots-clés : Bacterial Proteins, DBG, Gene Expression Regulation, Bacterial, Gram-Positive Bacteria, RNA, Bacterial, SRRB.

  • A. Breiman, S. Fieulaine, T. Meinnel, et C. Giglione, « The intriguing realm of protein biogenesis: Facing the green co-translational protein maturation networks », Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, vol. 1864, nᵒ 5, p. 531-550, 2016.
    Mots-clés : B3S, Co-translational folding, Co-translational modifications, Co-translational targeting, DBG, IMAPP, Macromolecular Substances, Plants, PROMTI, Protein Biosynthesis, Protein Folding, Protein Processing, Post-Translational, Proteolysis, Ribosome, Ribosome-associated protein biogenesis factors, Ribosomes.

  • P. Brézellec, I. Vallet-Gely, C. Possoz, S. Quevillon-Cheruel, et J. - L. Ferat, « DciA is an ancestral replicative helicase operator essential for bacterial replication initiation », Nature Communications, vol. 7, p. 13271, 2016.
    Résumé : Delivery of the replicative helicase onto DNA is an essential step in the initiation of replication. In bacteria, DnaC (in Escherichia coli) and DnaI (in Bacillus subtilis) are representative of the two known mechanisms that assist the replicative helicase at this stage. Here, we establish that these two strategies cannot be regarded as prototypical of the bacterial domain since dnaC and dnaI (dna[CI]) are present in only a few bacterial phyla. We show that dna[CI] was domesticated at least seven times through evolution in bacteria and at the expense of one gene, which we rename dciA (dna[CI] antecedent), suggesting that DciA and Dna[CI] share a common function. We validate this hypothesis by establishing in Pseudomonas aeruginosa that DciA possesses the attributes of the replicative helicase-operating proteins associated with replication initiation.
    Mots-clés : B3S, DBG, EMC2, FAAM, OCB.

  • 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.

  • R. Culerrier, M. Carraz, C. Mann, et M. Djabali, « MSK1 triggers the expression of the INK4AB/ARF locus in oncogene-induced senescence », Molecular Biology of the Cell, vol. 27, nᵒ 17, p. 2726-2734, 2016.
    Résumé : The tumor suppressor proteins p15(INK4B), p16(INK4A), and p14(ARF), encoded by the INK4AB/ARF locus, are crucial regulators of cellular senescence. The locus is epigenetically silenced by the repressive Polycomb complexes in growing cells but is activated in response to oncogenic stress. Here we show that the mitogen- and stress-activated kinase (MSK1) is up-regulated after RAF1 oncogenic stress and that the phosphorylated (activated) form of MSK1 is significantly increased in the nucleus and recruited to the INK4AB/ARF locus. We show that MSK1 mediates histone H3S28 phosphorylation at the INK4AB/ARF locus and contributes to the rapid transcriptional activation of p15(INK4B) and p16(INK4A) in human cells despite the presence of the repressive H3K27me3 mark. Furthermore, we show that upon MSK1 depletion in oncogenic RAF1-expressing cells, H3S28ph presence at the INK4 locus and p15(INK4B) and p16(INK4A) expression are reduced. Finally, we show that H3S28-MSK-dependent phosphorylation functions in response to RAF1 signaling and that ERK and p38α contribute to MSK1 activation in oncogene-induced senescence.
    Mots-clés : DBG, SEN.

  • M. de Dieuleveult, K. Yen, I. Hmitou, A. Depaux, F. Boussouar, D. Bou Dargham, S. Jounier, H. Humbertclaude, F. Ribierre, C. Baulard, N. P. Farrell, B. Park, C. Keime, L. Carrière, S. Berlivet, M. Gut, I. Gut, M. Werner, J. - F. Deleuze, R. Olaso, J. - C. Aude, S. Chantalat, B. F. Pugh, et M. Gérard, « Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells », Nature, vol. 530, nᵒ 7588, p. 113-116, 2016.
    Résumé : ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers target specific nucleosomes to regulate transcription is unclear. Here we present genome-wide remodeller-nucleosome interaction profiles for the chromatin remodellers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank micrococcal nuclease (MNase)-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites are nevertheless bound by non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and marked by H3K4me3 and H3K27ac modifications. RNA polymerase II therefore navigates hundreds of base pairs of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3' end of the NFR. Transcriptome analysis after remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers have either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs.
    Mots-clés : Animals, Chromatin Assembly and Disassembly, DBG, DNA Helicases, DNA-Binding Proteins, Gene Expression Regulation, Genome, GTR, Histones, Mice, Micrococcal Nuclease, Mouse Embryonic Stem Cells, Nuclear Proteins, Nucleosomes, Promoter Regions, Genetic, REMOD, RNA Polymerase II, Substrate Specificity, Trans-Activators, Transcription Factors, Transcription Initiation Site.

  • E. Deforzh, T. Vargas, J. Kropp, M. Vandamme, G. Pinna, et A. Polesskaya, « IMP-3 protects the mRNAs of cyclins D1 and D3 from GW182/AGO2-dependent translational repression », International Journal of Oncology, oct. 2016.
    Mots-clés : DBG, PARI, PF, RPTEG.

  • C. Denby Wilkes, O. Arnaiz, et L. Sperling, « ParTIES: a toolbox for Paramecium interspersed DNA elimination studies », Bioinformatics (Oxford, England), vol. 32, nᵒ 4, p. 599-601, 2016.
    Résumé : MOTIVATION: Developmental DNA elimination occurs in a wide variety of multicellular organisms, but ciliates are the only single-celled eukaryotes in which this phenomenon has been reported. Despite considerable interest in ciliates as models for DNA elimination, no standard methods for identification and characterization of the eliminated sequences are currently available. RESULTS: We present the Paramecium Toolbox for Interspersed DNA Elimination Studies (ParTIES), designed for Paramecium species, that (i) identifies eliminated sequences, (ii) measures their presence in a sequencing sample and (iii) detects rare elimination polymorphisms. AVAILABILITY AND IMPLEMENTATION: ParTIES is multi-threaded Perl software available at ParTIES is distributed under the GNU General Public Licence v3.
    Mots-clés : ANGE, Ciliophora Infections, DBG, DNA, Protozoan, Genome, Protozoan, Interspersed Repetitive Sequences, Paramecium, Protozoan Proteins, Software.

  • Y. Deng, C. Chen, Z. Zhao, J. Zhao, A. Jacq, X. Huang, et Y. Yang, « The RNA Chaperone Hfq Is Involved in Colony Morphology, Nutrient Utilization and Oxidative and Envelope Stress Response in Vibrio alginolyticus », PloS One, vol. 11, nᵒ 9, p. e0163689, 2016.
    Résumé : Hfq is a global regulator that is involved in environmental adaptation of bacteria and in pathogenicity. To gain insight into the role of Hfq in Vibrio alginolyticus, an hfq deletion mutant was constructed in V. alginolyticus ZJ-T strain and phenotypically characterized. Deletion of hfq led to an alteration of colony morphology and reduced extracellular polysaccharide production, a general impairment of growth in both rich medium and minimal media with different carbon sources or amino acids, enhanced sensitivity to oxidative stress and to several antibiotics. Furthermore, a differential transcriptomic analysis showed significant changes of transcript abundance for 306 protein coding genes, with 179 genes being up regulated and 127 down-regulated. Several of these changes could be related to the observed phenotypes of the mutant. Transcriptomic data also provided evidence for the induction of the extracytoplasmic stress response in absence of Hfq. Altogether, these findings point to broad regulatory functions for Hfq in V. alginolyticus cells, likely to underlie an important role in pathogenicity.
    Mots-clés : DBG, SRRB.

  • A. Devigne, P. Guérin, J. Lisboa, S. Quevillon-Cheruel, J. Armengaud, S. Sommer, C. Bouthier de la Tour, et P. Servant, « PprA Protein Is Involved in Chromosome Segregation via Its Physical and Functional Interaction with DNA Gyrase in Irradiated Deinococcus radiodurans Bacteria », mSphere, vol. 1, nᵒ 1, 2016.
    Résumé : PprA, a radiation-induced Deinococcus-specific protein, was previously shown to be required for cell survival and accurate chromosome segregation after exposure to ionizing radiation. Here, we used an in vivo approach to determine, by shotgun proteomics, putative PprA partners coimmunoprecipitating with PprA when cells were exposed to gamma rays. Among them, we found the two subunits of DNA gyrase and, thus, chose to focus our work on characterizing the activities of the deinococcal DNA gyrase in the presence or absence of PprA. Loss of PprA rendered cells hypersensitive to novobiocin, an inhibitor of the B subunit of DNA gyrase. We showed that treatment of bacteria with novobiocin resulted in induction of the radiation desiccation response (RDR) regulon and in defects in chromosome segregation that were aggravated by the absence of PprA. In vitro, the deinococcal DNA gyrase, like other bacterial DNA gyrases, possesses DNA negative supercoiling and decatenation activities. These two activities are inhibited in vitro by novobiocin and nalidixic acid, whereas PprA specifically stimulates the decatenation activity of DNA gyrase. Together, these results suggest that PprA plays a major role in chromosome decatenation via its interaction with the deinococcal DNA gyrase when D. radiodurans cells are recovering from exposure to ionizing radiation. IMPORTANCE D. radiodurans is one of the most radiation-resistant organisms known. This bacterium is able to cope with high levels of DNA lesions generated by exposure to extreme doses of ionizing radiation and to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Here, we identified partners of PprA, a radiation-induced Deinococcus-specific protein, previously shown to be required for radioresistance. Our study leads to three main findings: (i) PprA interacts with DNA gyrase after irradiation, (ii) treatment of cells with novobiocin results in defects in chromosome segregation that are aggravated by the absence of PprA, and (iii) PprA stimulates the decatenation activity of DNA gyrase. Our results extend the knowledge of how D. radiodurans cells survive exposure to extreme doses of gamma irradiation and point out the link between DNA repair, chromosome segregation, and DNA gyrase activities in the radioresistant D. radiodurans bacterium.
    Mots-clés : DBG, Deinococcus radiodurans, DNA decatenation, DNA gyrase, PprA, RBA.

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