UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2005

Carsten Janke, Krzysztof Rogowski, Dorota Wloga, Catherine Regnard, Andrey V Kajava, Jean-Marc Strub, Nevzat Temurak, Juliette van Dijk, Dominique Boucher, Alain van Dorsselaer, Swati Suryavanshi, Jacek Gaertig, Bernard Eddé (2005 Jun 17)

Tubulin polyglutamylase enzymes are members of the TTL domain protein family.

Science (New York, N.Y.) : 1758-62 : DOI : 10.1126/science.1113010 En savoir plus
Résumé

Polyglutamylation of tubulin has been implicated in several functions of microtubules, but the identification of the responsible enzyme(s) has been challenging. We found that the neuronal tubulin polyglutamylase is a protein complex containing a tubulin tyrosine ligase-like (TTLL) protein, TTLL1. TTLL1 is a member of a large family of proteins with a TTL homology domain, whose members could catalyze ligations of diverse amino acids to tubulins or other substrates. In the model protist Tetrahymena thermophila, two conserved types of polyglutamylases were characterized that differ in substrate preference and subcellular localization.

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Juan Reguera, Esther Grueso, Aura Carreira, Cristina Sánchez-Martínez, José M Almendral, Mauricio G Mateu (2005 May 6)

Functional relevance of amino acid residues involved in interactions with ordered nucleic acid in a spherical virus.

The Journal of biological chemistry : 17969-77 : DOI : 10.1074/jbc.M500867200 En savoir plus
Résumé

In the spherical virion of the parvovirus minute virus of mice, several amino acid side chains of the capsid were previously found to be involved in interactions with the viral single-stranded DNA molecule. We have individually truncated by mutation to alanine many (ten) of these side chains and analyzed the effects on capsid assembly, stability and conformation, viral DNA encapsidation, and virion infectivity. Mutation of residues Tyr-270, Asp-273, or Asp-474 led to a drastic reduction in infectivity. Mutant Y270A was defective in capsid assembly; mutant D273A formed stable capsids, but it was essentially unable to encapsidate the viral DNA or to externalize the N terminus of the capsid protein VP2, a connected conformational event. Mutation of residues Asp-58, Trp-60, Asn-183, Thr-267, or Lys-471 led to a moderate reduction in infectivity. None of these mutations had an effect on capsid assembly or stability, or on the DNA encapsidation process. However, those five mutant virions were substantially less stable than the parental virion in thermal inactivation assays. The results with this model spherical virus indicate that several capsid residues that are found to be involved in polar interactions or multiple hydrophobic contacts with the viral DNA molecule contribute to preserving the active conformation of the infectious viral particle. Their effect appears to be mediated by the non-covalent interactions they establish with the viral DNA. In addition, at least one acidic residue at each DNA-binding region is needed for DNA packaging.

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Sophie Bonnal, Frédéric Pileur, Cécile Orsini, Fabienne Parker, Françoise Pujol, Anne-Catherine Prats, Stéphan Vagner (2005 Feb 11)

Heterogeneous nuclear ribonucleoprotein A1 is a novel internal ribosome entry site trans-acting factor that modulates alternative initiation of translation of the fibroblast growth factor 2 mRNA.

The Journal of biological chemistry : 4144-53 : DOI : 10.1074/jbc.M411492200 En savoir plus
Résumé

Alternative initiation of translation of the human fibroblast growth factor 2 (FGF-2) mRNA at five in-frame CUG or AUG translation initiation codons requires various RNA cis-acting elements, including an internal ribosome entry site (IRES). Here we describe the purification of a trans-acting factor controlling FGF-2 mRNA translation achieved by several biochemical purification approaches. We have identified the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a factor that binds to the FGF-2 5′-leader RNA and that also complements defective FGF-2 translation in vitro in rabbit reticulocyte lysate. Recombinant hnRNP A1 stimulates in vitro translation at the four IRES-dependent initiation codons but has no effect on the cap-dependent initiation codon. Consistent with a role of hnRNP A1 in the control of alternative initiation of translation, short interfering RNA-mediated knock down of hnRNP A1 specifically inhibits translation at the four IRES-dependent initiation codons. Furthermore, hnRNP A1 binds to the FGF-2 IRES, implicating this interaction in the control of alternative initiation of translation.

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Année de publication : 2004

Emilie Bayart, Olga Grigorieva, Serge Leibovitch, Rosine Onclercq-Delic, Mounira Amor-Guéret (2004 Dec 15)

A major role for mitotic CDC2 kinase inactivation in the establishment of the mitotic DNA damage checkpoint.

Cancer research : 8954-9 : DOI : 10.1158/0008-5472.CAN-04-1613 En savoir plus
Résumé

Cdc2 kinase is inactivated when DNA damage occurs during the spindle assembly checkpoint. Here, we show that the level of mitotic Bloom syndrome protein phosphorylation reflects the level of cdc2 activity. A complete inactivation of cdc2 by either introduction of DNA double-strand breaks or roscovitine treatment prevents exit from mitosis. Thus, mitotic cdc2 inactivation plays a major role in the establishment of the mitotic DNA damage checkpoint. In response to mitotic cdc2 inactivation, the M/G(1) transition is delayed after releasing the drug block in nonmalignant cells, whereas tumor cells exit mitosis without dividing and rereplicate their DNA, which results in mitotic catastrophe. This opens the way for new chemotherapeutic strategies.

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Carsten Janke, Maria M Magiera, Nicole Rathfelder, Christof Taxis, Simone Reber, Hiromi Maekawa, Alexandra Moreno-Borchart, Georg Doenges, Etienne Schwob, Elmar Schiebel, Michael Knop (2004 Aug 5)

A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.

Yeast (Chichester, England) : 947-62 : DOI : 10.1002/yea.1142 En savoir plus
Résumé

Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.

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Christel Boutonnet, Olivier Boijoux, Sandra Bernat, Abdelhakkim Kharrat, Gilles Favre, Jean-Charles Faye, Stéphan Vagner (2004 Jul 1)

Pharmacological-based translational induction of transgene expression in mammalian cells.

EMBO reports : 721-7 : DOI : 10.1038/sj.embor.7400170 En savoir plus
Résumé

In the quest for the development of pharmacological switches that control gene expression, no system has been reported that regulates at the translational level. To permit small-molecule control of transgene translation, we have constructed a farnesyl transferase inhibitor-responsive translation initiation factor. This artificial protein is a three-component chimaera consisting of the ribosome recruitment core of the eIF4G1 eukaryotic translation initiation factor, the RNA-binding domain of the R17 bacteriophage coat protein and the plasma membrane localization CAAX motif of farnesylated H-Ras. This membrane-delocalized translation factor is inactive unless liberated in the cytosol. Farnesyl transferase inhibitor FTI-277 prevents the membrane association of the CAAX motif and thus increases the cytoplasmic levels of the eIF4G fusion protein, which is then capable of inducing translation of the second cistron of a bicistronic messenger RNA containing an R17-binding site in its intercistronic space. Such direct translational control by farnesyl transferase inhibitors provides a system for fast, graded and reversible regulation of transgene expression.

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Juan Reguera, Aura Carreira, Laura Riolobos, José María Almendral, Mauricio G Mateu (2004 Mar 2)

Role of interfacial amino acid residues in assembly, stability, and conformation of a spherical virus capsid.

Proceedings of the National Academy of Sciences of the United States of America : 2724-9 : DOI : 10.1073/pnas.0307748101 En savoir plus
Résumé

Twenty-eight amino acid residues involved in most noncovalent interactions between trimeric protein subunits in the capsid of the parvovirus minute virus of mice were truncated individually to alanine, and the effects on capsid assembly, thermostability, and conformation were analyzed. Only seven side chains were essential for protein subunit recognition. These side chains virtually corresponded with those that either buried a large hydrophobic surface on trimer association or formed buried intertrimer hydrogen bonds or salt bridges. The seven residues are evolutionarily conserved, and they define regularly spaced spots on a thin equatorial belt surrounding each trimer. Truncation of the many side chains that were dispensable for assembly, including those participating in solvent-accessible polar interactions, did not substantially affect capsid thermostability either. However, the interfacial residues located at the base of the pores delineating the capsid five-fold axes participated in a heat-induced conformational rearrangement associated with externalization of the capsid protein N terminus, and they were needed for infectivity. Thus, at the subunit interfaces of this model virus capsid, only key residues involved in the strongest interactions are critical for assembly and stability, but additional residues fulfill other important biological roles.

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Aura Carreira, Margarita Menéndez, Juan Reguera, José María Almendral, Mauricio G Mateu (2004 Feb 20)

In vitro disassembly of a parvovirus capsid and effect on capsid stability of heterologous peptide insertions in surface loops.

The Journal of biological chemistry : 6517-25 : DOI : 10.1074/jbc.M307662200 En savoir plus
Résumé

We have analyzed the in vitro disassembly of the capsid of the minute virus of mice, and the stability of capsid chimeras carrying heterologous epitope insertions. Upon heating in a physiological buffer, empty capsids formed by 60 copies of protein VP2 underwent first a reversible conformational change with a small enthalpy change detected by fluorescence. This change was associated with, but not limited to, externalization of the VP2 N terminus. Irreversible capsid dissociation as detected by changes in fluorescence, hemagglutination activity, and electrophoretic mobility occurred at much higher temperatures. Differential scanning calorimetry in the same conditions indicated that the dissociation/denaturation transition involved a high enthalpy change and proceeded through one or more intermediates. In contrast, in the presence of 1.5 M guanidinium chloride, heat-induced disassembly fitted a two-state irreversible process. Both thermally and chemically induced dissociation/denaturation yielded a form that had lost a part of the tertiary structure, but still retained the native secondary structure. Data from chemical dissociation indicates this form may correspond to a molten globule-like monomeric state of the capsid protein. All five antigenic peptide insertions attempted in exposed loops, despite being perhaps among the least disruptive, led to defects in folding/assembly of the capsid and, in most cases, to reduced capsid stability against thermal dissociation. The results with one of the simplest viral capsids reveal a complex pathway for disassembly, and a reduction in capsid assembly and stability upon insertion of peptides, even within the most exposed capsid loops.

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Année de publication : 2003

Rosine Onclercq-Delic, Patrick Calsou, Christine Delteil, Bernard Salles, Dora Papadopoulo, Mounira Amor-Guéret (2003 Nov 1)

Possible anti-recombinogenic role of Bloom’s syndrome helicase in double-strand break processing.

Nucleic acids research : 6272-82 : DOI : 10.1093/nar/gkg834 En savoir plus
Résumé

Bloom’s syndrome (BS) which associates genetic instability and predisposition to cancer is caused by mutations in the BLM gene encoding a RecQ family 3′-5′ DNA helicase. It has been proposed that the generation of genetic instability in BS cells could result from an aberrant non-homologous DNA end joining (NHEJ), one of the two main DNA double-strand break (DSB) repair pathways in mammalian cells, the second major pathway being homologous recombination (HR). Using cell extracts, we report first that Ku70/80 and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), key factors of the end-joining machinery, and BLM are located in close proximity on DNA and that BLM binds to DNA only in the absence of ATP. In the presence of ATP, BLM is phosphorylated and dissociates from DNA in a strictly DNA-PKcs-dependent manner. We also show that BS cells display, in vivo, an accurate joining of DSBs, reflecting thus a functional NHEJ pathway. In sharp contrast, a 5-fold increase of the HR-mediated DNA DSB repair in BS cells was observed. These results support a model in which NHEJ activation mediates BLM dissociation from DNA, whereas, under conditions where HR is favored, e.g. at the replication fork, BLM exhibits an anti-recombinogenic role.

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Catherine Regnard, Didier Fesquet, Carsten Janke, Dominique Boucher, Elisabeth Desbruyéres, Annette Koulakoff, Christine Insina, Pierre Travo, Bernard Eddé (2003 Oct 15)

Characterisation of PGs1, a subunit of a protein complex co-purifying with tubulin polyglutamylase.

Journal of cell science : 4181-90 : DOI : 10.1242/jcs.00743 En savoir plus
Résumé

Polyglutamylation is a post-translational modification initially discovered on tubulin. It has been implicated in multiple microtubule functions, including neuronal differentiation, axonemal beating and stability of the centrioles, and shown to modulate the interaction between tubulin and microtubule associated proteins. The enzymes catalysing this modification are not yet known. Starting with a partially purified fraction of mouse brain tubulin polyglutamylase, monoclonal antibodies were raised and used to further purify the enzyme by immunoprecipitation. The purified enzyme complex (Mr 360×103) displayed at least three major polypeptides of 32, 50 and 80×103, present in stochiometric amounts. We show that the 32×103 subunit is encoded by the mouse gene GTRGEO22, the mutation of which has recently been implicated in multiple defects in mice, including male sterility. We demonstrate that this subunit, called PGs1, has no catalytic activity on its own, but is implicated in the localisation of the enzyme at major sites of polyglutamylation, i.e. neurones, axonemes and centrioles.

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Sophie Bonnal, Céline Schaeffer, Laurent Créancier, Simone Clamens, Hervé Moine, Anne-Catherine Prats, Stéphan Vagner (2003 Oct 10)

A single internal ribosome entry site containing a G quartet RNA structure drives fibroblast growth factor 2 gene expression at four alternative translation initiation codons.

The Journal of biological chemistry : 39330-6 : DOI : 10.1074/jbc.M305580200 En savoir plus
Résumé

The 484-nucleotide (nt) alternatively translated region (ATR) of the human fibroblast growth factor 2 (FGF-2) mRNA contains four CUG and one AUG translation initiation codons. Although the 5′-end proximal CUG codon is initiated by a cap-dependent translation process, the other four initiation codons are initiated by a mechanism of internal entry of ribosomes. We undertook here a detailed analysis of the cis-acting elements defining the FGF-2 internal ribosome entry site (IRES). A thorough deletion analysis study within the 5′-ATR led us to define a 176-nt region as being necessary and sufficient for IRES function at four codons present in a downstream 308-nt RNA segment. Unexpectedly, a single IRES module is therefore responsible for translation initiation at four distantly localized codons. The determination of the FGF-2 5′-ATR RNA secondary structure by enzymatic and chemical probing experiments showed that the FGF-2 IRES contained two stem-loop regions and a G quartet motif that constitute novel structural determinants of IRES function.

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Christian Touriol, Stéphanie Bornes, Sophie Bonnal, Sylvie Audigier, Hervé Prats, Anne-Catherine Prats, Stéphan Vagner (2003 May 1)

Generation of protein isoform diversity by alternative initiation of translation at non-AUG codons.

Biology of the cell / under the auspices of the European Cell Biology Organization : 169-78 : DOI : 10.1016/S0248-4900(03)00033-9 En savoir plus
Résumé

The use of several translation initiation codons in a single mRNA, by expressing several proteins from a single gene, contributes to the generation of protein diversity. A small, yet growing, number of mammalian mRNAs initiate translation from a non-AUG codon, in addition to initiating at a downstream in-frame AUG codon. Translation initiation on such mRNAs results in the synthesis of proteins harbouring different amino terminal domains potentially conferring on these isoforms distinct functions. Use of non-AUG codons appears to be governed by several features, including the sequence context and the secondary structure surrounding the codon. Selection of the downstream initiation codon can occur by leaky scanning of the 43S ribosomal subunit, internal entry of ribosome or ribosomal shunting. The biological significance of non-AUG alternative initiation is demonstrated by the different subcellular localisations and/or distinct biological functions of the isoforms translated from the single mRNA as illustrated by the two main angiogenic factor genes encoding the fibroblast growth factor 2 (FGF2) and the vascular endothelial growth factor (VEGF). Consequently, the regulation of alternative initiation of translation might have a crucial role for the biological function of the gene product.

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Sophie Bonnal, Christel Boutonnet, Leonel Prado-Lourenço, Stéphan Vagner (2003 Jan 1)

IRESdb: the Internal Ribosome Entry Site database.

Nucleic acids research : 427-8 : DOI : 10.1093/nar/gkg003 En savoir plus
Résumé

Internal Ribosome Entry Sites (IRES) are cis-acting RNA sequences able to mediate internal entry of the 40S ribosomal subunit on some eukaryotic and viral messenger RNAs upstream of a translation initiation codon. These sequences are very diverse and are present in a growing list of mRNAs. Novel IRES sequences continue to be added to public databases every year and the list of unknown IRESes is certainly still very large. The IRES database is a comprehensive WWW resource for internal ribosome entry sites and presents currently available general information as well as detailed data for each IRES. It is a searchable, periodically updated collection of IRES RNA sequences. Sequences are presented in FASTA form and hotlinked to NCBI GenBank files. Several subsets of data are classified according to the viral taxon (for viral IRESes), to the gene product function (for cellular IRESes), to the possible cellular regulation or to the trans-acting factor that mediates IRES function. This database is accessible at http://ifr31w3.toulouse.inserm.fr/IRESdatabase/.

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Année de publication : 2002

Stefania Millevoi, Finola Geraghty, Bernadine Idowu, Jennifer L Y Tam, Michael Antoniou, Stéphan Vagner (2002 Sep 1)

A novel function for the U2AF 65 splicing factor in promoting pre-mRNA 3′-end processing.

EMBO reports : 869-74 : DOI : 10.1093/embo-reports/kvf173 En savoir plus
Résumé

Splicing and 3′-end processing (including cleavage and polyadenylation) of vertebrate pre-mRNAs are tightly coupled events that contribute to the extensive molecular network that coordinates gene expression. Sequences within the terminal intron of genes are essential to stimulate pre-mRNA 3′-end processing, although the factors mediating this effect are unknown. Here, we show that the pyrimidine tract of the last splice acceptor site of the human beta-globin gene is necessary to stimulate mRNA 3′-end formation in vivo and binds the U2AF 65 splicing factor. Naturally occurring beta-thalassaemia-causing mutations within the pyrimidine tract reduces both U2AF 65 binding and 3′-end cleavage efficiency. Significantly, a fusion protein containing U2AF 65, when tethered upstream of a cleavage/polyadenylation site, increases 3′-end cleavage efficiency in vitro and in vivo. Therefore, we propose that U2AF 65 promotes 3′-end processing, which contributes to 3′-terminal exon definition.

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Mouna Ababou, Virginie Dumaire, Yann Lécluse, Mounira Amor-Guéret (2002 Jul 1)

Cleavage of BLM and sensitivity of Bloom’s syndrome cells to hydroxurea and UV-C radiation.

Cell cycle (Georgetown, Tex.) : 262-6 : DOI : 10.4161/cc.1.4.136 En savoir plus
Résumé

Patients with Bloom’s syndrome (BS) show a strong genetic instability and a predisposition to all types of cancer. Here, we report that the Bloom’s syndrome protein (BLM) is cleaved in response to hydroxyurea (HU)- or UVC-induced apoptosis. The appearance and solubility of BLM proteolytic products differed according to whether proteolysis occurred in response to HU or UVC. One BS cell line homozygous for a null mutation in BLM was resistant to both UVC- and HU-induced apoptosis, while another one expressing a mutated BLM protein was resistant to HU-induced apoptosis but displayed normal sensitivity to UVC. Thus, UVC and HU appear to induce apoptosis through distinct pathways.

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