UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2007

Stephen M Lewis, Anne Veyrier, Nicoleta Hosszu Ungureanu, Sophie Bonnal, Stéphan Vagner, Martin Holcik (2007 Apr 1)

Subcellular relocalization of a trans-acting factor regulates XIAP IRES-dependent translation.

Molecular biology of the cell : 1302-11 : DOI : 10.1091/mbc.E06-06-0515 En savoir plus
Résumé

Translation of the X-linked inhibitor of apoptosis (XIAP) proceeds by internal ribosome entry site (IRES)-mediated initiation, a process that is physiologically important because XIAP expression is essential for cell survival under conditions of compromised cap-dependent translation, such as cellular stress. The regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the IRES element. We used RNA-affinity chromatography to identify XIAP ITAFs and isolated the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). We find that hnRNP A1 interacts with XIAP IRES RNA both in vitro and in vivo and that hnRNP A1 negatively regulates XIAP IRES activity. Moreover, XIAP IRES-dependent translation is significantly reduced when hnRNP A1 accumulates in the cytoplasm. Osmotic shock, a cellular stress that causes cytoplasmic accumulation of hnRNP A1, also leads to a decrease in XIAP levels that is abrogated by knockdown of hnRNP A1 expression. These results suggest that the subcellular localization of hnRNP A1 is an important determinant of its ability to negatively regulate XIAP IRES activity, suggesting that the subcellular distribution of ITAFs plays a critical role in regulating IRES-dependent translation. Our findings demonstrate that cytoplasmic hnRNP A1 is a negative regulator of XIAP IRES-dependent translation, indicating a novel function for the cytoplasmic form of this protein.

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Valérie Coiteux, Rosine Onclercq-Delic, Pierre Fenaux, Mounira Amor-Guéret (2007 Mar 1)

Predisposition to therapy-related acute leukemia with balanced chromosomal translocations does not result from a major constitutive defect in DNA double-strand break end joining.

Leukemia research : 353-8 : DOI : 10.1016/j.leukres.2006.06.003 En savoir plus
Résumé

The frequency of acute myeloid leukemia (AML) with balanced chromosomal translocations arising after anticancer therapy with DNA-damaging agents such as DNA topoisomerase II inhibitors has increased over the last two decades. However, factors that predispose to these therapy-related disorders are still poorly defined. It has been reported that DNA double-strand break (DSB) repair by the non-homologous end-joining (NHEJ) pathway is impaired in myeloid leukemia cells. This led us to hypothesize that therapy-related AML (t-AML) may result from individual differences in the repair of DSBs generated by the treatment. We show here that DSB repair is accurate, in vivo, in non-tumoral cells derived from patients who developed t-AML with t(9;11) or t(15;17) translocation after treatment for a first cancer with DNA topoisomerase II inhibitors. These results indicate that a major constitutive defect in the NHEJ pathway is unlikely to predispose to t-AML with balanced chromosomal translocations.

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Koji Ikegami, Robb L Heier, Midori Taruishi, Hiroshi Takagi, Masahiro Mukai, Shuichi Shimma, Shu Taira, Ken Hatanaka, Nobuhiro Morone, Ikuko Yao, Patrick K Campbell, Shigeki Yuasa, Carsten Janke, Grant R Macgregor, Mitsutoshi Setou (2007 Feb 27)

Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function.

Proceedings of the National Academy of Sciences of the United States of America : 3213-8 : DOI : 10.1073/pnas.0611547104 En savoir plus
Résumé

Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of alpha- and beta-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular « traffic sign » for motor proteins in neuronal cells. To investigate whether polyglutamylated alpha-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of alpha-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated alpha-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated alpha-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of alpha-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission.

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Année de publication : 2006

Stefania Millevoi, Clarisse Loulergue, Sabine Dettwiler, Sarah Zeïneb Karaa, Walter Keller, Michael Antoniou, Stéphan Vagner (2006 Oct 18)

An interaction between U2AF 65 and CF I(m) links the splicing and 3′ end processing machineries.

The EMBO journal : 4854-64 : DOI : 10.1038/sj.emboj.7601331 En savoir plus
Résumé

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3′ end processing of vertebrate pre-mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre-mRNA 3′ end processing. We identify an arginine/serine-rich region of U2AF 65 that mediates an interaction with an RS-like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF I(m)), an essential 3′ processing factor that functions at an early step in the recognition of the 3′ end processing signal. Tethered functional analysis shows that the U2AF 65/CF I(m) 59 interaction stimulates in vitro 3′ end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF I(m) 59 interaction in the functional coordination of splicing and 3′ end processing.

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C Carrasco, A Carreira, I A T Schaap, P A Serena, J Gómez-Herrero, M G Mateu, P J de Pablo (2006 Sep 12)

DNA-mediated anisotropic mechanical reinforcement of a virus.

Proceedings of the National Academy of Sciences of the United States of America : 13706-11 : DOI : 10.1073/pnas.0601881103 En savoir plus
Résumé

In this work, we provide evidence of a mechanism to reinforce the strength of an icosahedral virus by using its genomic DNA as a structural element. The mechanical properties of individual empty capsids and DNA-containing virions of the minute virus of mice are investigated by using atomic force microscopy. The stiffness of the empty capsid is found to be isotropic. Remarkably, the presence of the DNA inside the virion leads to an anisotropic reinforcement of the virus stiffness by approximately 3%, 40%, and 140% along the fivefold, threefold, and twofold symmetry axes, respectively. A finite element model of the virus indicates that this anisotropic mechanical reinforcement is due to DNA stretches bound to 60 concavities of the capsid. These results, together with evidence of biologically relevant conformational rearrangements of the capsid around pores located at the fivefold symmetry axes, suggest that the bound DNA may reinforce the overall stiffness of the viral particle without canceling the conformational changes needed for its infectivity.

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Emilie Bayart, Stéphanie Dutertre, Christian Jaulin, Rong-Bing Guo, Xu Guang Xi, Mounira Amor-Guéret (2006 Aug 1)

The Bloom syndrome helicase is a substrate of the mitotic Cdc2 kinase.

Cell cycle (Georgetown, Tex.) : 1681-6 : DOI : 10.4161/cc.5.15.3122 En savoir plus
Résumé

Bloom syndrome (BS) is a rare human autosomal recessive disorder characterized by marked genetic instability associated with greatly increased predisposition to a wide range of cancers affecting the general population. BS arises through mutations in both copies of the BLM gene which encodes a 3′-5′ DNA helicase identified as a member of the RecQ family. Several studies support a major role for BLM in the cellular response to DNA damage and stalled replication forks. However, the specific function(s) of BLM remain(s) unclear. The BLM protein is strongly expressed and phosphorylated during mitosis, but very little information is available about the origin and the significance of this phosphorylation. We show here that ATM kinase provides only a limited contribution to the mitotic phosphorylation of BLM. We also demonstrate that BLM is directly phosphorylated at multiple sites in vitro by the mitotic cdc2 kinase, and identify two new sites of mitotic BLM phosphorylation: Ser-714 and Thr-766. Our results identify BLM helicase as a new substrate for cdc2, which may have potential physiological implications for the role of BLM in mitosis.

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Aura Carreira, Mauricio G Mateu (2006 Jul 28)

Structural tolerance versus functional intolerance to mutation of hydrophobic core residues surrounding cavities in a parvovirus capsid.

Journal of molecular biology : 1081-93 : DOI : 10.1016/j.jmb.2006.05.013 En savoir plus
Résumé

The structural and functional relevance of amino acid residues surrounding cavities within the hydrophobic core of the protein subunits that form the capsid of parvoviruses has been investigated. Several of the evolutionarily conserved, hydrophobic residues that delimit these cavities in the capsid of the minute virus of mice were replaced by other hydrophobic residues that would affect the size and/or shape of the cavity. When four or more methylene-sized groups were introduced, or six or more groups removed, capsid assembly was drastically impaired. In contrast, the introduction or removal of up to three groups had no significant effect on capsid assembly or thermostability. However, many of these mutations affected a capsid conformational transition needed for viral infectivity. Replacement of some polar residues around the largest cavity showed that capsid assembly requires a carboxylate buried within this cavity, but both aspartate and glutamate are structurally accepted. Again, only the aspartate allowed the production of infectious viruses, because of a specific role in encapsidation of the viral genome. These observations provide evidence of a remarkable structural tolerance to mutation of the hydrophobic core of the protein subunits in a viral capsid, and of an involvement of core residues and internal cavities in capsid functions needed for infectivity.

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Mounira Amor-Guéret (2006 May 8)

Bloom syndrome, genomic instability and cancer: the SOS-like hypothesis.

Cancer letters : 1-12 : DOI : 10.1016/j.canlet.2005.04.023 En savoir plus
Résumé

Bloom syndrome (BS) displays one of the strongest known correlations between chromosomal instability and an increased risk of malignancy at an early age. The prevention of genomic instability and cancer depends on a complex network of pathways induced in response to DNA damage and stalled replication forks, including cell-cycle checkpoints, DNA repair, and apoptosis. Several studies have demonstrated that BLM is involved in the cellular response to DNA damage and stalled replication forks. BLM interacts physically and functionally with several proteins involved in the maintenance of genome integrity and BLM is redistributed and/or phosphorylated in response to several genotoxic stresses. The data concerning the relationship between BLM and these cellular pathways are summarized and the role of BLM in the rescue of arrested replication forks is discussed. Moreover, I speculate that BLM deficiency is lethal, and that BLM-deficient cells escaping apoptotic death do so by constitutively inducing a bacterial SOS-like response including the induction of alternative replication pathway(s) dependent on recombination, contributing to the mutator and hyper-Rec phenotypes characteristic of BS cells. This mechanism may be dependent on the RAD51 gene family, and involved in carcinogenesis in the general population.

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Irma G Gonzalez-Herrera, Leonel Prado-Lourenco, Frédéric Pileur, Caroline Conte, Aurélie Morin, Florence Cabon, Hervé Prats, Stephan Vagner, Francis Bayard, Sylvie Audigier, Anne-Catherine Prats (2006 Mar 1)

Testosterone regulates FGF-2 expression during testis maturation by an IRES-dependent translational mechanism.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology : 476-8 : DOI : 10.1096/fj.04-3314fje En savoir plus
Résumé

Spermatogenesis is a complex process involving cell proliferation, differentiation, and apoptosis. Fibroblast growth factor 2 (FGF-2) is involved in testicular function, but its role in spermatogenesis has not been fully documented. The control of FGF-2 expression particularly occurs at the translational level, by an internal ribosome entry site (IRES)-dependent mechanism driving the use of alternative initiation codons. To study IRES activity regulation in vivo, we have developed transgenic mice expressing a bicistronic construct coding for two luciferase genes. Here, we show that the FGF-2 IRES is age-dependently activated in mouse testis, whereas EMCV and c-myc IRESs are not. Real-time PCR confirms that this regulation is translational. By using immunohistological techniques, we demonstrate that FGF-2 IRES stimulation occurs in adult, but not in immature, type-A spermatogonias. This is correlated with activation of endogenous FGF-2 expression in spermatogonia; whereas FGF-2 mRNA transcription is known to decrease in adult testis. Interestingly, the FGF-2 IRES activation is triggered by testosterone and is partially inhibited by siRNA directed against the androgen receptor. Two-dimensional analysis of proteins bound to the FGF-2 mRNA 5’UTR after UV cross-linking reveals that testosterone treatment correlates with the binding of several proteins. These data suggest a paracrine loop where IRES-dependent FGF-2 expression, stimulated by Sertoli cells in response to testosterone produced by Leydig cells, would in turn activate Leydig function and testosterone production. In addition, nuclear FGF-2 isoforms could be involved in an intracrine function of FGF-2 in the start of spermatogenesis, mitosis, or meiosis initiation. This report demonstrates that mRNA translation regulation by an IRES-dependent mechanism participates in a physiological process.

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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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