UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2008

Cyrille Girard, Céline Verheggen, Henry Neel, Anne Cammas, Stephan Vagner, Johann Soret, Edouard Bertrand, Rémy Bordonné (2008 Jan 25)

Characterization of a short isoform of human Tgs1 hypermethylase associating with small nucleolar ribonucleoprotein core proteins and produced by limited proteolytic processing.

The Journal of biological chemistry : 2060-9 : DOI : 10.1074/jbc.M704209200 En savoir plus
Résumé

Tgs1 is the hypermethylase responsible for m(3)G cap formation of U small nuclear RNAs (U snRNAs) and small nucleolar RNAs (snoRNAs). In vertebrates, hypermethylation of snRNAs occurs in the cytoplasm, whereas this process takes place in the nucleus for snoRNAs. Accordingly, the hypermethylase is found in both compartments with a diffuse localization in the cytoplasm and a concentration in Cajal bodies in the nucleoplasm. In this study, we report that the Tgs1 hypermethylase exists as two species, a full-length cytoplasmic isoform and a shorter nuclear isoform of 65-70 kDa. The short isoform exhibits methyltransferase activity and associates with components of box C/D and H/ACA snoRNPs, pointing to a role of this isoform in hypermethylation of snoRNAs. We also show that production of the short Tgs1 isoform is inhibited by MG132, suggesting that it results from proteasomal limited processing of the full-length Tgs1 protein. Together, our results suggest that proteasome maturation constitutes a mechanism regulating Tgs1 function by generating Tgs1 species with different substrate specificities, subcellular localizations, and functions.

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Année de publication : 2007

Anne Cammas, Frédéric Pileur, Sophie Bonnal, Stephen M Lewis, Nicolas Lévêque, Martin Holcik, Stéphan Vagner (2007 Dec 1)

Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs.

Molecular biology of the cell : 5048-59 : DOI : 10.1091/mbc.E07-06-0603 En savoir plus
Résumé

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a nucleocytoplasmic shuttling protein that regulates gene expression through its action on mRNA metabolism and translation. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here, we show that hnRNP A1 is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5′ untranslated region of both the human rhinovirus-2 and the human apoptotic peptidase activating factor 1 (apaf-1) mRNAs, thereby regulating their translation. Furthermore, the cytoplasmic redistribution of hnRNP A1 after rhinovirus infection leads to enhanced rhinovirus IRES-mediated translation, whereas the cytoplasmic relocalization of hnRNP A1 after UVC irradiation limits the UVC-triggered translational activation of the apaf-1 IRES. Therefore, this study provides a direct demonstration that IRESs behave as translational enhancer elements regulated by specific trans-acting mRNA binding proteins in given physiological conditions. Our data highlight a new way to regulate protein synthesis in eukaryotes through the subcellular relocalization of a nuclear mRNA-binding protein.

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Rong-Bing Guo, Pascal Rigolet, Hua Ren, Bo Zhang, Xing-Dong Zhang, Shuo-Xing Dou, Peng-Ye Wang, Mounira Amor-Gueret, Xu Guang Xi (2007 Sep 18)

Structural and functional analyses of disease-causing missense mutations in Bloom syndrome protein.

Nucleic acids research : 6297-310 : DOI : 10.1093/nar/gkm536 En savoir plus
Résumé

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.

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Hua Ren, Shuo-Xing Dou, Pascal Rigolet, Ye Yang, Peng-Ye Wang, Mounira Amor-Gueret, Xu Guang Xi (2007 Aug 30)

The arginine finger of the Bloom syndrome protein: its structural organization and its role in energy coupling.

Nucleic acids research : 6029-41 : DOI : 10.1093/nar/gkm544 En savoir plus
Résumé

RecQ family helicases are essential in maintaining chromosomal DNA stability and integrity. Despite extensive studies, the mechanisms of these enzymes are still poorly understood. Crystal structures of many helicases reveal a highly conserved arginine residue located near the gamma-phosphate of ATP. This residue is widely recognized as an arginine finger, and may sense ATP binding and hydrolysis, and transmit conformational changes. We investigated the existence and role of the arginine finger in the Bloom syndrome protein (BLM), a RecQ family helicase, in ATP hydrolysis and energy coupling. Our studies by combination of structural modelling, site-directed mutagenesis and biochemical and biophysical approaches, demonstrate that mutations of residues interacting with the gamma-phosphate of ATP or surrounding the ATP-binding sites result in severe impairment in the ATPase activity of BLM. These mutations also impair BLM’s DNA-unwinding activities, but do not affect its ATP and DNA-binding abilities. These data allow us to identify R982 as the residue that functions as a BLM arginine finger. Our findings further indicate how the arginine finger is precisely positioned by the conserved motifs with respect to the gamma-phosphate.

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Sarah Lambert, Benoît Froget, Antony M Carr (2007 Jul 1)

Arrested replication fork processing: interplay between checkpoints and recombination.

DNA repair : 1042-61 : DOI : 10.1016/j.dnarep.2007.02.024 En savoir plus
Résumé

The arrest of DNA replication by DNA damage, nucleotide depletion, DNA-protein complexes or following clashes between transcription and replication factors all have the capacity to promote genome instability. In this review, we discuss how DNA replication is regulated by the checkpoint pathways that stabilise arrested replication forks and the recombination factors that process specific DNA structures resulting from fork arrest. We examine what is known about the interplay between the checkpoints and the recombination apparatus and review the evidence for a recombination-based fork restart pathway in eukaryotes.

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Hind Medyouf, Hélène Alcalde, Caroline Berthier, Marie Claude Guillemin, Nuno R dos Santos, Anne Janin, Didier Decaudin, Hugues de Thé, Jacques Ghysdael (2007 May 21)

Targeting calcineurin activation as a therapeutic strategy for T-cell acute lymphoblastic leukemia.

Nature medicine : 736-41 : DOI : 10.1038/nm1588 En savoir plus
Résumé

Calcineurin is a calcium-activated serine/threonine phosphatase critical to a number of developmental processes in the cardiovascular, nervous and immune systems. In the T-cell lineage, calcineurin activation is important for pre-T-cell receptor (TCR) signaling, TCR-mediated positive selection of thymocytes into mature T cells, and many aspects of the immune response. The critical role of calcineurin in the immune response is underscored by the fact that calcineurin inhibitors, such as cyclosporin A (CsA) and FK506, are powerful immunosuppressants in wide clinical use. We observed sustained calcineurin activation in human B- and T-cell lymphomas and in all mouse models of lymphoid malignancies analyzed. In intracellular NOTCH1 (ICN1)- and TEL-JAK2-induced T-cell lymphoblastic leukemia, two mouse models relevant to human malignancies, in vivo inhibition of calcineurin activity by CsA or FK506 induced apoptosis of leukemic cells and rapid tumor clearance, and substantially prolonged mouse survival. In contrast, ectopic expression of a constitutively activated mutant of calcineurin favored leukemia progression. Moreover, CsA treatment induced apoptosis in human lymphoma and leukemia cell lines. Thus, calcineurin activation is critical for the maintenance of the leukemic phenotype in vivo, identifying this pathway as a relevant therapeutic target in lymphoid malignancies.

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Juliette van Dijk, Krzysztof Rogowski, Julie Miro, Benjamin Lacroix, Bernard Eddé, Carsten Janke (2007 May 11)

A targeted multienzyme mechanism for selective microtubule polyglutamylation.

Molecular cell : 437-48 : DOI : 10.1016/j.molcel.2007.04.012 En savoir plus
Résumé

Polyglutamylases are enzymes that form polyglutamate side chains of variable lengths on proteins. Polyglutamylation of tubulin is believed to regulate interactions of microtubules (MTs) with MT-associated proteins and molecular motors. Subpopulations of MTs are differentially polyglutamylated, yet only one modifying enzyme has been discovered in mammals. In an attempt to better understand the heterogeneous appearance of tubulin polyglutamylation, we searched for additional enzymes and report here the identification of six mammalian polyglutamylases. Each of them has a characteristic mode of catalysis and generates distinct patterns of modification on MTs, which can be further diversified by cooperation of multiple enzymes. Polyglutamylases are restricted to confined tissues and subtypes of MTs by differential expression and localization. In conclusion, we propose a multienzyme mechanism of polyglutamylation that can explain how the diversity of polyglutamylation on selected types of MTs is controlled at the molecular level.

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Nuno R dos Santos, David S Rickman, Aurélien de Reynies, Françoise Cormier, Maryvonne Williame, Camille Blanchard, Marc-Henri Stern, Jacques Ghysdael (2007 May 1)

Pre-TCR expression cooperates with TEL-JAK2 to transform immature thymocytes and induce T-cell leukemia.

Blood : 3972-81 : DOI : 10.1182/blood-2006-09-048801 En savoir plus
Résumé

The TEL-JAK2 gene fusion, which has been identified in human leukemia, encodes a chimeric protein endowed with constitutive tyrosine kinase activity. TEL-JAK2 transgenic expression in the mouse lymphoid lineage results in fatal and rapid T-cell leukemia/lymphoma. In the present report we show that T-cell leukemic cells from EmuSRalpha-TEL-JAK2 transgenic mice present an aberrant CD8(+) differentiation phenotype, as determined by the expression of stage-specific cell surface markers and lineage-specific genes. TEL-JAK2 transforms immature CD4(-)CD8(-) double-negative thymocytes, as demonstrated by the development of T-cell leukemia with full penetrance in a Rag2-deficient genetic background. This disease is similar to the bona fide TEL-JAK2 disease as assessed by phenotypic and gene profiling analyses. Pre-TCR signaling synergizes with TEL-JAK2 to transform immature thymocytes and initiate leukemogenesis as shown by (1) the delayed leukemia onset in Rag2-, CD3epsilon- and pTalpha-deficient mice, (2) the occurrence of recurrent chromosomal alterations in pre-TCR-deficient leukemia, and (3) the correction of delayed leukemia onset in Rag2-deficient TEL-JAK2 mice by an H-Y TCRalphabeta transgene that mimics pre-TCR signaling. Although not affecting leukemia incidence and mouse survival, TCRalphabeta expression was shown to facilitate leukemic cell expansion in secondary lymphoid organs.

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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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