UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2008

Carsten Janke, Krzysztof Rogowski, Juliette van Dijk (2008 Jul 1)

Polyglutamylation: a fine-regulator of protein function? ‘Protein Modifications: beyond the usual suspects’ review series.

EMBO reports : 636-41 : DOI : 10.1038/embor.2008.114 En savoir plus
Résumé

Polyglutamylation is a post-translational modification in which glutamate side chains of variable lengths are formed on the modified protein. It is evolutionarily conserved from protists to mammals and its most prominent substrate is tubulin, the microtubule (MT) building block. Various polyglutamylation states of MTs can be distinguished within a single cell and they are also characteristic of specific cell types or organelles. Polyglutamylation has been proposed to be involved in the functional adaptation of MTs, as it occurs within the carboxy-terminal tubulin tails that participate directly in the binding of many structural and motor MT-associated proteins. The discovery of a new family of enzymes that catalyse this modification has brought new insight into the mechanism of polyglutamylation and now allows for direct functional studies of the role of tubulin polyglutamylation. Moreover, the recent identification of new substrates of polyglutamylation indicates that this post-translational modification could be a potential regulator of diverse cellular processes.

Replier
Mounira Amor-Guéret, Catherine Dubois-d'Enghien, Anthony Laugé, Rosine Onclercq-Delic, Abdelhamid Barakat, Elbekkay Chadli, Ahmed Aziz Bousfiha, Meriem Benjelloun, Elisabeth Flori, Bérénice Doray, Vincent Laugel, Maria Teresa Lourenço, Rui Gonçalves, Silvia Sousa, Jérôme Couturier, Dominique Stoppa-Lyonnet (2008 Jun 1)

Three new BLM gene mutations associated with Bloom syndrome.

Genetic testing : 257-61 : DOI : 10.1089/gte.2007.0119 En savoir plus
Résumé

Bloom’s syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.

Replier
Nassima Temime-Smaali, Lionel Guittat, Thomas Wenner, Emilie Bayart, Céline Douarre, Dennis Gomez, Marie-Josèphe Giraud-Panis, Arturo Londono-Vallejo, Eric Gilson, Mounira Amor-Guéret, Jean-François Riou (2008 May 21)

Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres.

The EMBO journal : 1513-24 : DOI : 10.1038/emboj.2008.74 En savoir plus
Résumé

Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIalpha depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIalpha expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIalpha is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function.

Replier
Juliette van Dijk, Julie Miro, Jean-Marc Strub, Benjamin Lacroix, Alain van Dorsselaer, Bernard Edde, Carsten Janke (2008 Feb 15)

Polyglutamylation is a post-translational modification with a broad range of substrates.

The Journal of biological chemistry : 3915-22 : DOI : 10.1074/jbc.M705813200 En savoir plus
Résumé

Polyglutamylation is a post-translational modification that generates lateral acidic side chains on proteins by sequential addition of glutamate amino acids. This modification was first discovered on tubulins, and it is important for several microtubule functions. Besides tubulins, only the nucleosome assembly proteins NAP1 and NAP2 have been shown to be polyglutamylated. Here, using a proteomic approach, we identify a large number of putative substrates for polyglutamylation in HeLa cells. By analyzing a selection of these putative substrates, we show that several of them can serve as in vitro substrates for two of the recently discovered polyglutamylases, TTLL4 and TTLL5. We further show that TTLL4 is the main polyglutamylase enzyme present in HeLa cells and that new substrates of polyglutamylation are indeed modified by TTLL4 in a cellular context. No clear consensus polyglutamylation site could be defined from the primary sequence of the here-identified new substrates of polyglutamylation. However, we demonstrate that glutamate-rich stretches are important for a protein to become polyglutamylated. Most of the newly identified substrates of polyglutamylation are nucleocytoplasmic shuttling proteins, including many chromatin-binding proteins. Our work reveals that polyglutamylation is a much more widespread post-translational modification than initially thought and thus that it might be a regulator of many cellular processes.

Replier
Hind Medyouf, Jacques Ghysdael (2008 Feb 1)

The calcineurin/NFAT signaling pathway: a novel therapeutic target in leukemia and solid tumors.

Cell cycle (Georgetown, Tex.) : 297-303 : DOI : 10.4161/cc.7.3.5357 En savoir plus
Résumé

The calcineurin/NFAT signaling pathway is unique to vertebrates and clear genetic evidences show that it plays critical roles in orchestrating the intricate cellular interactions that characterize vertebrate development and morphogenesis. In this setting, the transcriptional regulators of the NFAT family function as molecular integrators of specific calcium signals with other signaling pathways, including MAPkinase, WNT or NOTCH. Deregulation of calcineurin/NFAT signaling and/or abnormal expression of its components have recently been reported in solid tumors of epithelial origin, lymphoma and lymphoid leukemia. Our studies in mouse models of human T-ALL/lymphoma shows that persistent activation of calcineurin/NFAT signaling is pro-oncogenic in vivo and can be efficiently targeted by well-characterized calcineurin inhibitors. We further discuss facts and hypotheses concerning the molecular events that may act upstream and downstream of calcineurin and/or NFAT activation in different type of cancer cells.

Replier
Benoît Froget, Joël Blaisonneau, Sarah Lambert, Giuseppe Baldacci (2008 Feb 1)

Cleavage of stalled forks by fission yeast Mus81/Eme1 in absence of DNA replication checkpoint.

Molecular biology of the cell : 445-56 : DOI : 10.1091/mbc.E07-07-0728 En savoir plus
Résumé

During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase-specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.

Replier
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.

Replier

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.

Replier
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.

Replier
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.

Replier
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.

Replier
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.

Replier
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.

Replier
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.

Replier
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.

Replier