UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2020

Samah Matmati, Sarah Lambert, Vincent Géli, Stéphane Coulon (2020 Mar 12)

Telomerase Repairs Collapsed Replication Forks at Telomeres.

Cell reports : 3312-3322.e3 : DOI : S2211-1247(20)30233-3 En savoir plus
Résumé

Telomeres are difficult-to-replicate sites whereby replication itself may threaten telomere integrity. We investigate, in fission yeast, telomere replication dynamics in telomerase-negative cells to unmask problems associated with telomere replication. Two-dimensional gel analysis reveals that replication of telomeres is severely impaired and correlates with an accumulation of replication intermediates that arises from stalled and collapsed forks. In the absence of telomerase, Rad51, Mre11-Rad50-Nbs1 (MRN) complex, and its co-factor CtIP become critical to maintain telomeres, indicating that homologous recombination processes these intermediates to facilitate fork restart. We further show that a catalytically dead mutant of telomerase prevents Ku recruitment to telomeres, suggesting that telomerase and Ku both compete for the binding of telomeric-free DNA ends that are likely to originate from a reversed fork. We infer that Ku removal at collapsed telomeric forks allows telomerase to repair broken telomeres, thereby shielding telomeres from homologous recombination.

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Carsten Janke, Maria M Magiera (2020 Feb 27)

The tubulin code and its role in controlling microtubule properties and functions.

Nature reviews. Molecular cell biology : DOI : 10.1038/s41580-020-0214-3 En savoir plus
Résumé

Microtubules are core components of the eukaryotic cytoskeleton with essential roles in cell division, shaping, motility and intracellular transport. Despite their functional heterogeneity, microtubules have a highly conserved structure made from almost identical molecular building blocks: the tubulin proteins. Alternative tubulin isotypes and a variety of post-translational modifications control the properties and functions of the microtubule cytoskeleton, a concept known as the ‘tubulin code’. Here we review the current understanding of the molecular components of the tubulin code and how they impact microtubule properties and functions. We discuss how tubulin isotypes and post-translational modifications control microtubule behaviour at the molecular level and how this translates into physiological functions at the cellular and organism levels. We then go on to show how fine-tuning of microtubule function by some tubulin modifications can affect homeostasis and how perturbation of this fine-tuning can lead to a range of dysfunctions, many of which are linked to human disease.

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Satish Bodakuntla, Anne Schnitzler, Cristopher Villablanca, Christian Gonzalez-Billault, Ivan Bieche, Carsten Janke, Maria M Magiera (2020 Feb 13)

Tubulin polyglutamylation is a general traffic-control mechanism in hippocampal neurons.

Journal of cell science : DOI : jcs241802 En savoir plus
Résumé

Neurons are highly complex cells that heavily rely on intracellular transport to distribute a range of functionally essential cargoes within the cell. Post-translational modifications of tubulin are emerging as mechanisms for regulating microtubule functions, but their impact on neuronal transport is only marginally understood. Here, we have systematically studied the impact of post-translational polyglutamylation on axonal transport. In cultured hippocampal neurons, deletion of a single deglutamylase, CCP1 (also known as AGTPBP1), is sufficient to induce abnormal accumulation of polyglutamylation, i.e. hyperglutamylation. We next investigated how hyperglutamylation affects axonal transport of a range of functionally different neuronal cargoes: mitochondria, lysosomes, LAMP1 endosomes and BDNF vesicles. Strikingly, we found a reduced motility for all these cargoes, suggesting that polyglutamylation could act as a regulator of cargo transport in neurons. This, together with the recent discovery that hyperglutamylation induces neurodegeneration, makes it likely that perturbed neuronal trafficking could be one of the central molecular causes underlying this novel type of degeneration.This article has an associated First Person interview with the first author of the paper.

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Lambert, S. Borde, V. Charbonnier, J. B. Dantzer, F. Espeli, O. Guirouilh-Barbat, J. Llorente, B. Legube, G. Prioleau, M. N. Radicella, P. (2020 Feb 1)

Des mécanismes moléculaires aux applications cliniques. L’essentiel du Colloque Réplication-Réparation-Recombinaison 2019

Bull Cancer : 283-287 : DOI : 10.1016/j.bulcan.2020.01.003 En savoir plus
Yanzhang Luo, ShengQi Xiang, Peter Jan Hooikaas, Laura van Bezouwen, A S Jijumon, Carsten Janke, Friedrich Förster, Anna Akhmanova, Marc Baldus (2020 Jan 2)

Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy.

Nature communications : 18 : DOI : 10.1038/s41467-019-13876-x En savoir plus
Résumé

Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). While cryo-EM and X-ray crystallography have provided detailed views of interactions between MAPs with the microtubule lattice, little is known about how MAPs and their intrinsically disordered regions interact with the dynamic microtubule surface. NMR carries the potential to directly probe such interactions but so far has been precluded by the low tubulin yield. We present a protocol to produce [C, N]-labeled, functional microtubules (MTs) from human cells for solid-state NMR studies. This approach allowed us to demonstrate that MAPs can differently modulate the fast time-scale dynamics of C-terminal tubulin tails, suggesting distinct interaction modes. Our results pave the way for in-depth NMR studies of protein dynamics involved in MT assembly and their interactions with other cellular components.

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Année de publication : 2019

Shensi Shen, Sara Faouzi, Amandine Bastide, Sylvain Martineau, Hélène Malka-Mahieu, Yu Fu, Xiaoxiao Sun, Christine Mateus, Emilie Routier, Severine Roy, Laurent Desaubry, Fabrice André, Alexander Eggermont, Alexandre David, Jean-Yves Scoazec, Stéphan Vagner, Caroline Robert (2019 Dec 18)

An epitranscriptomic mechanism underlies selective mRNA translation remodelling in melanoma persister cells.

Nature communications : 5713 : DOI : 10.1038/s41467-019-13360-6 En savoir plus
Résumé

Cancer persister cells tolerate anticancer drugs and serve as the founders of acquired resistance and cancer relapse. Here we show that a subpopulation of BRAF mutant melanoma cells that tolerates exposure to BRAF and MEK inhibitors undergoes a reversible remodelling of mRNA translation that evolves in parallel with drug sensitivity. Although this process is associated with a global reduction in protein synthesis, a subset of mRNAs undergoes an increased efficiency in translation. Inhibiting the eIF4A RNA helicase, a component of the eIF4F translation initiation complex, abrogates this selectively increased translation and is lethal to persister cells. Translation remodelling in persister cells coincides with an increased N6-methyladenosine modification in the 5′-untranslated region of some highly translated mRNAs. Combination of eIF4A inhibitor with BRAF and MEK inhibitors effectively inhibits the emergence of persister cells and may represent a new therapeutic strategy to prevent acquired drug resistance.

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Année de publication : 2020

Aviel Even, Giovanni Morelli, Loïc Broix, Chiara Scaramuzzino, Silvia Turchetto, Ivan Gladwyn-Ng, Romain Le Bail, Michal Shilian, Stephen Freeman, Maria M Magiera, A S Jijumon, Nathalie Krusy, Brigitte Malgrange, Bert Brone, Paula Dietrich, Ioannis Dragatsis, Carsten Janke, Frédéric Saudou, Miguel Weil, Laurent Nguyen (2019 Dec 18)

ATAT1-enriched vesicles promote microtubule acetylation via axonal transport.

Science advances : eaax2705 : DOI : 10.1126/sciadv.aax2705 En savoir plus
Résumé

Microtubules are polymerized dimers of α- and β-tubulin that underlie a broad range of cellular activities. Acetylation of α-tubulin by the acetyltransferase ATAT1 modulates microtubule dynamics and functions in neurons. However, it remains unclear how this enzyme acetylates microtubules over long distances in axons. Here, we show that loss of ATAT1 impairs axonal transport in neurons in vivo, and cell-free motility assays confirm a requirement of α-tubulin acetylation for proper bidirectional vesicular transport. Moreover, we demonstrate that the main cellular pool of ATAT1 is transported at the cytosolic side of neuronal vesicles that are moving along axons. Together, our data suggest that axonal transport of ATAT1-enriched vesicles is the predominant driver of α-tubulin acetylation in axons.

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Année de publication : 2019

Satish Bodakuntla, A S Jijumon, Cristopher Villablanca, Christian Gonzalez-Billault, Carsten Janke (2019 Oct 29)

Microtubule-Associated Proteins: Structuring the Cytoskeleton.

Trends in cell biology : 804-819 : DOI : S0962-8924(19)30120-5 En savoir plus
Résumé

Microtubule-associated proteins (MAPs) were initially discovered as proteins that bind to and stabilize microtubules. Today, an ever-growing number of MAPs reveals a more complex picture of these proteins as organizers of the microtubule cytoskeleton that have a large variety of functions. MAPs enable microtubules to participate in a plethora of cellular processes such as the assembly of mitotic and meiotic spindles, neuronal development, and the formation of the ciliary axoneme. Although some subgroups of MAPs have been exhaustively characterized, a strikingly large number of MAPs remain barely characterized other than their interactions with microtubules. We provide a comprehensive view on the currently known MAPs in mammals. We discuss their molecular mechanisms and functions, as well as their physiological role and links to pathologies.

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Hannah L Klein, Giedrė Bačinskaja, Jun Che, Anais Cheblal, Rajula Elango, Anastasiya Epshtein, Devon M Fitzgerald, Belén Gómez-González, Sharik R Khan, Sandeep Kumar, Bryan A Leland, Léa Marie, Qian Mei, Judith Miné-Hattab, Alicja Piotrowska, Erica J Polleys, Christopher D Putnam, Elina A Radchenko, Anissia Ait Saada, Cynthia J Sakofsky, Eun Yong Shim, Mathew Stracy, Jun Xia, Zhenxin Yan, Yi Yin, Andrés Aguilera, Juan Lucas Argueso, Catherine H Freudenreich, Susan M Gasser, Dmitry A Gordenin, James E Haber, Grzegorz Ira, Sue Jinks-Robertson, Megan C King, Richard D Kolodner, Andrei Kuzminov, Sarah Ae Lambert, Sang Eun Lee, Kyle M Miller, Sergei M Mirkin, Thomas D Petes, Susan M Rosenberg, Rodney Rothstein, Lorraine S Symington, Pawel Zawadzki, Nayun Kim, Michael Lisby, Anna Malkova (2019 Jan 7)

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.

Microbial cell (Graz, Austria) : 1-64 : DOI : 10.15698/mic2019.01.664 En savoir plus
Résumé

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

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Année de publication : 2018

Athanasia Stoupa, Frédéric Adam, Dulanjalee Kariyawasam, Catherine Strassel, Sanjay Gawade, Gabor Szinnai, Alexandre Kauskot, Dominique Lasne, Carsten Janke, Kathiresan Natarajan, Alain Schmitt, Christine Bole-Feysot, Patrick Nitschke, Juliane Léger, Fabienne Jabot-Hanin, Frédéric Tores, Anita Michel, Arnold Munnich, Claude Besmond, Raphaël Scharfmann, François Lanza, Delphine Borgel, Michel Polak, Aurore Carré (2018 Nov 18)

TUBB1 mutations cause thyroid dysgenesis associated with abnormal platelet physiology.

EMBO molecular medicine : DOI : e9569 En savoir plus
Résumé

The genetic causes of congenital hypothyroidism due to thyroid dysgenesis (TD) remain largely unknown. We identified three novel gene mutations that co-segregated with TD in three distinct families leading to 1.1% of mutations in TD study cohort. (Tubulin, Beta 1 Class VI) encodes for a member of the β-tubulin protein family. gene is expressed in the developing and adult thyroid in humans and mice. All three mutations lead to non-functional α/β-tubulin dimers that cannot be incorporated into microtubules. In mice, knock-out disrupted microtubule integrity by preventing β1-tubulin incorporation and impaired thyroid migration and thyroid hormone secretion. In addition, mutations caused the formation of macroplatelets and hyperaggregation of human platelets after stimulation by low doses of agonists. Our data highlight unexpected roles for β1-tubulin in thyroid development and in platelet physiology. Finally, these findings expand the spectrum of the rare paediatric diseases related to mutations in tubulin-coding genes and provide new insights into the genetic background and mechanisms involved in congenital hypothyroidism and thyroid dysgenesis.

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Anissia Ait Saada, Sarah A E Lambert, Antony M Carr (2018 Aug 25)

Preserving replication fork integrity and competence via the homologous recombination pathway.

DNA repair : DOI : S1568-7864(18)30182-4 En savoir plus
Résumé

Flaws in the DNA replication process have emerged as a leading driver of genome instability in human diseases. Alteration to replication fork progression is a defining feature of replication stress and the consequent failure to maintain fork integrity and complete genome duplication within a single round of S-phase compromises genetic integrity. This includes increased mutation rates, small and large scale genomic rearrangement and deleterious consequences for the subsequent mitosis that result in the transmission of additional DNA damage to the daughter cells. Therefore, preserving fork integrity and replication competence is an important aspect of how cells respond to replication stress and avoid genetic change. Homologous recombination is a pivotal pathway in the maintenance of genome integrity in the face of replication stress. Here we review our recent understanding of the mechanisms by which homologous recombination acts to protect, restart and repair replication forks. We discuss the dynamics of these genetically distinct functions and their contribution to faithful mitoticsegregation.

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Maria M Magiera, Puja Singh, Carsten Janke (2018 May 31)

SnapShot: Functions of Tubulin Posttranslational Modifications.

Cell : 1552-1552.e1 : DOI : 10.1016/j.cell.2018.05.032 En savoir plus
Résumé

Post-translational modification of tubulin offers a mechanism for functional diversification of microtubules and regulation in a variety of physiological contexts. This SnapShot recaps the current state of understanding of tubulin posttranslational modifications and their functions in the regulation of biological processes. To view this SnapShot, open or download the PDF.

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Maria M Magiera, Puja Singh, Sudarshan Gadadhar, Carsten Janke (2018 May 31)

Tubulin Posttranslational Modifications and Emerging Links to Human Disease.

Cell : 1323-1327 : DOI : 10.1016/j.cell.2018.05.018 En savoir plus
Résumé

Tubulin posttranslational modifications are currently emerging as important regulators of the microtubule cytoskeleton and thus have a strong potential to be implicated in a number of disorders. Here, we review the latest advances in understanding the physiological roles of tubulin modifications and their links to a variety of pathologies.

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Sandrine M Caputo, Mélanie Léone, Francesca Damiola, Asa Ehlen, Aura Carreira, Pascaline Gaidrat, Alexandra Martins, Rita D Brandão, Ana Peixoto, Ana Vega, Claude Houdayer, Capucine Delnatte, Myriam Bronner, Danièle Muller, Laurent Castera, Marine Guillaud-Bataille, Inge Søkilde, Nancy Uhrhammer, Sophie Demontety, Hélène Tubeuf, Gaïa Castelain, , Uffe Birk Jensen, Ambre Petitalot, Sophie Krieger, Cédrick Lefol, Virginie Moncoutier, Nadia Boutry-Kryza, Henriette Roed Nielsen, Olga Sinilnikova, Dominique Stoppa-Lyonnet, Amanda B Spurdle, Manuel R Teixeira, Florence Coulet, Mads Thomassen, Etienne Rouleau (2018 Apr 3)

Full in-frame exon 3 skipping of BRCA2 confers high risk of breast and/or ovarian cancer.

Oncotarget : 17334-17348 : DOI : 10.18632/oncotarget.24671 En savoir plus
Résumé

Germline pathogenic variants in the gene are associated with a cumulative high risk of breast/ovarian cancer. Several variants result in complete loss of the exon-3 at the transcript level. The pathogenicity of these variants and the functional impact of loss of exon 3 have yet to be established. As a collaboration of the COVAR clinical trial group (France), and the ENIGMA consortium for investigating breast cancer gene variants, this study evaluated 8 variants resulting in complete deletion of exon 3. Clinical information for 39 families was gathered from Portugal, France, Denmark and Sweden. Multifactorial likelihood analyses were conducted using information from 293 patients, for 7 out of the 8 variants (including 6 intronic). For all variants combined the likelihood ratio in favor of causality was 4.39*10. These results provide convincing evidence for the pathogenicity of all examined variants that lead to a total exon 3 skipping, and suggest that other variants that result in complete loss of exon 3 at the molecular level could be associated with a high risk of cancer comparable to that associated with classical pathogenic variants in or gene. In addition, our functional study shows, for the first time, that deletion of exon 3 impairs the ability of cells to survive upon Mitomycin-C treatment, supporting lack of function for the altered BRCA2 protein in these cells. Finally, this study demonstrates that any variant leading to expression of only delta-exon 3 will be associated with an increased risk of breast and ovarian cancer.

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Catharina von Nicolai, Åsa Ehlén, Juan S Martinez, Aura Carreira (2018 Apr 1)

Dissecting the Recombination Mediator Activity of BRCA2 Using Biochemical Methods.

Methods in enzymology : 479-511 : DOI : S0076-6879(17)30356-7 En savoir plus
Résumé

Homologous recombination (HR) is an essential pathway to restart stalled replication forks, repair spontaneous DNA double-strand breaks, and generate genetic diversity. Together with genetic studies in model organisms, the development of purification protocols and biochemical assays has allowed investigators to begin to understand how the complex machinery of HR functions. At the core of the HR process is the recombination enzyme RecA in bacteria or RAD51 and DMC1 in eukaryotes. The main steps of HR can be reconstituted in vitro and involve: (1) The formation of a ssDNA-RAD51 complex into a helical structure termed the nucleoprotein filament after one DNA strand has been resected at the site of the break. (2) The homologous DNA pairing with an intact copy of the damaged chromatid to form a joint molecule also called displacement loop (D-loop). (3) The exchange of DNA strands and de novo DNA synthesis to restore the damaged/lost DNA. (4) The resolution of joint molecules by nucleolytic cleavage. The human tumor suppressor BRCA2 is a mediator of HR as it actively facilitates the DNA transactions of the recombination proteins RAD51 and DMC1 in a variety of ways: It stabilizes ssDNA-RAD51/DMC1 nucleoprotein filaments. It limits the assembly of RAD51 on dsDNA. It facilitates the replacement of replication protein A by RAD51. The result of these activities is a net increase of DNA strand exchange products as observed in vitro. Here, we describe some of the biochemical assays used to dissect the mediator activities of BRCA2.

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