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15 results on '"Cécile Robert"'

1. NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis

Author

Franck Vandermoere, Tiago M. Bandeiras, Ana C. F. Paiva, Edouard Bertrand, Bruno Charpentier, Xavier Manival, Céline Verheggen, Marie-Eve Chagot, Pedro M. F. Sousa, Marc Quinternet, Marie-Cécile Robert, Philippe Fort, Paulo E. Santo, Jonathan Bizarro, Yoann Abel, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Institut de génétique humaine (IGH), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Instituto de Biologia Experimental e Tecnológica (IBET), Universidade Nova de Lisboa = NOVA University Lisbon (NOVA), Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Ingénierie, Biologie et Santé en Lorraine (IBSLor), Université de Lorraine (UL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Génomique Fonctionnelle (IGF), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), CNRS, Université de Montpellier (UM), Université de Lorraine (UL), ANR, Ligue Nationale contre le cancer, Institut National du cancer, ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en Biologie cellulaire de Montpellier (CRBM), Verheggen, Céline, and Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID

Subjects
Proteomics, Saccharomyces cerevisiae Proteins, AcademicSubjects/SCI00010, Recombinant Fusion Proteins, [SDV]Life Sciences [q-bio], ATPase, Mutant, Chaperone, Cofactor, Gene Knockout Techniques, 03 medical and health sciences, Adenosine Triphosphate, 0302 clinical medicine, Protein Domains, Genes, Reporter, Ribonucleoproteins, Small Nucleolar, Protein Interaction Mapping, RNA and RNA-protein complexes, Genetics, Humans, RNP biogenesis, HSP90 Heat-Shock Proteins, Small nucleolar RNA, Eye Proteins, 030304 developmental biology, Multi-subunit complex assembly, 0303 health sciences, PAQosome, biology, DNA Helicases, Nuclear Proteins, Box C/D snoRNP, Hsp90, AAA proteins, Cell biology, [SDV] Life Sciences [q-bio], Multiprotein Complexes, Chaperone (protein), biology.protein, ATPases Associated with Diverse Cellular Activities, Carrier Proteins, 030217 neurology & neurosurgery, Biogenesis, HeLa Cells, Molecular Chaperones
Abstract

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.

Published
2020
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2. The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions

Author

Emeline Coleno, Marion Peter, Marie-Cécile Robert, Tianhong Wang, Xavier Pichon, Kazem Zibara, Arthur Imbert, Edouard Bertrand, Florian Mueller, Thomas Walter, Stavroula Mili, Racha Chouaib, Konstadinos Moissoglu, Institut de Génétique Moléculaire de Montpellier (IGMM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Franche-Comté Électronique Mécanique, Thermique et Optique - Sciences et Technologies (UMR 6174) (FEMTO-ST), Université de Technologie de Belfort-Montbeliard (UTBM)-Ecole Nationale Supérieure de Mécanique et des Microtechniques (ENSMM)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'économie et de sociologie du travail (LEST), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Hospices Civils de Lyon (HCL), Imagerie et Modélisation - Imaging and Modeling, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Equipe labellisée Ligue contre le Cancer, National Cancer Institute [Bethesda] (NCI-NIH), National Institutes of Health [Bethesda] (NIH), Institut de génétique humaine (IGH), Cancer et génome: Bioinformatique, biostatistiques et épidémiologie d'un système complexe, Institut Curie [Paris]-MINES ParisTech - École nationale supérieure des mines de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM), Lebanese University [Beirut] (LU), Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), This project was supported by France BioImaging (ANR-10-INBS-04), the Agence Nationale de la Recherche (ANR-11-BSV8-018-02, ANR-14-CE10-0018-01 and ANR-19-CE12-0007-03), and the Ligue Nationale Contre le Cancer and the Fondation pour la Recherche Médicale. This work was supported by the Labex EpiGenMed, from the framework 'Investissements d’avenir'. This work was supported in part by the Intramural Research Program of the National Cancer Institute, NIH., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-11-BSV8-0018,EJCbirth,Impacts de la transcription par l'ARN polymérase II sur l'assemblage de l'EJC(2011), ANR-14-CE10-0018,HI-FISH,Etude systématique de l'expression des gènes à l'échelle des molécules uniques d'ARN(2014), ANR-19-CE12-0007,TRANSFACT,Caracterisation des foyers de traduction: de nouvelles structures qui organisement finement le cytoplasme(2019), ANR-10-LABX-0012,EpiGenMed,From Genome and Epigenome to Molecular Medicine: turning new paradigms in biology into the therapeutic strategies of tomorrow(2010), Mines Paris - PSL (École nationale supérieure des mines de Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Cytoplasm, RNA localization, [SDV]Life Sciences [q-bio], Adenomatous Polyposis Coli Protein, RNA transport, Kinesins, Biology, Article, RNA Transport, 03 medical and health sciences, local translation, 0302 clinical medicine, Live cell imaging, MRNA transport, Animals, Humans, RNA, Messenger, Molecular Biology, cytoplasmic protrusions, 030304 developmental biology, 0303 health sciences, Messenger RNA, RNA, Cell biology, Kinesin, Cell Surface Extensions, 030217 neurology & neurosurgery, HeLa Cells
Abstract

International audience; RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two color imaging directly revealed single mRNAs transported by single KIF1C motors, with the 3’UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal a widespread RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role in transporting RNAs and clustering them within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA suggesting a possible feedback loop acting at the level of mRNA transport.

Published
2021
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3. A choreography of centrosomal mRNAs reveals a conserved localization mechanism involving active polysome transport

Author

Arthur Imbert, Marie-Cécile Robert, Hervé Le Hir, Emeline Coleno, Thierry Gostan, Virginie Georget, Adham Safieddine, Xavier Pichon, Frédéric Lionneton, Florian Mueller, Thomas Walter, Charles-Henri Lecellier, Racha Chouaib, Edouard Bertrand, Soha Salloum, Abdel-Meneem Traboulsi, Kazem Zibara, Marion Peter, Oh Sung Kwon, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Equipe labellisée Ligue contre le Cancer, Centre de Bioinformatique (CBIO), MINES ParisTech - École nationale supérieure des mines de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Institut Curie [Paris], Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), BioCampus Montpellier (BCM), Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Laboratory of Stem Cells [Lebanese, Beirut] (ER045-PRASE), Lebanese University [Beirut] (LU), Imagerie et Modélisation - Imaging and Modeling, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This project was supported by France BioImaging (ANR-10-INBS-04), by the Agence Nationale de la Recherche (ANR-11-BSV8-018-02 and ANR-14-CE10-0018-01), the Fondation pour la Recherche Médicale ('Bioinformatics' grant), the Institut Pasteur, the Ligue Nationale Contre le Cancer, and the Labex EpiGenMed from the framework 'Investissements d’avenir'. This work was also funded in part by the French government under the management of Agence Nationale de la Recherche as part of the 'Investissements d’avenir' program, reference ANR-19-P3IA-0001 (PRAIRIE 3IA Institute)., ANR-19-P3IA-0001,PRAIRIE,PaRis Artificial Intelligence Research InstitutE(2019), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-11-BSV8-0018,EJCbirth,Impacts de la transcription par l'ARN polymérase II sur l'assemblage de l'EJC(2011), ANR-14-CE10-0018,HI-FISH,Etude systématique de l'expression des gènes à l'échelle des molécules uniques d'ARN(2014), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Université Paris sciences et lettres (PSL), Institut de génétique humaine (IGH), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Mines Paris - PSL (École nationale supérieure des mines de Paris), Institut de biologie de l'ENS Paris (IBENS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), LE HIR, Hervé, PaRis Artificial Intelligence Research InstitutE - - PRAIRIE2019 - ANR-19-P3IA-0001 - P3IA - VALID, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, BLANC - Impacts de la transcription par l'ARN polymérase II sur l'assemblage de l'EJC - - EJCbirth2011 - ANR-11-BSV8-0018 - BLANC - VALID, Appel à projets générique - Etude systématique de l'expression des gènes à l'échelle des molécules uniques d'ARN - - HI-FISH2014 - ANR-14-CE10-0018 - Appel à projets générique - VALID, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, General Physics and Astronomy, ASPM, Cell Cycle Proteins, smFISH, RNA Transport, 0302 clinical medicine, local translation, Gene expression, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], centrosomes, Cycloheximide, Multidisciplinary, High-throughput screening, Translation (biology), Translocon, Cell biology, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Drosophila, Puromycin, Science, Mitosis, Molecular imaging, Spindle Apparatus, Biology, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Open Reading Frames, Microtubule, Polysome, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], conserved polysome choreography at centrosomes mRNA localization, Animals, Humans, RNA, Messenger, Centrosome, RNA metabolism, polysome imaging, cotranslational targeting, General Chemistry, NUMA1, 030104 developmental biology, Polyribosomes, 030217 neurology & neurosurgery, HeLa Cells
Abstract

Local translation allows for a spatial control of gene expression. Here, we use high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging reveals active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysome transport mediated by nascent proteins., Centrosomes function as microtubule organizing centers where several mRNAs accumulate. By employing high-throughput single molecule FISH screening, the authors discover that 8 human mRNAs localize to centrosomes with unique cell cycle dependent patterns using an active polysome targeting mechanism.

Published
2021
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4. Stochastic pausing at latent HIV-1 promoters generates transcriptional bursting

Author

Marie-Cécile Robert, Jean-Christophe Andrau, Eugenia Basyuk, Vera Slaninova, Kamalika Mukherjee, Rachel Topno, Yueyuxiao Yang, Ovidiu Radulescu, Meenakshi Basu-Shrivastava, Adèle L'Hostis, Florian Mueller, Alja Kozulic-Pirher, Nikolay Tsanov, Katjana Tantale, Edouard Bertrand, Thierry Gostan, Encar Garcia-Oliver, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), LPHI - Laboratory of Pathogen Host Interactions (LPHI), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)

Subjects
Time Factors, viruses, General Physics and Astronomy, HIV Infections, RNA polymerase II, medicine.disease_cause, [SHS]Humanities and Social Sciences, 0302 clinical medicine, Transcription (biology), Gene expression, Promoter Regions, Genetic, Polymerase, Transcriptional bursting, 0303 health sciences, Multidisciplinary, DNA-Directed RNA Polymerases, Virus Latency, 3. Good health, Cell biology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, tat Gene Products, Human Immunodeficiency Virus, Transcription, Algorithms, Transcriptional noise, Gene Expression Regulation, Viral, Viral protein, Science, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Humans, 030304 developmental biology, Stochastic Processes, Models, Genetic, Promoter, General Chemistry, Single-cell imaging, Cellular noise, medicine.disease, HIV-1, biology.protein, Virus Activation, 030217 neurology & neurosurgery, HeLa Cells
Abstract

Promoter-proximal pausing of RNA polymerase II is a key process regulating gene expression. In latent HIV-1 cells, it prevents viral transcription and is essential for latency maintenance, while in acutely infected cells the viral factor Tat releases paused polymerase to induce viral expression. Pausing is fundamental for HIV-1, but how it contributes to bursting and stochastic viral reactivation is unclear. Here, we performed single molecule imaging of HIV-1 transcription. We developed a quantitative analysis method that manages multiple time scales from seconds to days and that rapidly fits many models of promoter dynamics. We found that RNA polymerases enter a long-lived pause at latent HIV-1 promoters (>20 minutes), thereby effectively limiting viral transcription. Surprisingly and in contrast to current models, pausing appears stochastic and not obligatory, with only a small fraction of the polymerases undergoing long-lived pausing in absence of Tat. One consequence of stochastic pausing is that HIV-1 transcription occurs in bursts in latent cells, thereby facilitating latency exit and providing a rationale for the stochasticity of viral rebounds., The ability of HIV to alternate between acute and latent forms is thought to rely on a transcriptional feedback loop where polymerase pausing is released by the viral protein Tat. Here, the authors show that viral genome transcription can occur in a burst-like stochastic manner in the absence of Tat.

Published
2021
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5. A Dual Protein-mRNA Localization Screen Reveals Compartmentalized Translation and Widespread Co-translational RNA Targeting

Author

Thomas Walter, Emeline Coleno, Florian Mueller, Adham Safieddine, Xavier Pichon, Nikolay Tsanov, Arthur Imbert, Marie-Cécile Robert, Aubin Samacoits, Edouard Bertrand, Racha Chouaib, Anthony A. Hyman, Kazem Zibara, Christophe Zimmer, Matthias Peter, Ina Poser, Oh Sung Kwon, Hervé Le Hir, Abdel-Meneem Traboulsi, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Laboratory of Stem Cells [Lebanese, Beirut] (ER045-PRASE), Lebanese University [Beirut] (LU), Centre de Bioinformatique (CBIO), MINES ParisTech - École nationale supérieure des mines de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Cancer et génome: Bioinformatique, biostatistiques et épidémiologie d'un système complexe, Institut Curie [Paris]-MINES ParisTech - École nationale supérieure des mines de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Imagerie et Modélisation - Imaging and Modeling, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Max-Planck-Gesellschaft, Equipe labellisée Ligue contre le Cancer, This project was supported by France BioImaging (ANR-10-INBS-04), the Agence Nationale de la Recherche (ANR-11-BSV8-018-02 and ANR-14-CE10-0018-01), the Institut Pasteur, the Ligue Nationale Contre le Cancer, and the Fondation pour la Recherche Médicale (FRM grant to E.B.). Furthermore, this work was supported by the French government under management of Agence Nationale de la Recherche as part of the 'Investissements d'avenir' program, reference ANR-19-P3IA-0001 (PRAIRIE 3IA Institute) and by the Labex EpiGenMed from the framework 'Investissements d’avenir.', ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-11-BSV8-0018,EJCbirth,Impacts de la transcription par l'ARN polymérase II sur l'assemblage de l'EJC(2011), ANR-14-CE10-0018,HI-FISH,Etude systématique de l'expression des gènes à l'échelle des molécules uniques d'ARN(2014), ANR-19-P3IA-0001,PRAIRIE,PaRis Artificial Intelligence Research InstitutE(2019), ANR-10-LABX-0012,EpiGenMed,From Genome and Epigenome to Molecular Medicine: turning new paradigms in biology into the therapeutic strategies of tomorrow(2010), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Mines Paris - PSL (École nationale supérieure des mines de Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Zimmer, Christophe, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, BLANC - Impacts de la transcription par l'ARN polymérase II sur l'assemblage de l'EJC - - EJCbirth2011 - ANR-11-BSV8-0018 - BLANC - VALID, Appel à projets générique - Etude systématique de l'expression des gènes à l'échelle des molécules uniques d'ARN - - HI-FISH2014 - ANR-14-CE10-0018 - Appel à projets générique - VALID, PaRis Artificial Intelligence Research InstitutE - - PRAIRIE2019 - ANR-19-P3IA-0001 - P3IA - VALID, and Laboratoires d'excellence - From Genome and Epigenome to Molecular Medicine: turning new paradigms in biology into the therapeutic strategies of tomorrow - - EpiGenMed2010 - ANR-10-LABX-0012 - LABX - VALID

Subjects
RNA localization, Endosome, ASPM, translation factories, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biology, General Biochemistry, Genetics and Molecular Biology, smFISH, Cell Line, 03 medical and health sciences, 0302 clinical medicine, local translation, Polysome, Gene expression, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Humans, RNA, Messenger, Molecular Biology, Gene, 030304 developmental biology, Centrosome, 0303 health sciences, Messenger RNA, co-translational targeting Manuscript, Translation (biology), Cell Biology, Translocon, Cell biology, Protein Transport, Gene Expression Regulation, Polyribosomes, Protein Biosynthesis, Beta-catenin, RNA, RNA transport, co-translational targeting, 030217 neurology & neurosurgery, Developmental Biology
Abstract

International audience; Local translation allows spatial control of gene expression. Here, we performed a dual protein-mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. 32 mRNAs displayed specific cytoplasmic localizations with local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope, and centrosomes, the latter being cell-cycle-dependent. Automated classification of mRNA localization patterns revealed a high degree of intercellular heterogeneity. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For β-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

Published
2020
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6. A conserved choreography of mRNAs at centrosomes reveals a localization mechanism involving active polysome transport

Author

Hervé Le Hir, Abdel-Meneem Traboulsi, Virginie Georget, Emeline Coleno, Xavier Pichon, Kazem Zibara, Thierry Gostan, Racha Chouaib, Oh Sung Kwon, Edouard Bertrand, Soha Salloum, Charles Lecellier, Frédéric Lionneton, Marion Peter, Marie-Cécile Robert, Adham Safieddine, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), BioCampus Montpellier (BCM), Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Lebanese University [Beirut] (LU), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
polysome imaging, 0303 health sciences, [SDV]Life Sciences [q-bio], cotranslational targeting, ASPM, Translation (biology), Biology, NUMA1, Translocon, high-throughput screening, smFISH, Cell biology, 03 medical and health sciences, local translation, 0302 clinical medicine, Microtubule, Centrosome, Polysome, Gene expression, conserved polysome choreography at centrosomes mRNA localization, centrosomes, Mitosis, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

Local translation allows for a spatial control of gene expression. Here, we used high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses revealed a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging revealed active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysomes transport mediated by nascent proteins.; Local translation allows for a spatial control of gene expression. Here, we used high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses revealed a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging revealed active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysomes transport mediated by nascent proteins.

Published
2020
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7. Stochastic pausing at latent HIV-1 promoters generates transcriptional bursting

Author

A. Kozulic-Pirher, Marie-Cécile Robert, E. Garcia-Olivier, M. Basu, Edouard Bertrand, Ovidiu Radulescu, Katjana Tantale, A. L'Hostis, Jean-Christophe Andrau, Thierry Gostan, Y. Yang, F. Muller, Eugenia Basyuk, Institut de Génétique Moléculaire de Montpellier (IGMM), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)

Subjects
polymerase pausing, Transcriptional bursting, 0303 health sciences, [SDV]Life Sciences [q-bio], 030302 biochemistry & molecular biology, RNA, Promoter, Biology, 3. Good health, Cell biology, 03 medical and health sciences, Bursting, single molecule imaging, Transcription (biology), transcriptional noise, Gene expression, biology.protein, HIV-1 latency, bursting, Latency (engineering), Polymerase, 030304 developmental biology
Abstract

SummaryPromoter-proximal polymerase pausing is a key process regulating gene expression. In latent HIV-1 cells, it prevents viral transcription and is essential for latency maintenance, while in acutely infected cells the viral factor Tat releases paused polymerase to induce viral expression. Pausing is fundamental for HIV-1, but how it contributes to bursting and stochastic viral reactivation is unclear. Here, we performed single molecule imaging of HIV-1 transcription, and we developed a quantitative analysis method that manages multiple time scales from seconds to days, and that rapidly fits many models of promoter dynamics. We found that RNA polymerases enter a long-lived pause at latent HIV-1 promoters (>20 minutes), thereby effectively limiting viral transcription. Surprisingly and in contrast to current models, pausing appears stochastic and not obligatory, with only a small fraction of the polymerases undergoing long-lived pausing in absence of Tat. One consequence of stochastic pausing is that HIV-1 transcription occurs in bursts in latent cells, thereby facilitating latency exit and providing a rationale for the stochasticity of viral rebounds.

Published
2020
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8. New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells

Author

Robert H. Singer, Marie Cécile Robert, Edouard Bertrand, Evelina Tutucci, Xavier Pichon, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Université de Montpellier (UM), Heinlein, Manfred, Systems Bioinformatics, and AIMMS

Subjects
Single molecule, [SDV]Life Sciences [q-bio], Cell Culture Techniques, S. cerevisiae, Saccharomyces cerevisiae, Signal-To-Noise Ratio, Article, Cell Line, 03 medical and health sciences, 0302 clinical medicine, Transcription (biology), Live cell imaging, MS2-MCP system, Bacteriophage MS2, Gene expression, Image Processing, Computer-Assisted, Animals, Humans, Single cell, RNA, Messenger, In Situ Hybridization, Fluorescence, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Levivirus, Regulation of gene expression, 0303 health sciences, Messenger RNA, biology, mRNA labeling, RNA, Mammalian cells, biology.organism_classification, Yeast, Single Molecule Imaging, Cell biology, Luminescent Proteins, Capsid Proteins, mRNA localization, Single-Cell Analysis, Transcription, 030217 neurology & neurosurgery
Abstract

Live imaging of single RNA from birth to death brought important advances in our understanding of the spatiotemporal regulation of gene expression. These studies have provided a comprehensive understanding of RNA metabolism by describing the process step by step. Most of these studies used for live imaging a genetically encoded RNA-tagging system fused to fluorescent proteins. One of the best characterized RNA-tagging systems is derived from the bacteriophage MS2 and it allows single RNA imaging in real-time and live cells. This system has been successfully used to track the different steps of mRNA processing in many living organisms. The recent development of optimized MS2 and MCP variants now allows the labeling of endogenous RNAs and their imaging without modifying their behavior. In this chapter, we discuss the improvements in detecting single mRNAs with different variants of MCP and fluorescent proteins that we tested in yeast and mammalian cells. Moreover, we describe protocols using MS2-MCP systems improved for real-time imaging of single mRNAs and transcription dynamics in S. cerevisiae and mammalian cells, respectively.

Published
2020
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9. A localization screen reveals translation factories and widespread co-translational RNA targeting

Author

Ina Poser, Xavier Pichon, Edouard Bertrand, Nikolay Tsanov, Christophe Zimmer, Emeline Coleno, Hervé Le Hir, Oh Sung Kwon, Adham Safieddine, Thomas Walter, Marie-Cécile Robert, Marion Peter, Abdel-Meneem Traboulsi, Arthur Imbert, Kazem Zibara, Florian Mueller, Aubin Samacoits, Racha Chouaib, Anthony A. Hyman, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Equipe labelisée Ligue Contre le Cancer, Laboratory of Stem Cells [Lebanese, Beirut] (ER045-PRASE), Lebanese University [Beirut] (LU), Faculty of Sciences [Lebanese University], Equipe labellisée Ligue contre le Cancer, Centre de Bioinformatique (CBIO), MINES ParisTech - École nationale supérieure des mines de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Cancer et génome: Bioinformatique, biostatistiques et épidémiologie d'un système complexe, Institut Curie [Paris]-MINES ParisTech - École nationale supérieure des mines de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Imagerie et Modélisation - Imaging and Modeling, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Max-Planck-Gesellschaft, Université Paris sciences et lettres (PSL), This project was supported by France BioImaging (ANR-10-INBS-04), the Agence Nationale de la Recherche (ANR-11-BSV8-018-02 and ANR-14-CE10-0018-01), the Institut Pasteur, the Ligue Nationale Contre le Cancer and the Fondation pour la Recherche Médicale (FRM grant to EB). This work was supported by the Labex EpiGenMed, from the framework 'Investissements d’avenir'., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-14-CE10-0018,HI-FISH,Etude systématique de l'expression des gènes à l'échelle des molécules uniques d'ARN(2014), Lemesle, Marie, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, Appel à projets générique - Etude systématique de l'expression des gènes à l'échelle des molécules uniques d'ARN - - HI-FISH2014 - ANR-14-CE10-0018 - Appel à projets générique - VALID, Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Faculty of Sciences [Lebanese University] | Faculté des Sciences [Université Libanaise], Mines Paris - PSL (École nationale supérieure des mines de Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie de l'ENS Paris (IBENS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0303 health sciences, Messenger RNA, RNA localization, Translation (biology), translation factories, Biology, Translocon, smFISH, Cell biology, 03 medical and health sciences, 0302 clinical medicine, local translation, Cytoplasm, [SDV.SPEE] Life Sciences [q-bio]/Santé publique et épidémiologie, Polysome, co-translational targeting +Equal contributions, Gene expression, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, RNA transport, Gene, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

Publié sur BioRxiv le 21 mai 2020 : https://www.biorxiv.org/content/10.1101/2020.05.20.106989v1; Local translation allows a spatial control of gene expression. Here, we performed a dual protein/mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. A total of 32 mRNAs displayed specific cytoplasmic localizations, and we observed local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope and centrosomes, the latter being cell cycle dependent. Quantitation of mRNA distribution and automatic pattern classification revealed a high degree of localization heterogeneity between cells. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For β-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

Published
2020

10. A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting

Author

Xavier Darzacq, Marie-Cécile Robert, Edouard Bertrand, Katjana Tantale, Jean-Marc Victor, Florian Mueller, Serena Capozi, Christophe Zimmer, Julio Mateos-Langerak, Volker Bäcker, Alja Kozulic-Pirher, Eugenia Basyuk, Annick Lesne, Racha Chouaib, Institut de Génétique Moléculaire de Montpellier (IGMM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Imagerie et Modélisation - Imaging and Modeling, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physique Théorique de la Matière Condensée (LPTMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Laboratory of Stem Cells [Lebanese, Beirut] (ER045-PRASE), Lebanese University [Beirut] (LU), BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), F.M. was supported by a fellowship from the FRM and Institut Pasteur, S.C. by fellowships from the EEC ITN RNPnet and the LNCC, K.T. by a fellowship from ANRS. The work was supported by grants from SIDACTION, ANRS, ANR ‘Imagenex’ to Ed.B., and ANR EJCbirth to H Le Hir., We thank M. Lagha and N. Taylor for critical readings of the manuscript., ANR-10-BLAN-1222,IMAGenEx,Imagerie de l'expression génétique dans la cellule(2010), ANR-11-BSV8-0018,EJCbirth,Impacts de la transcription par l'ARN polymérase II sur l'assemblage de l'EJC(2011), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC), BioCampus Montpellier (BCM), Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Mueller, Florian, BLANC - Imagerie de l'expression génétique dans la cellule - - IMAGenEx2010 - ANR-10-BLAN-1222 - BLANC - VALID, BLANC - Impacts de la transcription par l'ARN polymérase II sur l'assemblage de l'EJC - - EJCbirth2011 - ANR-11-BSV8-0018 - BLANC - VALID, Institut de Génétique Moléculaire de Montpellier ( IGMM ), Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Imagerie et Modélisation, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de Physique Théorique de la Matière Condensée ( LPTMC ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratory of Stem Cells [Lebanese, Beirut] ( ER045-PRASE ), Lebanese University [Beirut], BioCampus Montpellier ( BCM ), Université Montpellier 1 ( UM1 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Institut de Génomique Fonctionnelle-Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de biologie de l'ENS Paris (UMR 8197/1024) ( IBENS ), École normale supérieure - Paris ( ENS Paris ) -École normale supérieure - Paris ( ENS Paris ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), ANR-10-BLAN-1222,IMAGenEx,Imagerie de l'expression génétique dans la cellule ( 2010 ), and ANR-11-BSV8-0018,EJCbirth,Impacts de la transcription par l'ARN polymérase II sur l'assemblage de l'EJC ( 2011 )

Subjects
0301 basic medicine, Transcription, Genetic, [SDV]Life Sciences [q-bio], General Physics and Astronomy, chemistry.chemical_compound, Models, Transcription (biology), Drosophila Proteins, tat, Promoter Regions, Genetic, Polymerase, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Transcriptional bursting, Genetics, Multidisciplinary, biology, DNA-Directed RNA Polymerases, TATA Box, Single Molecule Imaging, 3. Good health, Cell biology, [SDV] Life Sciences [q-bio], Enhancer Elements, Genetic, Gene Products, tat, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Drosophila, Transcription, Transcriptional Activation, Cell Survival, 1.1 Normal biological development and functioning, TATA box, Science, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Models, Biological, General Biochemistry, Genetics and Molecular Biology, Article, Promoter Regions, 03 medical and health sciences, Bursting, Genetic, Underpinning research, Transferases, Virology, Gene Products, Humans, Animals, [ SDV ] Life Sciences [q-bio], Base Sequence, TATA-Box Binding Protein, RNA, General Chemistry, Biological, Kinetics, 030104 developmental biology, chemistry, Gene Expression Regulation, Hela Cells, biology.protein, HIV-1, Generic health relevance, DNA, HeLa Cells
Abstract

Live-cell imaging has revealed unexpected features of gene expression. Here using improved single-molecule RNA microscopy, we show that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. During elongation, polymerases are spaced by few hundred nucleotides, and physical modelling suggests that DNA torsional stress may maintain polymerase spacing. We additionally observe that the HIV-1 promoter displays stochastic fluctuations on two time scales, which we refer to as multi-scale bursting. Each time scale is regulated independently: Mediator controls minute-scale fluctuation (convoys), while TBP-TATA-box interaction controls sub-hour fluctuations (long permissive/non-permissive periods). A cellular promoter also produces polymerase convoys and displays multi-scale bursting. We propose that slow, TBP-dependent fluctuations are important for phenotypic variability of single cells., HIV-1 viral gene expression stochastically switches between active and inactive states. Here, using improved single molecule RNA microscopy, the authors show that HIV-1 RNA stochastic transcription is achieved by groups of closely spaced polymerases, and is regulated by Mediator and TBP at different time scales.

Published
2016
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11. HSP90 and Its R2TP/Prefoldin-like Cochaperone Are Involved in the Cytoplasmic Assembly of RNA Polymerase II

Author

Dorothée Molle, Karim Azzag, Céline Verheggen, Séverine Boulon, Angus I. Lamond, Stéphanie Boireau, Edouard Bertrand, Marie Cécile Robert, Bérengère Pradet-Balade, Marya Georgieva, Henry Neel, Yasmeen Ahmad, Institut de Génétique Moléculaire de Montpellier (IGMM), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)

Subjects
Proteomics, Cytoplasm, Active Transport, Cell Nucleus, RNA-dependent RNA polymerase, RNA polymerase II, Article, 03 medical and health sciences, Genes, Reporter, RNA Polymerase I, Cell Line, Tumor, Protein Interaction Mapping, RNA polymerase I, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, HSP90 Heat-Shock Proteins, RNA, Small Interfering, Promoter Regions, Genetic, Molecular Biology, RNA polymerase II holoenzyme, Polymerase, 030304 developmental biology, Alpha-Amanitin, 0303 health sciences, biology, 030302 biochemistry & molecular biology, RNA, Cell Biology, Molecular biology, Cell biology, Protein Subunits, Multiprotein Complexes, biology.protein, HIV-1, RNA Polymerase II, Transcription factor II D, Protein Multimerization, Apoptosis Regulatory Proteins, Carrier Proteins, Small nuclear RNA, Molecular Chaperones, Protein Binding
Abstract

RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases.

Published
2010
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12. Nuclear retention prevents premature cytoplasmic appearance of mRNA

Author

Torben Heick Jensen, Edouard Bertrand, Marie-Cécile Robert, Thomas Beuchert Kallehauge, Institut de Génétique Moléculaire de Montpellier (IGMM), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)

Subjects
Cytoplasm, Saccharomyces cerevisiae Proteins, Genes, Fungal, Active Transport, Cell Nucleus, Saccharomyces cerevisiae, Biology, 03 medical and health sciences, 0302 clinical medicine, medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, Nuclear export signal, Molecular Biology, Gene, Heat-Shock Proteins, 030304 developmental biology, Adenosine Triphosphatases, Cell Nucleus, 0303 health sciences, Messenger RNA, Exosome Multienzyme Ribonuclease Complex, Cytoplasmic translation, Fungal genetics, RNA, RNA, Fungal, Cell Biology, Molecular biology, Cell nucleus, medicine.anatomical_structure, Mutation, 030217 neurology & neurosurgery
Abstract

In S. cerevisiae cells debilitated in mRNA nuclear export, transcripts are retained in nuclear foci ("dots"). The ultimate fate of dot-mRNA has remained elusive. Here, we use single molecule counting microscopy and (35)S-methionine pulse-labeling assays to quantify cytoplasmic HSP104 RNA levels and estimate HSP104 RNA translation status. HSP104 transcripts, retained in dots as a consequence of the mex67-5 mutation, are slowly released over time for cytoplasmic translation. Thus, dot-mRNA retains function. However, forcing its nuclear export, by overexpressing the Sub2p mRNA export factor, does not elevate Hsp104p protein levels but is instead paralleled by growth deficiency. Nuclear export and growth phenotypes are both counteracted by coexpressing the nuclear RNA quality control factor Rrp6p. Thus, prematurely released dot-mRNA is translationally inactive and possibly toxic. Accordingly, nuclear retention of mRNA may serve a precautionary role during stressful situations such as, e.g., decreased mRNA maturation competence.

Published
2012
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13. Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation

Author

Minoru Yoshida, Didier Auboeuf, Stuart Aitken, Ute Schmidt, Eugenia Basyuk, Marie-Cécile Robert, Jean-Philippe Villemin, Edouard Bertrand, Institut de Génétique Moléculaire de Montpellier (IGMM), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)

Subjects
Transcription, Genetic, Exonic splicing enhancer, macromolecular substances, Biology, Models, Biological, Ribonucleoprotein, U1 Small Nuclear, 03 medical and health sciences, Splicing factor, 0302 clinical medicine, Report, Cell Line, Tumor, Humans, snRNP, splice, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, Research Articles, 030304 developmental biology, Ribonucleoprotein, Genetics, 0303 health sciences, musculoskeletal, neural, and ocular physiology, Alternative splicing, Intron, Cell Biology, Cell biology, Molecular Imaging, Alternative Splicing, Kinetics, nervous system, RNA splicing, 030217 neurology & neurosurgery
Abstract

A combination of several rate-limiting steps allows for efficient control of alternative splicing., Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2–GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules.

Published
2011
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14. The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

Author

Nathalie Marmier-Gourrier, Tamás Kiss, Edouard Bertrand, Beáta E. Jády, Bruno Charpentier, Christina Pescia, Séverine Boulon, Céline Verheggen, Bérengère Pradet-Balade, Alain Krol, Benjamin Rothé, Barbara Bardoni, Laurence Wurth, Christine Allmang, Christiane Branlant, Marie-Cécile Robert, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Maturation des ARN et enzymologie moléculaire (MAEM), Cancéropôle du Grand Est-Université Henri Poincaré - Nancy 1 (UHP)-IFR111-Centre National de la Recherche Scientifique (CNRS), Architecture et réactivité de l'ARN (ARN), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie moléculaire eucaryote (LBME), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique Moléculaire de Montpellier ( IGMM ), Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Maturation des ARN et enzymologie moléculaire ( MAEM ), Cancéropôle du Grand Est-Université Henri Poincaré - Nancy 1 ( UHP ) -IFR111-Centre National de la Recherche Scientifique ( CNRS ), Architecture et réactivité de l'ARN ( ARN ), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de biologie moléculaire eucaryote du CNRS ( LBME ), Université Paul Sabatier - Toulouse 3 ( UPS ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de génétique et biologie moléculaire et cellulaire ( IGBMC ), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3)

Subjects
MESH: Signal Transduction, Protein Folding, MESH : Saccharomyces cerevisiae, MESH: DNA Helicases, Cell Cycle Proteins, MESH : Cell Cycle Proteins, MESH: Saccharomyces cerevisiae Proteins, Heterogeneous-Nuclear Ribonucleoprotein L, MESH : DNA Helicases, RUVBL2, MESH : Cell Proliferation, Conserved Sequence, Research Articles, MESH: Evolution, Molecular, Ribonucleoprotein, Adenosine Triphosphatases, 0303 health sciences, MESH: Conserved Sequence, biology, 030302 biochemistry & molecular biology, Nuclear Proteins, RNA-Binding Proteins, MESH : Protein Binding, MESH : HSP90 Heat-Shock Proteins, Hsp90, MESH: Saccharomyces cerevisiae, Cell biology, DNA-Binding Proteins, MESH : Heterogeneous-Nuclear Ribonucleoprotein L, MESH : DNA-Binding Proteins, MESH: Molecular Chaperones, Protein Binding, Signal Transduction, MESH : Protein Folding, Ribosomal Proteins, Saccharomyces cerevisiae Proteins, animal structures, MESH: Protein Folding, MESH : RNA-Binding Proteins, Saccharomyces cerevisiae, Article, Evolution, Molecular, MESH : Saccharomyces cerevisiae Proteins, 03 medical and health sciences, MESH: Cell Cycle Proteins, MESH : Conserved Sequence, MESH: Cell Proliferation, MESH: Adenosine Triphosphatases, MESH: HSP90 Heat-Shock Proteins, MESH: Heterogeneous-Nuclear Ribonucleoprotein L, MESH: Protein Binding, snRNP, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, HSP90 Heat-Shock Proteins, MESH : Evolution, Molecular, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, R2TP complex, Cell Proliferation, MESH : Signal Transduction, MESH : Adenosine Triphosphatases, DNA Helicases, MESH : Molecular Chaperones, RNA, MESH : Nuclear Proteins, Cell Biology, MESH: RNA-Binding Proteins, Chaperone (protein), biology.protein, MESH: Nuclear Proteins, Biogenesis, MESH: DNA-Binding Proteins, Molecular Chaperones, Transcription Factors
Abstract

International audience; RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.

Published
2008
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15. Processivity and Coupling in Messenger RNA Transcription

Author

Jean D. Beggs, Stuart Aitken, Igor Goryanin, Marie-Cécile Robert, Edouard Bertrand, and Ross D. Alexander

Subjects
Biophysics/Theory and Simulation, Transcription, Genetic, lcsh:Medicine, Biology, 03 medical and health sciences, 0302 clinical medicine, Transcription (biology), Gene expression, RNA, Messenger, Promoter Regions, Genetic, lcsh:Science, Biophysics/Transcription and Translation, Cell Biology/Gene Expression, 030304 developmental biology, Genetics, Stochastic Processes, 0303 health sciences, Messenger RNA, Multidisciplinary, lcsh:R, Minimum time, RNA, Processivity, Cell biology, lcsh:Q, 030217 neurology & neurosurgery, Research Article
Abstract

Background The complexity of messenger RNA processing is now being uncovered by experimental techniques that are capable of detecting individual copies of mRNA in cells, and by quantitative real-time observations that reveal the kinetics. This processing is commonly modelled by permitting mRNA to be transcribed only when the promoter is in the on state. In this simple on/off model, the many processes involved in active transcription are represented by a single reaction. These processes include elongation, which has a minimum time for completion and processing that is not captured in the model. Methodology In this paper, we explore the impact on the mRNA distribution of representing the elongation process in more detail. Consideration of the mechanisms of elongation leads to two alternative models of the coupling between the elongating polymerase and the state of the promoter: Processivity allows polymerases to complete elongation irrespective of the promoter state, whereas coupling requires the promoter to be active to produce a full-length transcript. We demonstrate that these alternatives have a significant impact on the predicted distributions. Models are simulated by the Gillespie algorithm, and the third and fourth moments of the resulting distribution are computed in order to characterise the length of the tail, and sharpness of the peak. By this methodology, we show that the moments provide a concise summary of the distribution, showing statistically-significant differences across much of the feasible parameter range. Conclusions We conclude that processivity is not fully consistent with the on/off model unless the probability of successfully completing elongation is low—as has been observed. The results also suggest that some form of coupling between the promoter and a rate-limiting step in transcription may explain the cell's inability to maintain high mRNA levels at low noise—a prediction of the on/off model that has no supporting evidence.

Published
2010
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