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1. RNA polymerase I mutant affects ribosomal RNA processing and ribosomal DNA stability

Author: Christophe Normand, Christophe Dez, Lise Dauban, Sophie Queille, Sarah Danché, Sarra Abderrahmane, Frederic Beckouet, and Olivier Gadal
Subjects: ribosomal RNA, Transcription, Genome stability, S.cerevisiae, Genetics, QH426-470
Abstract: Transcription is a major contributor to genomic instability. The ribosomal RNA (rDNA) gene locus consists of a head-to-tail repeat of the most actively transcribed genes in the genome. RNA polymerase I (RNAPI) is responsible for massive rRNA production, and nascent rRNA is co-transcriptionally assembled with early assembly factors in the yeast nucleolus. In Saccharomyces cerevisiae, a mutant form of RNAPI bearing a fusion of the transcription factor Rrn3 with RNAPI subunit Rpa43 (CARA-RNAPI) has been described previously. Here, we show that the CARA-RNAPI allele results in a novel type of rRNA processing defect, associated with rDNA genomic instability. A fraction of the 35S rRNA produced in CARA-RNAPI mutant escapes processing steps and accumulates. This accumulation is increased in mutants affecting exonucleolytic activities of the exosome complex. CARA-RNAPI is synthetic lethal with monopolin mutants that are known to affect the rDNA condensation. CARA-RNAPI strongly impacts rDNA organization and increases rDNA copy number variation. Reduced rDNA copy number suppresses lethality, suggesting that the chromosome segregation defect is caused by genomic rDNA instability. We conclude that a constitutive association of Rrn3 with transcribing RNAPI results in the accumulation of rRNAs that escape normal processing, impacting rDNA organization and affecting rDNA stability.
Published: 2024
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2. The dual life of disordered lysine-rich domains of snoRNPs in rRNA modification and nucleolar compaction

Author: Carine Dominique, Nana Kadidia Maiga, Alfonso Méndez-Godoy, Benjamin Pillet, Hussein Hamze, Isabelle Léger-Silvestre, Yves Henry, Virginie Marchand, Valdir Gomes Neto, Christophe Dez, Yuri Motorin, Dieter Kressler, Olivier Gadal, Anthony K. Henras, and Benjamin Albert
Subjects: Science
Abstract: Abstract Intrinsically disordered regions (IDRs) are highly enriched in the nucleolar proteome but their physiological role in ribosome assembly remains poorly understood. Our study reveals the functional plasticity of the extremely abundant lysine-rich IDRs of small nucleolar ribonucleoprotein particles (snoRNPs) from protists to mammalian cells. We show in Saccharomyces cerevisiae that the electrostatic properties of this lysine-rich IDR, the KKE/D domain, promote snoRNP accumulation in the vicinity of nascent rRNAs, facilitating their modification. Under stress conditions reducing the rate of ribosome assembly, they are essential for nucleolar compaction and sequestration of key early-acting ribosome biogenesis factors, including RNA polymerase I, owing to their self-interaction capacity in a latent, non-rRNA-associated state. We propose that such functional plasticity of these lysine-rich IDRs may represent an ancestral eukaryotic regulatory mechanism, explaining how nucleolar morphology is continuously adapted to rRNA production levels.
Published: 2024
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3. Coupling Between Production of Ribosomal RNA and Maturation: Just at the Beginning

Author: Chaima Azouzi, Mariam Jaafar, Christophe Dez, Raghida Abou Merhi, Annick Lesne, Anthony K. Henras, and Olivier Gadal
Subjects: RNA polymerase I (Pol I), ribosomal RNA (rRNA) processing, transcription, termination of transcription, ribosomal RNA (rRNA) genes, RNA folding, Biology (General), QH301-705.5
Abstract: Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35S/47S in yeast/human) is achieved by up to hundreds of RNA polymerase I (Pol I) enzymes simultaneously transcribing a single rRNA gene. In this review, we present recent advances in understanding the coupling between rRNA production and nascent rRNA folding. Mapping of the distribution of Pol I along ribosomal DNA at nucleotide resolution, using either native elongating transcript sequencing (NET-Seq) or crosslinking and analysis of cDNAs (CRAC), revealed frequent Pol I pausing, and CRAC results revealed a direct coupling between pausing and nascent RNA folding. High density of Pol I per gene imposes topological constraints that establish a defined pattern of polymerase distribution along the gene, with a persistent spacing between transcribing enzymes. RNA folding during transcription directly acts as an anti-pausing mechanism, implying that proper folding of the nascent rRNA favors elongation in vivo. Defects in co-transcriptional folding of rRNA are likely to induce Pol I pausing. We propose that premature termination of transcription, at defined positions, can control rRNA production in vivo.
Published: 2021
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4. Non-Coding, RNAPII-Dependent Transcription at the Promoters of rRNA Genes Regulates Their Chromatin State in S. cerevisiae

Author: Emma Lesage, Jorge Perez-Fernandez, Sophie Queille, Christophe Dez, Olivier Gadal, and Marta Kwapisz
Subjects: UPS, RNA polymerase I (RNAPI), RNA polymerase II (RNAPII), ribosomal DNA (rDNA), ribosomal RNA (rRNA), long non-coding RNA (lncRNA), Genetics, QH426-470
Abstract: Pervasive transcription is widespread in eukaryotes, generating large families of non-coding RNAs. Such pervasive transcription is a key player in the regulatory pathways controlling chromatin state and gene expression. Here, we describe long non-coding RNAs generated from the ribosomal RNA gene promoter called UPStream-initiating transcripts (UPS). In yeast, rDNA genes are organized in tandem repeats in at least two different chromatin states, either transcribed and largely depleted of nucleosomes (open) or assembled in regular arrays of nucleosomes (closed). The production of UPS transcripts by RNA Polymerase II from endogenous rDNA genes was initially documented in mutants defective for rRNA production by RNA polymerase I. We show here that UPS are produced in wild-type cells from closed rDNA genes but are hidden within the enormous production of rRNA. UPS levels are increased when rDNA chromatin states are modified at high temperatures or entering/leaving quiescence. We discuss their role in the regulation of rDNA chromatin states and rRNA production.
Published: 2021
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5. Excessive rDNA Transcription Drives the Disruption in Nuclear Homeostasis during Entry into Senescence in Budding Yeast

Author: Sandrine Morlot, Jia Song, Isabelle Léger-Silvestre, Audrey Matifas, Olivier Gadal, and Gilles Charvin
Subjects: Biology (General), QH301-705.5
Abstract: Summary: Budding yeast cells undergo a limited number of divisions before they enter senescence and die. Despite recent mechanistic advances, whether and how molecular events are temporally and causally linked during the transition to senescence remain elusive. Here, using real-time observation of the accumulation of extrachromosomal rDNA circles (ERCs) in single cells, we provide evidence that ERCs build up rapidly with exponential kinetics well before any physiological decline. We then show that ERCs fuel a massive increase in ribosomal RNA (rRNA) levels in the nucleolus, which do not mature into functional ribosomes. This breakdown in nucleolar coordination is followed by a loss of nuclear homeostasis, thus defining a chronology of causally related events leading to cell death. A computational analysis supports a model in which a series of age-independent processes lead to an age-dependent increase in cell mortality, hence explaining the emergence of aging in budding yeast. : The accumulation of extrachromosomal rDNA circles (ERCs) is a hallmark of aging in budding yeast. Morlot et al. show that ERCs accumulate ahead of senescence onset and fuel an excessive rDNA transcription, ultimately leading to impaired nuclear homeostasis and an irreversible cell cycle slowdown. Keywords: live cell imaging, microfluidics, yeast replicative aging, ribosome biogenesis, nucleolus, rDNA, ERCs
Published: 2019
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6. Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I.

Author: Tommy Darrière, Michael Pilsl, Marie-Kerguelen Sarthou, Adrien Chauvier, Titouan Genty, Sylvain Audibert, Christophe Dez, Isabelle Léger-Silvestre, Christophe Normand, Anthony K Henras, Marta Kwapisz, Olga Calvo, Carlos Fernández-Tornero, Herbert Tschochner, and Olivier Gadal
Subjects: Genetics, QH426-470
Abstract: Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.
Published: 2019
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7. A ribosome assembly stress response regulates transcription to maintain proteome homeostasis

Author: Benjamin Albert, Isabelle C Kos-Braun, Anthony K Henras, Christophe Dez, Maria Paula Rueda, Xu Zhang, Olivier Gadal, Martin Kos, and David Shore
Subjects: ribosome biogenesis, ribosomal protein, heat shock factor 1, Ifh1, topoisomerase, protein aggregation, Medicine, Science, Biology (General), QH301-705.5
Abstract: Ribosome biogenesis is a complex and energy-demanding process requiring tight coordination of ribosomal RNA (rRNA) and ribosomal protein (RP) production. Given the extremely high level of RP synthesis in rapidly growing cells, alteration of any step in the ribosome assembly process may impact growth by leading to proteotoxic stress. Although the transcription factor Hsf1 has emerged as a central regulator of proteostasis, how its activity is coordinated with ribosome biogenesis is unknown. Here, we show that arrest of ribosome biogenesis in the budding yeast Saccharomyces cerevisiae triggers rapid activation of a highly specific stress pathway that coordinately upregulates Hsf1 target genes and downregulates RP genes. Activation of Hsf1 target genes requires neo-synthesis of RPs, which accumulate in an insoluble fraction and presumably titrate a negative regulator of Hsf1, the Hsp70 chaperone. RP aggregation is also coincident with that of the RP gene activator Ifh1, a transcription factor that is rapidly released from RP gene promoters. Our data support a model in which the levels of newly synthetized RPs, imported into the nucleus but not yet assembled into ribosomes, work to continuously balance Hsf1 and Ifh1 activity, thus guarding against proteotoxic stress during ribosome assembly.
Published: 2019
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8. Non-Coding, RNAPII-Dependent Transcription at the Promoters of rRNA Genes Regulates Their Chromatin State in S. cerevisiae

Author: Olivier Gadal, Christophe Dez, Marta Kwapisz, Jorge Perez-Fernandez, Sophie Queille, Emma Lesage, Unité de biologie moléculaire, cellulaire et du développement (MCD), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-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)
Subjects: RNA polymerase II, QH426-470, Biology, UPS, Biochemistry, long non-coding RNA (lncRNA), 03 medical and health sciences, Transcription (biology), RNA polymerase II (RNAPII), [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Gene expression, Genetics, RNA polymerase I, ribosomal DNA (rDNA), Molecular Biology, Gene, 030304 developmental biology, 0303 health sciences, ribosomal RNA (rRNA), 030302 biochemistry & molecular biology, Promoter, Ribosomal RNA, Chromatin, biology.protein, RNA polymerase I (RNAPI)
Abstract: International audience; Pervasive transcription is widespread in eukaryotes, generating large families of non- coding RNAs. Such pervasive transcription is a key player in the regulatory pathways controlling chromatin state and gene expression. Here, we describe long non-coding RNAs generated from the ribosomal RNA gene promoter called UPStream-initiating transcripts (UPS). In yeast, rDNA genes are organized in tandem repeats in at least two different chromatin states, either transcribed and largely depleted of nucleosomes (open) or assembled in regular arrays of nucleosomes (closed). The production of UPS transcripts by RNA Polymerase II from endogenous rDNA genes was initially documented in mutants defective for rRNA production by RNA polymerase I. We show here that UPS are produced in wild-type cells from closed rDNA genes but are hidden within the enormous production of rRNA. UPS levels are increased when rDNA chromatin states are modified at high temperatures or entering/leaving quiescence. We discuss their role in the regulation of rDNA chromatin states and rRNA production.
Published: 2021
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9. Nuclear envelope expansion in budding yeast is independent of cell growth and does not determine nuclear volume

Author: Orna Cohen-Fix, Carolyn A. Larabell, Jian-Hua Chen, Alison D. Walters, Gerry McDermott, Renjie Wang, Olivier Gadal, Kwabena Amoateng, and Spang, Anne
Subjects: Nucleolus, Nuclear Envelope, 1.1 Normal biological development and functioning, Biology, Endoplasmic Reticulum, Medical and Health Sciences, Fluorescence, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Underpinning research, Spindle pole body separation, medicine, Nuclear membrane, Cycloheximide, Molecular Biology, Tomography, Secretory pathway, Phospholipids, 030304 developmental biology, Cell Proliferation, 0303 health sciences, Secretory Pathway, Cell growth, Nuclear Functions, Cell Cycle, Fatty Acids, Cell Biology, Articles, Biological Sciences, Cell cycle, medicine.anatomical_structure, Phenotype, chemistry, Cell Nucleus Size, Mutation, Saccharomycetales, Biophysics, Generic health relevance, Nucleus, 030217 neurology & neurosurgery, DNA, Developmental Biology
Abstract: Most cells exhibit a constant ratio between nuclear and cell volume. The mechanism dictating this constant ratio and the nuclear component(s) that scale with cell size are not known. To address this, we examined the consequences to the size and shape of the budding yeast nucleus when cell expansion is inhibited by down-regulating components of the secretory pathway. We find that under conditions where cell size increase is restrained, the nucleus becomes bilobed, with the bulk of the DNA in one lobe and the nucleolus in the other. The formation of bilobed nuclei is dependent on fatty acid and phospholipid synthesis, suggesting that it is associated with nuclear membrane expansion. Bilobed nuclei appeared predominantly after spindle pole body separation, suggesting that nuclear envelope expansion follows cell-cycle cues rather than cell size. Importantly, cells with bilobed nuclei had the same nuclear:cell volume ratio as cells with round nuclei. Therefore, the bilobed nucleus could be a consequence of continued NE expansion as cells traverse the cell cycle without an accompanying increase in nuclear volume due to the inhibition of cell growth. Our data suggest that nuclear volume is not determined by nuclear envelope availability but by one or more nucleoplasmic factors.
Published: 2019

10. Regulation of Cohesin-Mediated Chromosome Folding by Eco1 and Other Partners

Author: Romain Koszul, Luciana Lazar-Stefanita, Olivier Gadal, Nathalie Bastié, Agnès Thierry, Rémi Montagne, Lise Dauban, Axel Cournac, Frédéric Beckouët, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Régulation spatiale des Génomes - Spatial Regulation of Genomes, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), L.D. was supported by ARC fellowships. L.L.S. was partially supported by a fellowship from Fondation pour la Recherche Médicale (SPE201801005113). This research was supported by funding to R.K. from the European Research Council under the Horizon 2020 Program (ERC grant agreement 260822)., We thank T.U. Tanaka, Kerstin Bystricky, and K. Nasmyth for sharing strains and K. Shiahige for the Smc3 antibody. We thank Aurèle Piazza, Dave Lane, Luis Aragon, Julien Mozziconacci, Christophe Chapard, Olivier Cuvier, Tony Marchal, and O.G. and R.K. teams for discussions and comments on the manuscript., European Project: 260822,EC:FP7:ERC,ERC-2010-StG_20091118,DICIG(2011), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Saccharomyces cerevisiae Proteins, Chromosomal Proteins, Non-Histone, [SDV]Life Sciences [q-bio], Pds5, Cell Cycle Proteins, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biology, Chromatids, 03 medical and health sciences, 0302 clinical medicine, Acetyltransferases, Hi-C, Wapl, Chromosome Segregation, Centromere, Translocase, Sister chromatids, saccharomyces cerevisiae, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Eco1, Molecular Biology, Mitosis, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, mitosis, 0303 health sciences, chromatin loop, [SDV.GEN]Life Sciences [q-bio]/Genetics, loop extrusion, Cohesin, Chromosome, Nuclear Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Cell biology, Chromatin, chromosome organization, biology.protein, Chromatin Loop, cell cycle, biological phenomena, cell phenomena, and immunity, Chromosomes, Fungal, 030217 neurology & neurosurgery
Abstract: International audience; Cohesin, a member of the SMC complex family, holds sister chromatids together but also shapes chromosomes by promoting the formation of long-range intra-chromatid loops, a process proposed to be mediated by DNA loop extrusion. Here we describe the roles of three cohesin partners, Pds5, Wpl1, and Eco1, in loop formation along either unreplicated or mitotic Saccharomyces cerevisiae chromosomes. Pds5 limits the size of DNA loops via two different pathways: the canonical Wpl1-mediated releasing activity and an Eco1-dependent mechanism. In the absence of Pds5, the main barrier to DNA loop expansion appears to be the centromere. Our data also show that Eco1 acetyl-transferase inhibits the translocase activity that powers loop formation and contributes to the positioning of loops through a mechanism that is distinguishable from its role in cohesion establishment. This study reveals that the mechanisms regulating cohesin-dependent chromatin loops are conserved among eukaryotes while promoting different functions.
Published: 2020
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11. A major role for Eco1 in regulating cohesin-mediated mitotic chromosome folding

Author: Romain Koszul, Olivier Gadal, Lise Dauban, Axel Cournac, Luciana Lazar-Stefanita, Frédéric Beckouët, Rémi Montagne, Agnès Thierry, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay, Régulation spatiale des Génomes - Spatial Regulation of Genomes, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0303 health sciences, Cohesin complex, Cohesin, [SDV]Life Sciences [q-bio], Chromosome, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Biology, Chromatin, Cell biology, 03 medical and health sciences, 0302 clinical medicine, Centromere, Sister chromatids, Chromatid, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, biological phenomena, cell phenomena, and immunity, Mitosis, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract: Understanding how chromatin organizes spatially into chromatid and how sister chromatids are maintained together during mitosis is of fundamental importance in chromosome biology. Cohesin, a member of the Structural Maintenance of Chromosomes (SMC) complex family, holds sister chromatids together 1–3 and promotes long-range intra-chromatid DNA looping 4,5. These cohesin-mediated DNA loops are important for both higher-order mitotic chromatin compaction6,7 and, in some organisms, compartmentalization of chromosomes during interphase into topologically associating domains (TADs) 8,9. Our understanding of the mechanism(s) by which cohesin generates large DNA loops remains incomplete. It involves a combination of molecular partners and active expansion/extrusion of DNA loops. Here we dissect the roles on loop formation of three partners of the cohesin complex: Pds5 10, Wpl1 11 and Eco1 acetylase 12, during yeast mitosis. We identify a new function for Eco1 in negatively regulating cohesin translocase activity, which powers loop extrusion. In the absence of negative regulation, the main barrier to DNA loop expansion appears to be the centromere. Those results provide new insights on the mechanisms regulating cohesin dependent DNA looping.
Published: 2019

12. Quantification of the dynamic behaviour of ribosomal DNA genes and nucleolus during yeast Saccharomyces cerevisiae cell cycle

Author: Alain Kamgoue, Olivier Gadal, Sylvain Cantaloube, Frédéric Beckouët, Lise Dauban, Renjie Wang, Isabelle Léger-Silvestre, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Centre de Biologie Intégrative (CBI), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie et de Technologies de Saclay (IBITECS), Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Institut de pharmacologie et de biologie structurale (IPBS)
Subjects: Saccharomyces cerevisiae Proteins, Cohesin complex, Nucleolus, Chromosomal Proteins, Non-Histone, [SDV]Life Sciences [q-bio], Saccharomyces cerevisiae, Cell Cycle Proteins, Biology, DNA, Ribosomal, 03 medical and health sciences, Structural Biology, Mitosis, Metaphase, Ribosomal DNA, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Cohesin, 030302 biochemistry & molecular biology, Cell Cycle, Nuclear Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, Cell biology, Interphase, Cell Division, Cell Nucleolus
Abstract: International audience; Spatial organisation of chromosomes is a determinant of genome stability and is required for proper mitotic segregation. However, visualization of individual chromatids in living cells and quantification of their geometry, remains technically challenging. Here, we used live cell imaging to quantitate the three-dimensional conformation of yeast Saccharomyces cerevisiae ribosomal DNA (rDNA). rDNA is confined within the nucleolus and is composed of about 200 copies representing about 10% of the yeast genome. To fluorescently label rDNA in living cells, we generated a set of nucleolar proteins fused to GFP or made use of a tagged rDNA, in which lacO repetitions were inserted in each repeat unit. We could show that nucleolus is not modified in appearance, shape or size during interphase while rDNA is highly reorganized. Computationally tracing 3D rDNA paths allowed us to quantitatively assess rDNA size, shape and geometry. During interphase, rDNA was progressively reorganized from a zigzag segmented line of small size (5,5 µm) to a long, homogeneous, line-like structure of 8,7 µm in metaphase. Most importantly, whatever the cell-cycle stage considered, rDNA fibre could be decomposed in subdomains, as previously suggested for 3D chromatin organisation. Finally, we could determine that spatial reorganisation of these subdomains and establishment of rDNA mitotic organisation is under the control of the cohesin complex.
Published: 2019
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13. Rouse model with transient intramolecular contacts on a timescale of seconds recapitulates folding and fluctuation of yeast chromosomes

Author: Kerstin Bystricky, Aurélien Bancaud, Renjie Wang, Jean-Marc Victor, Cédric Vaillant, Marius Socol, Pascal Carrivain, Daniel Jost, Olivier Gadal, Christophe Normand, Eric Le Cam, Vincent Dahirel, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Équipe Micro-Nanofluidique pour les sciences de la vie et de l’environnement (LAAS-MILE), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Laboratoire de biologie moléculaire eucaryote (LBME), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Henan University of Science and Technology, Biologie Computationnelle et Mathématique (TIMC-IMAG-BCM), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de Physique de l'ENS Lyon (Phys-ENS), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), PHysicochimie des Electrolytes et Nanosystèmes InterfaciauX (PHENIX), Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Nutrition et Alimentation des Populations aux Suds (NutriPass), Université Montpellier 1 (UM1)-Institut de Recherche pour le Développement (IRD)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Laboratoire de Physique Théorique de la Matière Condensée (LPTMC), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire des matériaux et du génie physique (LMGP), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique de Grenoble (INPG), Université Toulouse III - Paul Sabatier (UPS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes (UGA), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université Paris-Saclay, Université Pierre et Marie Curie - Paris 6 (UPMC)-ESPCI ParisTech-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UPS), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Université Toulouse 1 Capitole (UT1), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), École normale supérieure - Lyon (ENS Lyon)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), and Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)
Subjects: [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], DNA Folding, Chromatin and Epigenetics, Chromosome conformation capture, 03 medical and health sciences, Motion, 0302 clinical medicine, Genetics, Nucleosome, Gene Regulation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, DNA, Fungal, 030304 developmental biology, 0303 health sciences, biology, Models, Genetic, Dynamics (mechanics), Gene regulation, Chromatin and Epigenetics, Chromatin, Nucleosomes, Folding (chemistry), Kinetics, Histone, Chemical physics, biology.protein, Chromosomes, Fungal, Parametrization, 030217 neurology & neurosurgery
Abstract: DNA folding and dynamics along with major nuclear functions are determined by chromosome structural properties, which remain, thus far, elusive in vivo. Here, we combine polymer modeling and single particle tracking experiments to determine the physico-chemical parameters of chromatin in vitro and in living yeast. We find that the motion of reconstituted chromatin fibers can be recapitulated by the Rouse model using mechanical parameters of nucleosome arrays deduced from structural simulations. Conversely, we report that the Rouse model shows some inconsistencies to analyze the motion and structural properties inferred from yeast chromosomes determined with chromosome conformation capture techniques (specifically, Hi-C). We hence introduce the Rouse model with Transient Internal Contacts (RouseTIC), in which random association and dissociation occurs along the chromosome contour. The parametrization of this model by fitting motion and Hi-C data allows us to measure the kinetic parameters of the contact formation reaction. Chromosome contacts appear to be transient; associated to a lifetime of seconds and characterized by an attractive energy of –0.3 to –0.5 kBT. We suggest attributing this energy to the occurrence of histone tail-DNA contacts and notice that its amplitude sets chromosomes in ‘theta’ conditions, in which they are poised for compartmentalization and phase separation.
Published: 2019
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14. Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

Author: Christophe Dez, Carlos Fernández-Tornero, Isabelle Léger-Silvestre, Marie-Kerguelen Sarthou, Michael Pilsl, Anthony K. Henras, Olga Calvo, Olivier Gadal, Marta Kwapisz, Christophe Normand, Sylvain Audibert, Titouan Genty, Adrien Chauvier, Tommy Darrière, Herbert Tschochner, Agencia Estatal de Investigación (España), Agence Nationale de la Recherche (France), Université de Toulouse, Ministerio de Economía y Competitividad (España), Fundación Ramón Areces, Ministerio de Ciencia, Innovación y Universidades (España), Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Universität Regensburg, Biochemie-Zentrum Regensburg (BZR), Lehrstuhl Biochemie III, Regensburg, Germany, Département de biologie [Sherbrooke] (UdeS), Faculté des sciences [Sherbrooke] (UdeS), Université de Sherbrooke (UdeS)-Université de Sherbrooke (UdeS), Instituto de Biología Funcional y Genómica, IBFG-CSIC, Universidad de Salamanca, Salamanca, Spain, and Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Subjects: Cancer Research, Heredity, Transcription, Genetic, viruses, Mutant, QH426-470, Biochemistry, Polymerases, Suppressor Genes, chemistry.chemical_compound, Genetic Suppression, 0302 clinical medicine, Transcription (biology), RNA Polymerase I, RNA polymerase, RNA Precursors, Super-active mutant, ComputingMilieux_MISCELLANEOUS, Genetics (clinical), Polymerase, Gel Electrophoresis, 0303 health sciences, biology, RNA Polymerase, Eukaryota, Cell biology, Nucleic acids, Ribosomal RNA, Rpa49, Pol1 Transcription Initiation Complex Proteins, Research Article, Cellular structures and organelles, Saccharomyces cerevisiae Proteins, Saccharomyces cerevisiae, Research and Analysis Methods, DNA, Ribosomal, 03 medical and health sciences, Electrophoretic Techniques, Genetic, ARN polimerasa I, Gene Types, DNA-binding proteins, RNA polymerase I, Genetics, Point Mutation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Non-coding RNA, Gene, Molecular Biology, Transcription factor, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, [SDV.GEN]Life Sciences [q-bio]/Genetics, Biology and life sciences, 2409 Genética, 3201.02 Genética Clínica, Organisms, Fungi, RNA, Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Yeast, chemistry, RNA, Ribosomal, Enzyme, Mutation, Biochemical analysis, biology.protein, Ribosomes, 030217 neurology & neurosurgery, DNA, Transcription Factors
Abstract: Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that wild-type RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I., This work was supported by the Agence Nationale de la Recherche (ANDY) and the IDEX of Toulouse University (Clemgene and Nudgene). We thank Margaux Cescato, Lilian Guillot and Jorge Perez-Fernandez for strain and plasmid constructions. YCp50-26 carrying RPA49 was kindly provided by P. Thuriaux. We also thank Melanie Panarotto for the Miller spread analysis. This work benefited from exchanges with all unit members, including Anthony Henras, Frederic Beckouët, Lise Dauban, and Marta Kwapisz. We thank Stephanie Balor and Vanessa Soldan from the METI platform for the TEM acquisitions. CFT was supported by grants BFU2013-48374-P and BFU2017-87397-P of the Spanish Ministry of Economy and Competitiveness, and by the Ramón Areces Foundation.
Published: 2019
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15. High resolution microscopy reveals the nuclear shape of budding yeast during cell cycle and in various biological states

Author: Christophe Normand, Alain Kamgoue, Olivier Gadal, Thomas Mangeat, Renjie Wang, and Isabelle Léger-Silvestre
Subjects: 0301 basic medicine, Cell Nucleus Shape, Localization microscopy, Nucleolus, Nuclear Envelope, Population, Biology, Bioinformatics, Spindle pole body, 03 medical and health sciences, Imaging, Three-Dimensional, medicine, Nuclear pore, education, Interphase, education.field_of_study, Microscopy, Confocal, Super-resolution microscopy, Cell Cycle, Cell Biology, G1 Phase Cell Cycle Checkpoints, Carbon, Nuclear pore complex, Nuclear Pore Complex Proteins, 030104 developmental biology, medicine.anatomical_structure, Nuclear geometry, Super resolution microscopy, Saccharomycetales, Biophysics, Nucleus, Research Article
Abstract: How spatial organization of the genome depends on nuclear shape is unknown, mostly because accurate nuclear size and shape measurement is technically challenging. In large cell populations of the yeast Saccharomyces cerevisiae, we assessed the geometry (size and shape) of nuclei in three dimensions with a resolution of 30 nm. We improved an automated fluorescence localization method by implementing a post-acquisition correction of the spherical microscopic aberration along the z-axis, to detect the three dimensional (3D) positions of nuclear pore complexes (NPCs) in the nuclear envelope. Here, we used a method called NucQuant to accurately estimate the geometry of nuclei in 3D throughout the cell cycle. To increase the robustness of the statistics, we aggregated thousands of detected NPCs from a cell population in a single representation using the nucleolus or the spindle pole body (SPB) as references to align nuclei along the same axis. We could detect asymmetric changes of the nucleus associated with modification of nucleolar size. Stereotypical modification of the nucleus toward the nucleolus further confirmed the asymmetric properties of the nuclear envelope., Summary: This novel method to explore 3D geometry of the nuclear envelope with enhanced resolution and post-acquisition correction of z-axis aberration revealed increased NPC density near the SPB and the nucleolus.
Published: 2016

16. Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III–transcribed genes in budding yeast

Author: Christophe Normand, Ashutosh Shukla, Christophe Dez, Olivier Gadal, Purnima Bhargava, Renjie Wang, Isabelle Léger-Silvestre, and Praveen Belagal
Subjects: 0301 basic medicine, Saccharomyces cerevisiae Proteins, Transcription, Genetic, Nucleolus, viruses, Centromere, Biology, RNA polymerase III, Chromosomes, 03 medical and health sciences, chemistry.chemical_compound, RNA polymerase, Molecular Biology, Gene, Genetics, Cell Nucleus, Nuclear Functions, Optical Imaging, RNA Polymerase III, Cell Biology, Articles, Telomere, Budding yeast, 030104 developmental biology, chemistry, Saccharomycetales, Interphase, Cell Nucleolus, Peptide Termination Factors
Abstract: In budding yeast, RNA polymerase III–transcribed genes preferentially associate with the nucleolar and nuclear periphery when permitted by the Rabl-like orientation of interphase chromosomes., The association of RNA polymerase III (Pol III)–transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III–transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements—centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III–transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III–transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III–dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III–transcribed genes required active transcription. We conclude that the association of Pol III–transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization.
Published: 2016

17. Nucleolar stress causes the entry into replicative senescence in budding yeast

Author: Isabelle Léger-Silvestre, Sandrine Morlot, Olivier Gadal, Gilles Charvin, Jia Song, and Audrey Matifas
Subjects: Senescence, Programmed cell death, Cell division, Transition (genetics), Nucleolus, Extrachromosomal DNA, Mutant, Cell cycle, Biology, Cell biology
Abstract: SummaryThe accumulation of Extrachromosomal rDNA Circles (ERCs) and their asymmetric segregation upon division have been hypothesized to be responsible for replicative senescence in mother yeasts and rejuvenation in daughter cells. However, it remains unclear by which molecular mechanisms ERCs would trigger the irreversible cell cycle slow-down leading to cell death. We show that ERCs accumulation is concomitant with a nucleolar stress, characterized by a massive accumulation of pre-rRNAs in the nucleolus, leading to a loss of nucleus-to-cytoplasm ratio, decreased growth rate and cell-cycle slow-down. This nucleolar stress, observed in old mothers, is not inherited by rejuvenated daughters. Unlike WT, in the long-lived mutant fob1∆, a majority of cells is devoid of nucleolar stress and does not experience replicative senescence before death. Our study provides a unique framework to order the successive steps that govern the transition to replicative senescence and highlights the causal role of nucleolar stress in cellular aging.
Published: 2018
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18. Capturing Chromosome Structural Properties From Their Spatial and Temporal Fluctuations

Author: Kerstin Bystricky, Olivier Gadal, Aurélien Bancaud, Équipe Micro-Nanofluidique pour les sciences de la vie et de l’environnement (LAAS-MILE), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Laboratoire de biologie moléculaire eucaryote (LBME), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, 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)
Subjects: Genetics, Physics, 0303 health sciences, Systems biology, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Nuclear organization, Computational biology, Genome, Chromatin, Folding (chemistry), 03 medical and health sciences, 0302 clinical medicine, Chromosome (genetic algorithm), Live cell imaging, Chromatin conformation, 030217 neurology & neurosurgery, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract: Chromosome folding and nuclear organization are influenced by cellular processes while regulating genome functions. Intense efforts based on interdisciplinary research at the forefront of physics, mathematics, molecular and cellular biology, and biochemistry have contributed to a better understanding of the physical properties and mechanisms of chromosome conformation. In this chapter, we focus on technologies for capturing chromosome spatial and temporal fluctuations based on the realization that chromatin conformation is highly dynamic at every length and time scale. We describe state-of-the-art chromatin labeling technologies, focusing on optimizing yield of signal-to-noise ratio, high-throughput automated imaging techniques, and physical modeling. Systems biology approaches to map chromosome conformation using single or multiple labeled loci are illustrated, and implications of chromosome structural fluctuations on biological functions are discussed.
Published: 2017

19. Principles of chromatin organization in yeast: relevance of polymer models to describe nuclear organization and dynamics

Author: Aurélien Bancaud, Renjie Wang, Julien Mozziconacci, Olivier Gadal, Laboratoire de biologie moléculaire eucaryote (LBME), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-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), Équipe Micro-Nanofluidique pour les sciences de la vie et de l’environnement (LAAS-MILE), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), and Université Fédérale Toulouse Midi-Pyrénées
Subjects: Genetics, Polymers, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Nuclear organization, Saccharomyces cerevisiae, Computational biology, Cell Biology, Biology, biology.organism_classification, Genome, Models, Biological, Yeast, Chromatin, Chromosomes, Chromosome conformation capture, Yeast chromosome, Animals, Humans, Relevance (information retrieval), Genome, Fungal, ComputingMilieux_MISCELLANEOUS
Abstract: Nuclear organization can impact on all aspects of the genome life cycle. This organization is thoroughly investigated by advanced imaging and chromosome conformation capture techniques, providing considerable amount of datasets describing the spatial organization of chromosomes. In this review, we will focus on polymer models to describe chromosome statics and dynamics in the yeast Saccharomyces cerevisiae. We suggest that the equilibrium configuration of a polymer chain tethered at both ends and placed in a confined volume is consistent with the current literature, implying that local chromatin interactions play a secondary role in yeast nuclear organization. Future challenges are to reach an integrated multi-scale description of yeast chromosome organization, which is crucially needed to improve our understanding of the regulation of genomic transaction.
Published: 2015
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20. High-throughput chromatin motion tracking in living yeast reveals the flexibility of the fiber throughout the genome

Author: Houssam Hajjoul, Pascal Carrivain, Aurélien Bancaud, Julien Mozziconacci, Isabelle Goiffon, Julien Mathon, Hubert Ranchon, Kerstin Bystricky, Olivier Gadal, Jean-Marc Victor, Benjamin Albert, Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT), Laboratoire de Physique Théorique de la Matière Condensée (LPTMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie moléculaire eucaryote (LBME), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Équipe Nano Ingénierie et Intégration des Systèmes (LAAS-N2IS), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées, Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), and Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1)
Subjects: Models, Molecular, BUDDING YEAST, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Saccharomyces cerevisiae, ORGANIZATION, Molecular Dynamics Simulation, Biology, Genome, SACCHAROMYCES-CEREVISIAE, 03 medical and health sciences, 0302 clinical medicine, MAMMALIAN-CELLS, Genetics, medicine, CEREVISIAE LINKER HISTONE, Genetics (clinical), 030304 developmental biology, Cell Nucleus, Persistence length, 0303 health sciences, Research, Dynamics (mechanics), Chromosome, Telomere, CHROMOSOME DYNAMICS, biology.organism_classification, Chromatin, High-Throughput Screening Assays, MODEL, medicine.anatomical_structure, Genetic Loci, PRINCIPLES, Biophysics, Brownian dynamics, DOMAIN PROTEIN MPS3, Chromosomes, Fungal, Genome, Fungal, INTERPHASE NUCLEUS, Nucleus, 030217 neurology & neurosurgery
Abstract: Chromosome dynamics are recognized to be intimately linked to genomic transactions, yet the physical principles governing spatial fluctuations of chromatin are still a matter of debate. Using high-throughput single-particle tracking, we recorded the movements of nine fluorescently labeled chromosome loci located on chromosomes III, IV, XII, and XIV of Saccharomyces cerevisiae over an extended temporal range spanning more than four orders of magnitude (10−2–103 sec). Spatial fluctuations appear to be characterized by an anomalous diffusive behavior, which is homogeneous in the time domain, for all sites analyzed. We show that this response is consistent with the Rouse polymer model, and we confirm the relevance of the model with Brownian dynamics simulations and the analysis of the statistical properties of the trajectories. Moreover, the analysis of the amplitude of fluctuations by the Rouse model shows that yeast chromatin is highly flexible, its persistence length being qualitatively estimated to
Published: 2013
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21. Systematic characterization of the conformation and dynamics of budding yeast chromosome XII

Author: Purnima Bhargava, Ashutosh Shukla, Aurélien Bancaud, Olivier Gadal, Benjamin Albert, Hua Wong, Alain Kamgoue, Hicham Saad, Julien Mathon, Christophe Zimmer, Christophe Normand, Julien Mozziconacci, David Villa, Isabelle Léger-Silvestre, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées, Council of Scientific and Industrial Research [India] (CSIR), Laboratoire de Physique Théorique de la Matière Condensée (LPTMC), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC), GDR 3536 Architecture et Dynamique Nucléaires (ADN), Université Pierre et Marie Curie - Paris 6 (UPMC), Imagerie et Modélisation, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by an ATIP-plus grant from Centre National de la Recherche Scientifique, by the Agence Nationale de la Recherche (Nucleopol, Ribeuc, and program JCJC), Jeune équipe from FRM, and University Paul Sabatier (a fellowship for J. Mathon). This work is a French-Indian collaborative effort funded by Indo-French Centre for the Promotion of Advanced Research (IFCPAR) Project 4103 between the laboratories of Dr. Bhargava and Dr. Gadal., ANR-08-PCVI-0036,NUCLEOPOL,Integrated structural biology of rDNA transcription(2008), ANR-06-BLAN-0037,RIBEUC,Ribosome biogenesis in eukaryotes: mechanisms and connections with other pathways(2006), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-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), Laboratoire d'analyse et d'architecture des systèmes [Toulouse] (LAAS), Institut National Polytechnique [Toulouse] (INP) - Institut National des Sciences Appliquées - Toulouse (INSA Toulouse) - Université Paul Sabatier - Toulouse 3 (UPS) - Centre National de la Recherche Scientifique (CNRS), Council of Scientific & Industrial Research [India] (CSIR), Université Pierre et Marie Curie - Paris 6 (UPMC) - Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] - Centre National de la Recherche Scientifique (CNRS), ANR-08-PCVI-0036, NUCLEOPOL, Integrated structural biology of rDNA transcription(2008), and ANR-06-BLAN-0037, RIBEUC, Ribosome biogenesis in eukaryotes: mechanisms and connections with other pathways(2006)
Subjects: Transcription, Genetic, Nucleolus, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Saccharomyces cerevisiae, Genome, DNA, Ribosomal, Time-Lapse Imaging, 03 medical and health sciences, 0302 clinical medicine, Report, Computer Simulation, DNA, Fungal, Ribosomal DNA, Research Articles, 030304 developmental biology, Genetics, Sirolimus, 0303 health sciences, biology, [PHYS.PHYS.PHYS-BIO-PH] Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chromosome, Chromosome Mapping, Cell Biology, Telomere, biology.organism_classification, Chromatin, High-Throughput Screening Assays, Saccharomycetales, Genetic Loci, Chromosomes, Fungal, Genome, Fungal, 030217 neurology & neurosurgery, Cell Nucleolus
Abstract: Comprehensive analysis of the intranuclear territories and motion of budding yeast chromosome XII loci suggests that long-range chromosome architecture is mainly determined by the physical principles of polymers., Chromosomes architecture is viewed as a key component of gene regulation, but principles of chromosomal folding remain elusive. Here we used high-throughput live cell microscopy to characterize the conformation and dynamics of the longest chromosome of Saccharomyces cerevisiae (XII). Chromosome XII carries the ribosomal DNA (rDNA) that defines the nucleolus, a major hallmark of nuclear organization. We determined intranuclear positions of 15 loci distributed every ∼100 kb along the chromosome, and investigated their motion over broad time scales (0.2–400 s). Loci positions and motions, except for the rDNA, were consistent with a computational model of chromosomes based on tethered polymers and with the Rouse model from polymer physics, respectively. Furthermore, rapamycin-dependent transcriptional reprogramming of the genome only marginally affected the chromosome XII internal large-scale organization. Our comprehensive investigation of chromosome XII is thus in agreement with recent studies and models in which long-range architecture is largely determined by the physical principles of tethered polymers and volume exclusion.
Published: 2013
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22. Nuclear organization and chromatin dynamics in yeast: Biophysical models or biologically driven interactions?

Author: Christophe Normand, Isabelle Léger-Silvestre, Olivier Gadal, Benjamin Albert, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Biologie Cellulaire du Noyau (BCN), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Saccharomyces cerevisiae, Biophysics, Computational biology, Biochemistry, Genome, Biophysical Phenomena, Chromosomes, Diffusion, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Gene Expression Regulation, Fungal, Genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Epigenetics, Nuclear pore, Scaffold/matrix attachment region, Molecular Biology, Gene, 030304 developmental biology, Cell Nucleus, 0303 health sciences, biology, Models, Theoretical, Chromatin Assembly and Disassembly, biology.organism_classification, Chromatin, Nuclear transport, 030217 neurology & neurosurgery
Abstract: International audience; Over the past decade, tremendous progress has been made in understanding the spatial organization of genes and chromosomes. Nuclear organization can be thought of as information that is not encoded in DNA, but which nevertheless impacts gene expression. Nuclear organizational influences can be cell-specific and are potentially heritable. Thus, nuclear organization fulfills all the criteria necessary for it to be considered an authentic level of epigenetic information. Chromosomal nuclear organization is primarily dictated by the biophysical properties of chromatin. Diffusion models of polymers confined in the crowded nuclear space accurately recapitulate experimental observation. Diffusion is a Brownian process, which implies that the positions of chromosomes and genes are not defined deterministically but are likely to be dictated by the laws of probability. Despite the small size of their nuclei, budding yeast have been instrumental in discovering how epigenetic information is encoded in the spatial organization of the genome. The relatively simple organization of the yeast nucleus and the very high number of genetically identical cells that can be observed under fluorescent microscopy allow statistically robust definitions of the gene and chromosome positions in the nuclear space to be constructed. In this review, we will focus on how the spatial organization of the chromatin in the yeast nucleus might impact transcription. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
Published: 2012
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23. The nucleolar protein Nop19p interacts preferentially with Utp25p and Dhr2p and is essential for the production of the 40S ribosomal subunit in Saccharomyces cerevisiae

Author: Elodie Choque, Christophe Dez, Odile Burlet-Schiltz, Marlène Marcellin, Olivier Gadal, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-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 pharmacologie et de biologie structurale (IPBS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées
Subjects: Saccharomyces cerevisiae Proteins, Nucleolus, Protein subunit, Saccharomyces cerevisiae, Immunoblotting, Ribosome biogenesis, Biology, DEAD-box RNA Helicases, RNA Precursors, RNA, Ribosomal, 18S, Immunoprecipitation, Eukaryotic Small Ribosomal Subunit, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Nuclear protein, RNA Processing, Post-Transcriptional, Molecular Biology, Ribosome Subunits, Small, Eukaryotic, Nuclear Proteins, RNA, Fungal, Cell Biology, Ribosomal RNA, biology.organism_classification, RNA Helicase A, Molecular biology, Cell biology, RNA, Ribosomal, Mutation, Carrier Proteins, Cell Nucleolus, Research Paper, Protein Binding
Abstract: International audience; In eukaryotes, ribosome biogenesis is a process of major interest that requires more than 200 factors acting coordinately in time and space. Using genetic and proteomic studies, most of the components have now been identified. Based on its nucleolar localization, we characterized the protein encoded by the open reading frame YGR251W, we renamed Nop19p as playing an essential role in ribosome biogenesis. Depletion of the Nop19p in yeast impairs pre-rRNA processing at sites A₀, A₁ and A₂, leading to a strong decrease in 18S rRNA and 40S subunit levels. Nop19p is a component of 90S preribosomes which assembly is believed to result from stepwise incorporation of UTP modules. We show that Nop19p depletion does not impair the incorporation of UTP subcomplexes on preribosomes and conversely that depletion of UTP subcomplexes does not affect Nop19p recruitment on 90S preribosomes. TAP experiments under stringent conditions revealed that Nop19p interacts preferentially with the DEAH-box RNA helicase Dhr2p and Utp25p, both required for A 0, A 1 and A 2 cleavages. Nop19p appeared essential for the incorporation of Utp25p in preribosomes. In addition, our results suggest that in absence of Nop19p, Dhr2p remains trapped within aberrant preribosomes.
Published: 2011
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24. RNA polymerase I-specific subunits promote polymerase clustering to enhance the rRNA gene transcription cycle

Author: Christophe Normand, Isabelle Léger-Silvestre, Benjamin Albert, Jorge Perez-Fernandez, Kostya I. Panov, Joost C. B. M. Zomerdijk, Patrick Schultz, Martin K. Ostermaier, Olivier Gadal, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)
Subjects: Transcription, Genetic, MESH: RNA Polymerase I, viruses, Nuclear Localization Signals, RNA polymerase II, MESH: Nuclear Localization Signals, 0302 clinical medicine, RNA Polymerase I, Gene Expression Regulation, Fungal, Transcriptional regulation, Polymerase, Research Articles, Conserved Sequence, 0303 health sciences, MESH: Genetic Complementation Test, MESH: Conserved Sequence, biology, MESH: Protein Multimerization, MESH: Cell Nucleus Shape, MESH: Transcription Factors, MESH: Protein Subunits, MESH: Saccharomyces cerevisiae, MESH: Schizosaccharomyces, MESH: Cell Nucleolus, Cell Nucleolus, MESH: Gene Expression Regulation, Fungal, Cell Nucleus Shape, Saccharomyces cerevisiae, RNA polymerase III, Article, 03 medical and health sciences, Schizosaccharomyces, MESH: Genes, rRNA, RNA polymerase I, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, MESH: Humans, MESH: Transcription, Genetic, Genetic Complementation Test, Genes, rRNA, Cell Biology, Processivity, Ribosomal RNA, Molecular biology, Protein Subunits, biology.protein, DNA polymerase I, Protein Multimerization, 030217 neurology & neurosurgery, Transcription Factors
Abstract: The Pol I subunits Rpa34 and Rpa49, required for elongation and 35S rRNA production, promote clustering of neighboring Pol I complexes to enhance the rRNA gene transcription cycle., RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I–specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.
Published: 2011
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25. Cell cycle-dependent kinetochore localization of condensin complex in Saccharomyces cerevisiae

Author: Olivier Gadal, Gaëlle Bourout, Ulf Nehrbass, Sophie Bachellier-Bassi, Biologie Cellulaire du Noyau (BCN), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Saccharomyces cerevisiae Proteins, Chromosomal Proteins, Non-Histone, Condensin, Mitosis, Cell Cycle Proteins, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Cell cycle, DNA, Ribosomal, Spindle pole body, 03 medical and health sciences, Condensin complex, 0302 clinical medicine, Structural Biology, Centromere, Kinetochores, 030304 developmental biology, Anaphase, Adenosine Triphosphatases, Centromeres, 0303 health sciences, Cohesin, Kinetochore, Nuclear Proteins, Cell biology, DNA-Binding Proteins, SUMO, Multiprotein Complexes, biology.protein, 030217 neurology & neurosurgery
Abstract: International audience; In budding yeast mitosis is endonuclear and associated with a very limited condensation of the chromosomes. Despite this partial chromosomal condensation, condensin is conserved and essential for the Saccharomyces cerevisiae mitotic cycle. Here, we investigate the localization of condensin during the mitotic cycle. In addition to a constitutive association with rDNA, we have discovered that condensin is localized to the kinetochore in a cell cycle-dependent manner. Shortly after duplication of the spindle pole body, the yeast equivalent of the centrosome, we observed a local enrichment of condensin colocalizing with kinetochore components. This specific association is consistent with mutant phenotypes of chromosome loss and defective sister chromatid separation at anaphase. During a short period of the cell cycle, we observed, at the single cell level, a spatial proximity of condensin and a cohesin rosette, without colocalization. Furthermore, using a genetic screen we demonstrated that condensin localization at kinetochores is specifically impaired in a mutant for ulp2/smt4, an abundant SUMO protease. In conclusion, during chromosome segregation, we established a SUMO-dependent cell cycle-specific condensin concentration colocalizing with kinetochores.
Published: 2008
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26. Hmo1 Is Required for TOR-Dependent Regulation of Ribosomal Protein Gene Transcription▿ †

Author: Alain Jacquier, Olivier Gadal, Ulf Nehrbass, Laurence Decourty, Axel B. Berger, Gwenael Badis, Biologie Cellulaire du Noyau (BCN), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Génétique des Intéractions Macromoléculaires, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), and Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Ribosomal Proteins, Saccharomyces cerevisiae Proteins, Transcription, Genetic, DNA Mutational Analysis, Ribosome biogenesis, RNA polymerase II, Saccharomyces cerevisiae, Protein Serine-Threonine Kinases, DNA, Ribosomal, 03 medical and health sciences, 0302 clinical medicine, MESH: Saccharomyces cerevisiae Proteins, Ribosomal protein, Transcription (biology), Gene Expression Regulation, Fungal, Transcriptional regulation, RNA polymerase I, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: DNA Mutational Analysis, MESH: Antibiotics, Antineoplastic, Promoter Regions, Genetic, Molecular Biology, 030304 developmental biology, Genetics, Sirolimus, 0303 health sciences, MESH: DNA, Ribosomal, Antibiotics, Antineoplastic, biology, MESH: Transcription, Genetic, High Mobility Group Proteins, Promoter, Cell Biology, MESH: Transcription Factors, Articles, DNA-Directed RNA Polymerases, Ribosomal RNA, MESH: High Mobility Group Proteins, MESH: Ribosomal Proteins, MESH: Saccharomyces cerevisiae, MESH: DNA-Directed RNA Polymerases, MESH: Promoter Regions (Genetics), RNA, Ribosomal, biology.protein, MESH: RNA, Ribosomal, MESH: Sirolimus, 030217 neurology & neurosurgery, MESH: Gene Expression Regulation, Fungal, Transcription Factors
Abstract: International audience; Ribosome biogenesis requires equimolar amounts of four rRNAs and all 79 ribosomal proteins (RP). Coordinated regulation of rRNA and RP synthesis by eukaryotic RNA polymerases (Pol) I, III, and II is a key requirement for growth control. Using a novel global genetic approach, we showed that the absence of Hmo1 becomes lethal when combined with mutations of components of either the RNA Pol II or Pol I transcription machineries, of specific RP, or of the TOR pathway. Hmo1 directly interacts with both the region transcribed by Pol I and a subset of RP gene promoters. Down-regulation of Hmo1 expression affects RP gene expression. Upon TORC1 inhibition, Hmo1 dissociates from ribosomal DNA (rDNA) and some RP gene promoters simultaneously. Finally, in the absence of Hmo1, TOR-dependent repression of RP genes is alleviated. Therefore, we show here that Saccharomyces cerevisiae Hmo1 is directly involved in coordinating rDNA transcription by Pol I and RP gene expression by Pol II under the control of the TOR pathway.
Published: 2007
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27. RNA polymerase I-specific subunit CAST/hPAF49 has a role in the activation of transcription by upstream binding factor

Author: Kaori Nishiyama, Jackie Russell, Joost C. B. M. Zomerdijk, Kostya I. Panov, Takashi Saito, Tatiana B. Panova, Olivier Gadal, Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee, Biologie Cellulaire du Noyau (BCN), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, RIKEN Research Center for Allergy and Immunology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), and Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Transcriptional Activation, MESH: RNA Polymerase I, Protein subunit, In Vitro Techniques, Biology, Models, Biological, MESH: Tyrosine, 03 medical and health sciences, RNA Polymerase I, Transcription (biology), MESH: Intracellular Signaling Peptides and Proteins, RNA polymerase I, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Phosphorylation, MESH: Pol1 Transcription Initiation Complex Proteins, Molecular Biology, Transcription factor, Gene, Polymerase, 030304 developmental biology, 0303 health sciences, MESH: Humans, MESH: Phosphorylation, 030302 biochemistry & molecular biology, Intracellular Signaling Peptides and Proteins, MESH: Models, Biological, RNA, Articles, Cell Biology, MESH: Protein Subunits, Molecular biology, MESH: Hela Cells, Protein Subunits, Pol1 Transcription Initiation Complex Proteins, MESH: Trans-Activation (Genetics), biology.protein, Tyrosine, HeLa Cells
Abstract: International audience; Eukaryotic RNA polymerases are large complexes, 12 subunits of which are structurally or functionally homologous across the three polymerase classes. Each class has a set of specific subunits, likely targets of their cognate transcription factors. We have identified and characterized a human RNA polymerase I (Pol I)-specific subunit, previously identified as ASE-1 (antisense of ERCC1) and as CD3epsilon-associated signal transducer (CAST), and here termed CAST or human Pol I-associated factor of 49 kDa (hPAF49), after mouse orthologue PAF49. We provide evidence for growth-regulated Tyr phosphorylation of CAST/hPAF49, specifically in initiation-competent Pol Ibeta complexes in HeLa cells, at a conserved residue also known to be important for signaling during T-cell activation. CAST/hPAF49 can interact with activator upstream binding factor (UBF) and, weakly, with selectivity factor 1 (SL1) at the rDNA (ribosomal DNA repeat sequence encoding the 18S, 5.8S, and 28S rRNA genes) promoter. CAST/hPAF49-specific antibodies and excess CAST/hPAF49 protein, which have no effect on basal Pol I transcription, inhibit UBF-activated transcription following functional SL1-Pol I-rDNA complex assembly and disrupt the interaction of UBF with CAST/hPAF49, suggesting that interaction of this Pol I-specific subunit with UBF is crucial for activation. Drawing on parallels between mammalian and Saccharomyces cerevisiae Pol I transcription machineries, we advance one model for CAST/hPAF49 function in which the network of interactions of Pol I-specific subunits with UBF facilitates conformational changes of the polymerase, leading to stabilization of the Pol I-template complex and, thereby, activation of transcription.
Published: 2006
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28. SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope

Author: Jean-Christophe Olivo-Marin, Annick Lesne, Henri Buc, Susana Rodríguez-Navarro, Ulf Nehrbass, Eduard C. Hurt, Christophe Zimmer, Olivier Gadal, Frank Feuerbach-Fournier, Auguste Genovesio, Ghislain G. Cabal, Biologie Cellulaire du Noyau (BCN), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Analyse d'Images Quantitative (AIQ), Biochemie-Zentrum (BZH), Universität Heidelberg [Heidelberg] = Heidelberg University, Laboratoire de Physique Théorique des Liquides (LPTL), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Département de Biologie cellulaire et infection - Department of Cell Biology and infection (BCI), Institut Pasteur [Paris] (IP), Cell Biology Programme, EMBL Heidelberg, Institut Pasteur Korea - Institut Pasteur de Corée, Réseau International des Instituts Pasteur (RIIP), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Heidelberg Universit, and Institut Pasteur [Paris]
Subjects: Saccharomyces cerevisiae Proteins, Histone acetyltransferase complex, Transcription, Genetic, Nuclear Envelope, Saccharomyces cerevisiae, Genes, Fungal, Diffusion, 03 medical and health sciences, 0302 clinical medicine, Transcription (biology), Genes, Reporter, Gene Expression Regulation, Fungal, medicine, Nuclear pore, Gene, 030304 developmental biology, Genetics, 0303 health sciences, Reporter gene, Multidisciplinary, biology, Models, Genetic, Chemistry, RNA, RNA, Fungal, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, Cell biology, Cell nucleus, medicine.anatomical_structure, Mutation, Trans-Activators, 030217 neurology & neurosurgery, Protein Binding
Abstract: Changes in the transcriptional state of genes have been correlated with their repositioning within the nuclear space. Tethering reporter genes to the nuclear envelope alone can impose repression and recent reports have shown that, after activation, certain genes can also be found closer to the nuclear periphery. The molecular mechanisms underlying these phenomena have remained elusive. Here, with the use of dynamic three-dimensional tracking of a single locus in live yeast (Saccharomyces cerevisiae) cells, we show that the activation of GAL genes (GAL7, GAL10 and GAL1) leads to a confinement in dynamic motility. We demonstrate that the GAL locus is subject to sub-diffusive movement, which after activation can become constrained to a two-dimensional sliding motion along the nuclear envelope. RNA-fluorescence in situ hybridization analysis after activation reveals a higher transcriptional activity for the peripherally constrained GAL genes than for loci remaining intranuclear. This confinement was mediated by Sus1 and Ada2, members of the SAGA histone acetyltransferase complex, and Sac3, a messenger RNA export factor, physically linking the activated GAL genes to the nuclear-pore-complex component Nup1. Deleting ADA2 or NUP1 abrogates perinuclear GAL confinement without affecting GAL1 transcription. Accordingly, transcriptional activation is necessary but not sufficient for the confinement of GAL genes at the nuclear periphery. The observed real-time dynamic mooring of active GAL genes to the inner side of the nuclear pore complex is in accordance with the 'gene gating' hypothesis.
Published: 2006
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29. Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing

Author: Elisabeth Petfalski, David Tollervey, Nicole Gas, Pierre-Emmanuel Gleizes, Olivier Gadal, Ed Hurt, and Daniela Strauss
Subjects: Ribosomal Proteins, Nucleolus, Green Fluorescent Proteins, Molecular Sequence Data, Biology, Article, Fungal Proteins, 03 medical and health sciences, Ribosomal protein, Yeasts, RNA Precursors, Granular component, Amino Acid Sequence, RNA Processing, Post-Transcriptional, RRNA processing, Ribosomal DNA, Conserved Sequence, 030304 developmental biology, 0303 health sciences, Eukaryotic Large Ribosomal Subunit, 030302 biochemistry & molecular biology, Temperature, RNA, Fungal, Cell Biology, Ribosomal RNA, rRNA processing, ribosome biogenesis, ribosome export, nucleolus, preribosome, Molecular biology, Protein Structure, Tertiary, Luminescent Proteins, Amino Acid Substitution, RNA, Ribosomal, Mutation, Nucleolus organizer region, Sequence Alignment, Cell Nucleolus
Abstract: Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.
Published: 2002
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30. The Nucle(ol)ar Tif6p and Efl1p Are Required for a Late Cytoplasmic Step of Ribosome Synthesis

Author: Franco Fasiolo, Olivier Gadal, Michael Breitenbach, Jean-Marie Garnier, Jean-Sebastien Graindorge, Eduard C. Hurt, Bruno Senger, Ambaliou Sanni, Alain Camasses, Denis L. J. Lafontaine, Architecture et réactivité de l'ARN (ARN), Centre National de la Recherche Scientifique (CNRS)-Université Louis Pasteur - Strasbourg I, GDR 3536 Architecture et Dynamique Nucléaires (ADN), Université Pierre et Marie Curie - Paris 6 (UPMC), Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Laboratoire de Biochimie et de Biologie Moléculaire (Université d'Abomey Calavi, Cotonou, Bénin) (LBBM), University of Abomey Calavi (UAC), Centre européen de recherche et d'enseignement des géosciences de l'environnement (CEREGE), Aix Marseille Université (AMU)-Institut national des sciences de l'Univers (INSU - CNRS)-Collège de France (CdF (institution))-Institut de Recherche pour le Développement (IRD)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Heidelberg University, Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université d’Abomey-Calavi = University of Abomey Calavi (UAC), Institut de Recherche pour le Développement (IRD)-Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Collège de France (CdF (institution))-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Collège de France (CdF)-Institut national des sciences de l'Univers (INSU - CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), and University of Heidelberg
Subjects: Cytoplasm, Saccharomyces cerevisiae Proteins, [SDV]Life Sciences [q-bio], GTPase, Saccharomyces cerevisiae, Biology, Ribosome, GTP Phosphohydrolases, 03 medical and health sciences, Genes, Reporter, Homologous chromosome, RNA Precursors, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA Processing, Post-Transcriptional, Molecular Biology, Conserved Sequence, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Cell Nucleus, 0303 health sciences, Eukaryotic Large Ribosomal Subunit, 030302 biochemistry & molecular biology, Biological Transport, RNA, Fungal, Cell Biology, Ribosomal RNA, In vitro, Cell biology, Enzyme Activation, Molecular Weight, Protein Subunits, Phenotype, Biochemistry, EIF6, RNA, Ribosomal, Ribosomes, Cell Division, Cell Nucleolus, Gene Deletion
Abstract: Deletion of elongation factor-like 1 (Efl1p), a cytoplasmic GTPase homologous to the ribosomal translocases EF-G/EF-2, results in nucle(ol)ar pre-rRNA processing and pre-60S subunits export defects. Efl1p interacts genetically with Tif6p, a nucle(ol)ar protein stably associated with pre-60S subunits and required for their synthesis and nuclear exit. In the absence of Efl1p, 50% of Tif6p is relocated to the cytoplasm. In vitro, the GTPase activity of Efl1p is stimulated by 60S, and Efl1p promotes the dissociation of Tif6p-60S complexes. We propose that Tif6p binds to the pre-60S subunits in the nucle(ol)us and escorts them to the cytoplasm where the GTPase activity of Efl1p triggers a late structural rearrangement, which facilitates the release of Tif6p and its recycling to the nucle(ol)us.
Published: 2001
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31. Nuclear Export of 60S Ribosomal Subunits Depends on Xpo1p and Requires a Nuclear Export Sequence-Containing Factor, Nmd3p, That Associates with the Large Subunit Protein Rpl10p

Author: Bernard L. Trumpower, Olivier Gadal, Jacques J. Kessl, Ed Hurt, Daniela Strauß, and David Tollervey
Subjects: Ribosomal Proteins, Saccharomyces cerevisiae Proteins, Protein subunit, Receptors, Cytoplasmic and Nuclear, Saccharomyces cerevisiae, Biology, Karyopherins, Ribosome, Fungal Proteins, Ribosomal protein, Gene Expression Regulation, Fungal, Nucleocytoplasmic Communication, Nuclear protein, Nuclear export signal, Molecular Biology, Eukaryotic Large Ribosomal Subunit, Nuclear cap-binding protein complex, Nuclear Proteins, RNA-Binding Proteins, Cell Biology, Molecular biology, Cell biology, Mutation, Nucleoporin, Carrier Proteins, Ribosomes
Abstract: Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rp125p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits, We find that thermosensitive nmd3 mutants are impaired ill large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We conclude that nuclear export of 60S subunits requires the nuclear export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p.
Published: 2001
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32. A protein–protein interaction map of yeast RNA polymerase III

Author: André Sentenac, V. Van Mullem, Jean-François Briand, Christophe Carles, Christian Marck, Liudmilla Rubbi, Jean-Christophe Andrau, M. Goussot, Amando Flores, Claire Boschiero, Michel Werner, Olivier Gadal, and Pierre Thuriaux
Subjects: Transcription, Genetic, Macromolecular Substances, Protein subunit, Recombinant Fusion Proteins, RNA polymerase II, Saccharomyces cerevisiae, Biology, RNA polymerase III, Conserved sequence, chemistry.chemical_compound, Open Reading Frames, Peptide Library, RNA Polymerase I, Transcription Factors, TFIII, RNA polymerase, RNA polymerase I, Conserved Sequence, Genetics, Multidisciplinary, Binding Sites, RNA Polymerase III, Processivity, Biological Sciences, Biochemistry, chemistry, Transcription preinitiation complex, biology.protein, RNA Polymerase II, Transcription Factors
Abstract: The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein–protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10α, and ABC10β), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.
Published: 1999

33. RNA polymerase I mutant affects ribosomal RNA processing and ribosomal DNA stability.

Author: Normand, Christophe, Dez, Christophe, Dauban, Lise, Queille, Sophie, Danché, Sarah, Abderrahmane, Sarra, Beckouet, Frederic, and Gadal, Olivier
Abstract: Transcription is a major contributor to genomic instability. The ribosomal RNA (rDNA) gene locus consists of a head-to-tail repeat of the most actively transcribed genes in the genome. RNA polymerase I (RNAPI) is responsible for massive rRNA production, and nascent rRNA is co-transcriptionally assembled with early assembly factors in the yeast nucleolus. In Saccharomyces cerevisiae, a mutant form of RNAPI bearing a fusion of the transcription factor Rrn3 with RNAPI subunit Rpa43 (CARA-RNAPI) has been described previously. Here, we show that the CARA-RNAPI allele results in a novel type of rRNA processing defect, associated with rDNA genomic instability. A fraction of the 35S rRNA produced in CARA-RNAPI mutant escapes processing steps and accumulates. This accumulation is increased in mutants affecting exonucleolytic activities of the exosome complex. CARA-RNAPI is synthetic lethal with monopolin mutants that are known to affect the rDNA condensation. CARA-RNAPI strongly impacts rDNA organization and increases rDNA copy number variation. Reduced rDNA copy number suppresses lethality, suggesting that the chromosome segregation defect is caused by genomic rDNA instability. We conclude that a constitutive association of Rrn3 with transcribing RNAPI results in the accumulation of rRNAs that escape normal processing, impacting rDNA organization and affecting rDNA stability. [ABSTRACT FROM AUTHOR]
Published: 2024
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34. Meeting report from the first European OddPols meeting: Toulouse 2018.

Author: Dauban, Lise, Cerezo, Emilie, Henras, Anthony, and Gadal, Olivier
Subjects: *GENETIC transcription, *RNA polymerases
Abstract: The eleventh international conference on transcription by RNA polymerases I, III, IV and V (the OddPols) was held from June 26th to June 29th 2018 at the Museum of Natural History of Toulouse, France and organized by Anthony Henras and Olivier Gadal. The scientific committee was composed of David Engelke, Joachim Griesenbeck, Ross Hannan, Deborah Johnson, Richard Maraia, Christoph Müller, Craig Pikaard, David Schneider and Ian Willis. The organizers are grateful to the " Centre de Biologie Intégrative de Toulouse ", for support in the organization of the event. Participants from 13 different countries presented their newest exciting results during the scientific sessions and during extended conversation hours in the cosy atmosphere of the botanic garden of the Museum. Here we present the highlights of all oral presentations. [ABSTRACT FROM AUTHOR]
Published: 2019
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35. The conserved RNA-binding protein Seb1 promotes cotranscriptional ribosomal RNA processing by controlling RNA polymerase I progression.

Author: Duval, Maxime, Yague-Sanz, Carlo, Turowski, Tomasz W., Petfalski, Elisabeth, Tollervey, David, and Bachand, François
Subjects: RNA-binding proteins, RIBOSOMAL RNA, ORGANELLE formation, EUKARYOTIC cells, CONDITIONED response
Abstract: Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. We show here that the conserved fission yeast RNA-binding protein Seb1 associates with the RNAPI transcription machinery and promotes RNAPI pausing states along the rDNA. The overall faster progression of RNAPI at the rDNA in Seb1-deficient cells impaired cotranscriptional pre-rRNA processing and the production of mature rRNAs. Given that Seb1 also influences pre-mRNA processing by modulating RNAPII progression, our findings unveil Seb1 as a pause-promoting factor for RNA polymerases I and II to control cotranscriptional RNA processing. Ribosome biogenesis is influenced by the rate of RNAPI progression. Yet, mechanisms that control RNAPI elongation have remained elusive. Here, the authors show that the conserved protein Seb1 promotes cotranscriptional rRNA processing by controlling RNAPI progression. [ABSTRACT FROM AUTHOR]
Published: 2023
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36. Acknowledgment to the Reviewers of Cancers in 2022.

Published: 2023
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37. Nuclear ingression of cytoplasmic bodies accompanies a boost in autophagy.

Author: Garcia, Manon, Kumanski, Sylvain, Ellas-Villalobos, Alberto, Cazevieille, Chantal, Soulet, Caroline, and Moriel-Carretero, Maria
Published: 2022
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38. Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast.

Author: Jaafar, Mariam, Contreras, Julia, Dominique, Carine, Martín-Villanueva, Sara, Capeyrou, Régine, Vitali, Patrice, Rodríguez-Galán, Olga, Velasco, Carmen, Humbert, Odile, Watkins, Nicholas J., Villalobo, Eduardo, Bohnsack, Katherine E., Bohnsack, Markus T., Henry, Yves, Merhi, Raghida Abou, de la Cruz, Jesús, and Henras, Anthony K.
Subjects: RNA helicase, RIBOSOMAL proteins, ORGANELLE formation, BIOCHEMICAL genetics, RIBOSOMAL RNA, YEAST
Abstract: Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit. The molecular events underlying the assembly and maturation of the early pre-60S particles during eukaryotic ribosome synthesis are not well understood. Here, the authors combine yeast genetics and biochemical experiments to characterise the functions of two important players of eukaryotic ribosome biogenesis, the box C/D snoRNP snR190 and the helicase Dbp7, which both interact. They show that the snR190 snoRNA acts as a RNA chaperone that assists the structuring of the 25S rRNA during the maturation of early pre-60S particles and that Dbp7 is important for facilitating remodeling events in the peptidyl transferase center region of the 25S rRNAs during the maturation of early pre-60S particles. [ABSTRACT FROM AUTHOR]
Published: 2021
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39. Non-Coding, RNAPII-Dependent Transcription at the Promoters of rRNA Genes Regulates Their Chromatin State in S. cerevisiae.

Author: Lesage, Emma, Perez-Fernandez, Jorge, Queille, Sophie, Dez, Christophe, Gadal, Olivier, and Kwapisz, Marta
Subjects: RNA polymerase II, RIBOSOMAL RNA, RNA polymerases, NON-coding RNA, RIBOSOMAL DNA, LINCRNA, CHROMATIN
Abstract: Pervasive transcription is widespread in eukaryotes, generating large families of noncoding RNAs. Such pervasive transcription is a key player in the regulatory pathways controlling chromatin state and gene expression. Here, we describe long non-coding RNAs generated from the ribosomal RNA gene promoter called UPStream-initiating transcripts (UPS). In yeast, rDNA genes are organized in tandem repeats in at least two different chromatin states, either transcribed and largely depleted of nucleosomes (open) or assembled in regular arrays of nucleosomes (closed). The production of UPS transcripts by RNA Polymerase II from endogenous rDNA genes was initially documented in mutants defective for rRNA production by RNA polymerase I. We show here that UPS are produced in wild-type cells from closed rDNA genes but are hidden within the enormous production of rRNA. UPS levels are increased when rDNA chromatin states are modified at high temperatures or entering/leaving quiescence. We discuss their role in the regulation of rDNA chromatin states and rRNA production. [ABSTRACT FROM AUTHOR]
Published: 2021
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40. Impact of DNA methylation on 3D genome structure

Author: Buitrago, Diana, Labrador, Mireia, Arcon, Juan Pablo, Lema, Rafael, Flores, Oscar, Esteve-Codina, Anna, Blanc, Julie, Villegas, Nuria, Bellido, David, Gut, Marta, Dans, Pablo D., Heath, Simon C., Gut, Ivo G., Brun Heath, Isabelle, and Orozco, Modesto
Published: 2021
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41. Rouse model with transient intramolecular contacts on a timescale of seconds recapitulates folding and fluctuation of yeast chromosomes.

Author: Socol, Marius, Wang, Renjie, Jost, Daniel, Carrivain, Pascal, Vaillant, Cédric, Le Cam, Eric, Dahirel, Vincent, Normand, Christophe, Bystricky, Kerstin, Victor, Jean-Marc, Gadal, Olivier, and Bancaud, Aurélien
Published: 2019
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42. Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I.

Author: Darrière, Tommy, Pilsl, Michael, Sarthou, Marie-Kerguelen, Chauvier, Adrien, Genty, Titouan, Audibert, Sylvain, Dez, Christophe, Léger-Silvestre, Isabelle, Normand, Christophe, Henras, Anthony K., Kwapisz, Marta, Calvo, Olga, Fernández-Tornero, Carlos, Tschochner, Herbert, and Gadal, Olivier
Subjects: RNA polymerase I, POINT mutation (Biology), DNA-binding proteins, SUPPRESSOR mutation, RIBOSOMAL RNA
Abstract: Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I. [ABSTRACT FROM AUTHOR]
Published: 2019
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43. Turnover of aberrant pre-40S pre-ribosomal particles is initiated by a novel endonucleolytic decay pathway.

Author: Choque, Elodie, Schneider, Claudia, Gadal, Olivier, and Dez, Christophe
Published: 2018
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44. From dynamic chromatin architecture to DNA damage repair and back.

Author: Fabre, Emmanuelle and Zimmer, Christophe
Subjects: CHROMATIN, DNA repair, DNA damage, CELL nuclei, GENOMES
Abstract: Maintaining the integrity of the genome in the face of DNA damage is crucial to ensure the survival of the cell and normal development. DNA lesions and repair occur in the context of the chromatin fiber, whose 3D organization and movements in the restricted volume of the nucleus are under intense scrutiny. Here, we highlight work from our and other labs that addresses how the dynamic organization of the chromatin fiber affects the repair of damaged DNA and how, conversely, DNA damage and repair affect the structure and dynamics of chromatin in the budding yeast nucleus. [ABSTRACT FROM AUTHOR]
Published: 2018
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45. High resolution microscopy reveals the nuclear shape of budding yeast during cell cycle and in various biological states.

Author: Renjie Wang, Kamgoue, Alain, Normand, Christophe, Léger-Silvestre, Isabelle, Mangeat, Thomas, and Gadal, Olivier
Subjects: SACCHAROMYCES cerevisiae, FUNGAL cell cycle, NUCLEAR shapes
Abstract: How spatial organization of the genome depends on nuclear shape is unknown, mostly because accurate nuclear size and shape measurement is technically challenging. In large cell populations of the yeast Saccharomyces cerevisiae, we assessed the geometry (size and shape) of nuclei in three dimensions with a resolution of 30 nm. We improved an automated fluorescence localization method by implementing a post-acquisition correction of the spherical microscopic aberration along the z-axis, to detect the three dimensional (3D) positions of nuclear pore complexes (NPCs) in the nuclear envelope. Here, we used a method called NucQuant to accurately estimate the geometry of nuclei in 3D throughout the cell cycle. To increase the robustness of the statistics, we aggregated thousands of detected NPCs from a cell population in a single representation using the nucleolus or the spindle pole body (SPB) as references to align nuclei along the same axis. We could detect asymmetric changes of the nucleus associated with modification of nucleolar size. Stereotypical modification of the nucleus toward the nucleolus further confirmed the asymmetric properties of the nuclear envelope. [ABSTRACT FROM AUTHOR]
Published: 2016
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46. Studying Coxiella burnetii Type IV Substrates in the Yeast Saccharomyces cerevisiae: Focus on Subcellular Localization and Protein Aggregation.

Author: Rodríguez-Escudero, María, Cid, Víctor J., Molina, María, Schulze-Luehrmann, Jan, Lührmann, Anja, and Rodríguez-Escudero, Isabel
Subjects: Q fever, COXIELLA burnetii, GRAM-negative bacteria, BIOCHEMICAL substrates, SACCHAROMYCES cerevisiae, BIOLOGICAL aggregation
Abstract: Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors. [ABSTRACT FROM AUTHOR]
Published: 2016
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47. Structural basis for binding the TREX2 complex to nuclear pores, GAL1 localisation and mRNA export.

Author: Jani, Divyang, Valkov, Eugene, and Stewart, Murray
Published: 2014
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48. SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope

Author: Cabal, Ghislain G., Genovesio, Auguste, Rodriguez-Navarro, Susana, Zimmer, Christophe, Gadal, Olivier, Lesne, Annick, Buc, Henri, Feuerbach-Fournier, Frank, Olivo-Marin, Jean-Christophe, Hurt, Eduard C., and Nehrbass, Ulf
Subjects: Environmental issues, Science and technology, Zoology and wildlife conservation
Abstract: Author(s): Ghislain G. Cabal [1, 8]; Auguste Genovesio [2, 8, 7]; Susana Rodriguez-Navarro [4, 7]; Christophe Zimmer [2]; Olivier Gadal [1]; Annick Lesne [5]; Henri Buc [3]; Frank Feuerbach-Fournier [1]; [...]
Published: 2006
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49. Structure-function analysis of Hmo1 unveils an ancestral organization of HMG-Box factors involved in ribosomal DNA transcription from yeast to human.

Author: Albert, Benjamin, Colleran, Christine, Léger-Silvestre, Isabelle, Berger, Axel B., Dez, Christophe, Normand, Christophe, Perez-Fernandez, Jorge, McStay, Brian, and Gadal, Olivier
Published: 2013
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50. The nucleolar protein Nop19p interacts preferentially with Utp25p and Dhr2p and is essential for the production of the 40S ribosomal subunit in Saccharomyces cerevisiae.

Author: Choque, Elodie, Marcellin, Marlène, Burlet-Schiltz, Odile, Gadal, Olivier, and Dez, Christophe
Published: 2011
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