Author: "Mireille Bétermier" - Searchworks@Jio Institute Digital Library Search Results

Your search keyword '"Mireille Bétermier"' showing total 67 results

Start Over Author "Mireille Bétermier"

67 results on '"Mireille Bétermier"'

1. Uncoupling programmed DNA cleavage and repair scrambles the Paramecium somatic genome

Author: Julien Bischerour, Olivier Arnaiz, Coralie Zangarelli, Vinciane Régnier, Florence Iehl, Virginie Ropars, Jean-Baptiste Charbonnier, and Mireille Bétermier
Subjects: CP: Molecular biology, CP: Genomics, Biology (General), QH301-705.5
Abstract: Summary: In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double-strands breaks (DSBs) introduced by the PiggyMac endonuclease are repaired in a highly concerted manner by the non-homologous end joining (NHEJ) pathway, illustrated by complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show that expression of a DNA-binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but leads to the accumulation of unrepaired DSBs. We uncoupled DNA cleavage and repair by co-expressing wild-type and mutant Ku70. High-throughput sequencing of the developing macronucleus genome in these conditions identifies the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. Coupling the two steps of IES excision ensures that both extremities are held together throughout the process, suggesting that DSB repair proteins are essential for assembly of a synaptic precleavage complex.
Published: 2024
Full Text: View/download PDF

2. The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases

Author: Marc Guérineau, Luiza Bessa, Séverine Moriau, Ewen Lescop, François Bontems, Nathalie Mathy, Eric Guittet, Julien Bischerour, Mireille Bétermier, and Nelly Morellet
Subjects: Domesticated transposase, Zinc finger structure, Genome rearrangements, Ciliates, Histones, Genetics, QH426-470
Abstract: Abstract Background Transposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive. Results We provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state. Conclusions The present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.
Published: 2021
Full Text: View/download PDF

3. Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination.

Author: Arthur Abello, Vinciane Régnier, Olivier Arnaiz, Romain Le Bars, Mireille Bétermier, and Julien Bischerour
Subjects: Genetics, QH426-470
Abstract: Gene duplication and diversification drive the emergence of novel functions during evolution. Because of whole genome duplications, ciliates from the Paramecium aurelia group constitute a remarkable system to study the evolutionary fate of duplicated genes. Paramecium species harbor two types of nuclei: a germline micronucleus (MIC) and a somatic macronucleus (MAC) that forms from the MIC at each sexual cycle. During MAC development, ~45,000 germline Internal Eliminated Sequences (IES) are excised precisely from the genome through a 'cut-and-close' mechanism. Here, we have studied the P. tetraurelia paralogs of KU80, which encode a key DNA double-strand break repair factor involved in non-homologous end joining. The three KU80 genes have different transcription patterns, KU80a and KU80b being constitutively expressed, while KU80c is specifically induced during MAC development. Immunofluorescence microscopy and high-throughput DNA sequencing revealed that Ku80c stably anchors the PiggyMac (Pgm) endonuclease in the developing MAC and is essential for IES excision genome-wide, providing a molecular explanation for the previously reported Ku-dependent licensing of DNA cleavage at IES ends. Expressing Ku80a under KU80c transcription signals failed to complement a depletion of endogenous Ku80c, indicating that the two paralogous proteins have distinct properties. Domain-swap experiments identified the α/β domain of Ku80c as the major determinant for its specialized function, while its C-terminal part is required for excision of only a small subset of IESs located in IES-dense regions. We conclude that Ku80c has acquired the ability to license Pgm-dependent DNA cleavage, securing precise DNA elimination during programmed rearrangements. The present study thus provides novel evidence for functional diversification of genes issued from a whole-genome duplication.
Published: 2020
Full Text: View/download PDF

4. Improved methods and resources for paramecium genomics: transcription units, gene annotation and gene expression

Author: Olivier Arnaiz, Erwin Van Dijk, Mireille Bétermier, Maoussi Lhuillier-Akakpo, Augustin de Vanssay, Sandra Duharcourt, Erika Sallet, Jérôme Gouzy, and Linda Sperling
Subjects: Ciliate, Cap-Seq, TSS, RNA-Seq, Gene annotation, Autogamy, Biotechnology, TP248.13-248.65, Genetics, QH426-470
Abstract: Abstract Background The 15 sibling species of the Paramecium aurelia cryptic species complex emerged after a whole genome duplication that occurred tens of millions of years ago. Given extensive knowledge of the genetics and epigenetics of Paramecium acquired over the last century, this species complex offers a uniquely powerful system to investigate the consequences of whole genome duplication in a unicellular eukaryote as well as the genetic and epigenetic mechanisms that drive speciation. High quality Paramecium gene models are important for research using this system. The major aim of the work reported here was to build an improved gene annotation pipeline for the Paramecium lineage. Results We generated oriented RNA-Seq transcriptome data across the sexual process of autogamy for the model species Paramecium tetraurelia. We determined, for the first time in a ciliate, candidate P. tetraurelia transcription start sites using an adapted Cap-Seq protocol. We developed TrUC, multi-threaded Perl software that in conjunction with TopHat mapping of RNA-Seq data to a reference genome, predicts transcription units for the annotation pipeline. We used EuGene software to combine annotation evidence. The high quality gene structural annotations obtained for P. tetraurelia were used as evidence to improve published annotations for 3 other Paramecium species. The RNA-Seq data were also used for differential gene expression analysis, providing a gene expression atlas that is more sensitive than the previously established microarray resource. Conclusions We have developed a gene annotation pipeline tailored for the compact genomes and tiny introns of Paramecium species. A novel component of this pipeline, TrUC, predicts transcription units using Cap-Seq and oriented RNA-Seq data. TrUC could prove useful beyond Paramecium, especially in the case of high gene density. Accurate predictions of 3′ and 5′ UTR will be particularly valuable for studies of gene expression (e.g. nucleosome positioning, identification of cis regulatory motifs). The P. tetraurelia improved transcriptome resource, gene annotations for P. tetraurelia, P. biaurelia, P. sexaurelia and P. caudatum, and Paramecium-trained EuGene configuration are available through ParameciumDB ( http://paramecium.i2bc.paris-saclay.fr ). TrUC software is freely distributed under a GNU GPL v3 licence ( https://github.com/oarnaiz/TrUC ).
Published: 2017
Full Text: View/download PDF

5. International Congress on Transposable elements (ICTE 2016) in Saint Malo: mobile elements under the sun of Brittany

Author: Pascale Lesage, Mireille Bétermier, Antoine Bridier-Nahmias, Michael Chandler, Séverine Chambeyron, Gael Cristofari, Nicolas Gilbert, Hadi Quesneville, Chantal Vaury, and Jean-Nicolas Volff
Subjects: Transposable elements, Evolution of transposable elements, Impact on genomes, Control of transposition, Mechanism of transposition, Transposon-based gene therapy, Genetics, QH426-470
Abstract: Abstract The third international conference on Transposable Elements (ICTE) was held 16–19 April 2016 in Saint Malo, France. Organized by the French Transposition Community (Research group of the CNRS: “Mobile genetic elements: from mechanism to populations, an integrative approach”) and the French Society of Genetics, the conference’s goal was to bring together researchers who study transposition in diverse organisms, using multiple experimental approaches. The meeting gathered 180 participants from all around the world. Most of them contributed through poster presentations, invited talks and short talks selected from poster abstracts. The talks were organized into six scientific sessions: “Taming mobile DNA: self and non-self recognition”; “Trans-generational inheritance”; “Mobile DNA genome structure and organization, from molecular mechanisms to applications”; “Remembrance of (retro)transposon past: mobile DNA in genome evolution”; and finally “The yin and the yang of mobile DNA in human health”.
Published: 2016
Full Text: View/download PDF

6. Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium

Author: Julien Bischerour, Simran Bhullar, Cyril Denby Wilkes, Vinciane Régnier, Nathalie Mathy, Emeline Dubois, Aditi Singh, Estienne Swart, Olivier Arnaiz, Linda Sperling, Mariusz Nowacki, and Mireille Bétermier
Subjects: transposées domestication, piggyBacv, DNA elimination, ciliates, IES, Medicine, Science, Biology (General), QH301-705.5
Abstract: The domestication of transposable elements has repeatedly occurred during evolution and domesticated transposases have often been implicated in programmed genome rearrangements, as remarkably illustrated in ciliates. In Paramecium, PiggyMac (Pgm), a domesticated PiggyBac transposase, carries out developmentally programmed DNA elimination, including the precise excision of tens of thousands of gene-interrupting germline Internal Eliminated Sequences (IESs). Here, we report the discovery of five groups of distant Pgm-like proteins (PgmLs), all able to interact with Pgm and essential for its nuclear localization and IES excision genome-wide. Unlike Pgm, PgmLs lack a conserved catalytic site, suggesting that they rather have an architectural function within a multi-component excision complex embedding Pgm. PgmL depletion can increase erroneous targeting of residual Pgm-mediated DNA cleavage, indicating that PgmLs contribute to accurately position the complex on IES ends. DNA rearrangements in Paramecium constitute a rare example of a biological process jointly managed by six distinct domesticated transposases.
Published: 2018
Full Text: View/download PDF

7. TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements.

Author: Kamila Maliszewska-Olejniczak, Julita Gruchota, Robert Gromadka, Cyril Denby Wilkes, Olivier Arnaiz, Nathalie Mathy, Sandra Duharcourt, Mireille Bétermier, and Jacek K Nowak
Subjects: Genetics, QH426-470
Abstract: Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs). Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium, and establishes for the first time a specific role of TFIIS in non-coding transcription in eukaryotes.
Published: 2015
Full Text: View/download PDF

8. Ku-mediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia.

Author: Antoine Marmignon, Julien Bischerour, Aude Silve, Clémentine Fojcik, Emeline Dubois, Olivier Arnaiz, Aurélie Kapusta, Sophie Malinsky, and Mireille Bétermier
Subjects: Genetics, QH426-470
Abstract: During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage factory, enabling tight coupling between DSB introduction and repair during PGR.
Published: 2014
Full Text: View/download PDF

9. Is non-homologous end-joining really an inherently error-prone process?

Author: Mireille Bétermier, Pascale Bertrand, and Bernard S Lopez
Subjects: Genetics, QH426-470
Abstract: DNA double-strand breaks (DSBs) are harmful lesions leading to genomic instability or diversity. Non-homologous end-joining (NHEJ) is a prominent DSB repair pathway, which has long been considered to be error-prone. However, recent data have pointed to the intrinsic precision of NHEJ. Three reasons can account for the apparent fallibility of NHEJ: 1) the existence of a highly error-prone alternative end-joining process; 2) the adaptability of canonical C-NHEJ (Ku- and Xrcc4/ligase IV-dependent) to imperfect complementary ends; and 3) the requirement to first process chemically incompatible DNA ends that cannot be ligated directly. Thus, C-NHEJ is conservative but adaptable, and the accuracy of the repair is dictated by the structure of the DNA ends rather than by the C-NHEJ machinery. We present data from different organisms that describe the conservative/versatile properties of C-NHEJ. The advantages of the adaptability/versatility of C-NHEJ are discussed for the development of the immune repertoire and the resistance to ionizing radiation, especially at low doses, and for targeted genome manipulation.
Published: 2014
Full Text: View/download PDF

10. The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

Author: Olivier Arnaiz, Nathalie Mathy, Céline Baudry, Sophie Malinsky, Jean-Marc Aury, Cyril Denby Wilkes, Olivier Garnier, Karine Labadie, Benjamin E Lauderdale, Anne Le Mouël, Antoine Marmignon, Mariusz Nowacki, Julie Poulain, Malgorzata Prajer, Patrick Wincker, Eric Meyer, Sandra Duharcourt, Laurent Duret, Mireille Bétermier, and Linda Sperling
Subjects: Genetics, QH426-470
Abstract: Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated.
Published: 2012
Full Text: View/download PDF

11. Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining.

Author: Aurélie Kapusta, Atsushi Matsuda, Antoine Marmignon, Michael Ku, Aude Silve, Eric Meyer, James D Forney, Sophie Malinsky, and Mireille Bétermier
Subjects: Genetics, QH426-470
Abstract: During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi-mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5'-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3' ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a "cut-and-close" mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics.
Published: 2011
Full Text: View/download PDF

12. Inter-generational nuclear crosstalk links the control of gene expression to programmed genome rearrangements during theParameciumsexual cycle

Author: Mélanie Bazin-Gélis, Evangelia Eleftheriou, Coralie Zangarelli, Gaëlle Lelandais, Linda Sperling, Olivier Arnaiz, and Mireille Bétermier
Abstract: Multinucleate cells are found in many eukaryotes, but how multiple nuclei coordinate their functions is still poorly understood. In the cytoplasm of the ciliateParamecium tetraurelia, two micronuclei (MIC) serving sexual reproduction coexist with a somatic macronucleus (MAC) dedicated to gene expression. During sexual processes, the MAC is progressively destroyed while still ensuring transcription and new MACs develop from copies of the zygotic MIC. Several gene clusters are successively induced and switched off before vegetative growth resumes. Concomitantly, programmed genome rearrangements (PGR) remove transposons and their relics from the new MACs. Development of the new MACs is controlled by the old MAC, since the latter expresses genes involved in PGR, including thePGMgene encoding the essential PiggyMac endonuclease that cleaves the ends of eliminated sequences. Using RNA deep sequencing and transcriptome analysis, we show that impairing PGR up-deregulates key PGR genes, together with ∼600 other genes possibly also involved in PGR. Among these genes, 42% are no longer induced when no new MACs are formed, including 180 genes that are co-expressed withPGMunder all tested conditions. We propose that bi-directional crosstalk between the two coexisting generations of MACs links gene expression to the progression of MAC development.
Published: 2023

13. GC content, but not nucleosome positioning, directly contributes to intron splicing efficiency in Paramecium

Author: Stefano Gnan, Mélody Matelot, Marion Weiman, Olivier Arnaiz, Frédéric Guérin, Linda Sperling, Mireille Bétermier, Claude Thermes, Chun-Long Chen, and Sandra Duharcourt
Subjects: Genetics, Genetics (clinical)
Abstract: Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur cotranscriptionally, and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We show that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, among the smallest periodicities reported. Our analysis has revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint favoring introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for nonsense-mediated decay effectors, we show that introns located at the edge of nucleosomes display higher splicing efficiency than those at the center. However, multiple regression analysis indicates that the low GC content of introns, rather than nucleosome positioning, is associated with high splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning, and intron evolution in Paramecium.
Published: 2022

14. Developmental timing of programmed DNA elimination in $Paramecium\ tetraurelia$ recapitulates germline transposon evolutionary dynamics

Author: Coralie Zangarelli, Olivier Arnaiz, Mickaël Bourge, Kevin Gorrichon, Yan Jaszczyszyn, Nathalie Mathy, Loïc Escoriza, Mireille Bétermier, Vinciane Régnier, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Reproduction et développement des plantes (RDP), 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)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Universitaire du Cancer de Toulouse - Oncopole (IUCT Oncopole - UMR 1037), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Institut National de la Santé et de la Recherche Médicale (INSERM), UFR Sciences du Vivant [Sciences] - Université Paris Cité (UFR SDV UPCité), Université Paris Cité (UPCité), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Bétermier, Mireille
Subjects: [SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], Genetics, Genetics (clinical)
Abstract: With its nuclear dualism, the ciliate Paramecium constitutes an original model to study how host genomes cope with transposable elements (TEs). P. tetraurelia harbors two germline micronuclei (MIC) and a polyploid somatic macronucleus (MAC) that develops from the MIC at each sexual cycle. Throughout evolution, the MIC genome has been continuously colonized by TEs and related sequences that are removed from the somatic genome during MAC development. Whereas TE elimination is generally imprecise, excision of ∼45,000 TE-derived Internal Eliminated Sequences (IESs) is precise, allowing for functional gene assembly. Programmed DNA elimination is concomitant with genome amplification. It is guided by non-coding RNAs and repressive chromatin marks. A subset of IESs is excised independently of this epigenetic control, raising the question of how IESs are targeted for elimination. To gain insight into the determinants of IES excision, we established the developmental timing of DNA elimination genome-wide by combining fluorescence-assisted nuclear sorting with next-generation sequencing. Essentially all IESs are excised within only one endoreplication round (32C to 64C), while TEs are eliminated at a later stage. We show that time, rather than replication, controls the progression of DNA elimination. We defined four IES classes according to excision timing. The earliest excised IESs tend to be independent of epigenetic factors, display strong sequence signals at their ends and originate from the most ancient integration events. We conclude that old IESs have been optimized during evolution for early and accurate excision, by acquiring stronger sequence determinants and escaping epigenetic control.
Published: 2022

15. The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases

Author: Julien Bischerour, Nathalie Mathy, Eric Guittet, Séverine Moriau, Marc Guérineau, Mireille Bétermier, Nelly Morellet, Luiza Mamigonian Bessa, Ewen Lescop, François Bontems, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie des Substances Naturelles (ICSN), Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Reproduction et développement des plantes (RDP), 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)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Chimie des Substances NaturellesIR-RMN-THC Fr3050 CNRS, ANR-14-CE10-0005,PIGGYPACK,Domestication de transposases et épigénétique: Impact sur la dynamique des génomes(2014), Institute for Integrative Biology of the Cell, and École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL)
Subjects: Transposable element, [SDV]Life Sciences [q-bio], Genome rearrangements, QH426-470, Histones, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Gene, Transposase, Domesticated transposase, 030304 developmental biology, Zinc finger, 0303 health sciences, Ciliates, biology, Research, fungi, Tetrahymena, biology.organism_classification, Zinc finger structure, Chromatin, Histone, chemistry, biology.protein, 030217 neurology & neurosurgery, DNA
Abstract: BackgroundTransposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliateParamecium tetraureliais a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive.ResultsWe provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase fromTrichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases frompiggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state.ConclusionsThe present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases fromParameciumandTetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.
Published: 2021

16. GC content, but not nucleosome positioning, directly contributes to intron splicing efficiency in

Author: Stefano, Gnan, Mélody, Matelot, Marion, Weiman, Olivier, Arnaiz, Frédéric, Guérin, Linda, Sperling, Mireille, Bétermier, Claude, Thermes, Chun-Long, Chen, and Sandra, Duharcourt
Subjects: Base Composition, Paramecium, RNA Splicing, Exons, Introns, Nucleosomes
Abstract: Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of
Published: 2021

17. GC content but not nucleosome positioning directly contributes to intron-splicing efficiency in Paramecium

Author: Olivier Arnaiz, Frédéric Guerin, Sandra Duharcourt, Mélody Matelot, Mireille Bétermier, Claude Thermes, Marion Weiman, Stefano Gnan, Chun-Long Chen, Linda Sperling, Dynamique de l'information génétique : bases fondamentales et cancer (DIG CANCER), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Sorbonne Université (SU), Institut Jacques Monod (IJM (UMR_7592)), Université de Paris (UP)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)
Subjects: 0303 health sciences, biology, [SDV]Life Sciences [q-bio], Intron, Computational biology, biology.organism_classification, Genome, Chromatin, 03 medical and health sciences, 0302 clinical medicine, RNA splicing, Nucleosome, Paramecium tetraurelia, Paramecium, 030217 neurology & neurosurgery, GC-content, 030304 developmental biology
Abstract: Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the >90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur co-transcriptionally and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We showed that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, amongst the smallest periodicities reported. Our analysis revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint to locate introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for the nonsense-mediated decay effectors, we showed that introns located at the edge of nucleosomes display higher splicing efficiency than those at the centre. However, multiple regression analysis indicated that the GC content, rather than nucleosome positioning, directly contributes to intron splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning and intron evolution in Paramecium.
Published: 2021

18. Coupling DNA Damage and Repair: an Essential Safeguard during Programmed DNA Double-Strand Breaks?

Author: Mireille Bétermier, Valérie Borde, Jean-Pierre de Villartay, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut Curie [Paris], Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), BORDE, Valerie, Dynamique de l'information génétique : bases fondamentales et cancer (DIG CANCER), Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratory of Genome Dynamics in the Immune System, Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), ANR-18-CE12-0005,LaMarque,Transitions évolutives dans la reconnaissance du 'soi' génomique: petits ARN, facteurs spécifiques de séquence et méthylation de l'ADN(2018), ANR-18-CE12-0018,CreaDiv,Mécanisme de la recombinaison mitotique et méiotique(2018), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Institut Curie-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Sorbonne Université (SU), Institute for Integrative Biology of the Cell [Gif-sur-Yvette] (I2BC), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP)
Subjects: Genome instability, Spo11, DNA damage, DNA recombination, [SDV]Life Sciences [q-bio], DNA repair, [SDV.GEN] Life Sciences [q-bio]/Genetics, Models, Biological, Recombination-activating gene, Genomic Instability, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, programmed DNA double-strand break (prDSB), [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Recombinase, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, DNA Breaks, Double-Stranded, Transposase, 030304 developmental biology, Recombination, Genetic, 0303 health sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, biology, fungi, Cell Biology, Cell biology, [SDV] Life Sciences [q-bio], Meiosis, chemistry, biology.protein, Homologous recombination, 030217 neurology & neurosurgery, DNA, genome stability
Abstract: International audience; DNA double-strand breaks (DSBs) are the most toxic DNA lesions given their oncogenic potential. Nevertheless, programmed DSBs (prDSBs) contribute to several biological processes. Formation of prDSBs is the 'price to pay' to achieve these essential biological functions. Generated by domesticated PiggyBac transposases, prDSBs have been integrated in the life cycle of ciliates. Created by Spo11 during meiotic recombination, they constitute a driving force of evolution and ensure balanced chromosome content for successful reproduction. Produced by the RAG1/2 recombinase, they are required for the development of the adaptive immune system in many species. The coevolution of processes that couple introduction of prDSBs to their accurate repair may constitute an effective safeguard against genomic instability.
Published: 2019

19. Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase

Author: Nadine Assrir, Nathalie Mathy, Silke Andrea Wieninger, Michael Nilges, Fred Dyda, Julien Bischerour, Eric Guittet, Ewen Lescop, Xianghong Li, Benjamin Bardiaux, Séverine Moriau, Alison B. Hickman, Nancy L. Craig, Jennifer L Taylor, Mireille Bétermier, Nelly Morellet, Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Howard Hughes Medical Institute (HHMI), Johns Hopkins University (JHU), Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), National Institutes of Health [Bethesda] (NIH), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Intramural Research Program of the NIH and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (to J.L.T., A.B.H., F.D.), The Centre National de la Recherche Scientifique (CNRS) and the Agence Nationale de la Recherche (ANR) [ANR-14-CE10-0005-01 (PIGGYPACK) to N.M., E.L., N.A., E.G., N. Mathy, J.B., M.B.], The Institut de Chimie des Substances Naturelles (ICSN) (to S.M), European Union [FP7-IDEAS-ERC 294809 to M.N., Howard Hughes Medical Institute, Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine (to X.L. N.L.C.). Funding for open access charge: Laboratoire de Biologie et Chimie Structurales, Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Université Paris-Saclay, 91198 Gif sur Yvette cedex, France., ANR-14-CE10-0005,PIGGYPACK,Domestication de transposases et épigénétique: Impact sur la dynamique des génomes(2014), European Project: 294809,EC:FP7:ERC,ERC-2011-ADG_20110310,BAYCELLS(2012), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Transposable element, [SDV]Life Sciences [q-bio], Mutant, Transposases, Computational biology, Biology, DNA sequencing, Genome engineering, 03 medical and health sciences, Protein Domains, Structural Biology, Transcription (biology), Sequence-specific DNA binding, Genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Transposase, Zinc finger, Base Sequence, Zinc Fingers, DNA, DNA-Binding Proteins, Molecular Docking Simulation, Zinc, 030104 developmental biology, Mutation, DNA Transposable Elements, Protein Binding
Abstract: International audience; The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.
Published: 2018

20. Identification of a miniature Sae2/Ctp1/CtIP ortholog from Paramecium tetraurelia required for sexual reproduction and DNA double-strand break repair

Author: Antoine Marmignon, Julia Godau, Anika Trenner, Christine von Aesch, Emeline Dubois, Aurélie Kapusta, Mireille Bétermier, Lauriane Simon, Lorenza P. Ferretti, Alessandro A. Sartori, Raphael Guerois, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Génétique, Reproduction et Développement (GReD ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), Department of Human Genetics [Salt Lake City], University of Utah, Assemblage moléculaire et intégrité du génome (AMIG), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Réarrangements programmés du génome (MICMAC), Département Biologie des Génomes (DBG), University of Zurich, Bétermier, Mireille, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])
Subjects: 1303 Biochemistry, DNA Repair, sae2, [SDV]Life Sciences [q-bio], Mutant, Amino Acid Motifs, Protozoan Proteins, Biochemistry, endonuclease, 1307 Cell Biology, Endonuclease, chemistry.chemical_compound, 0302 clinical medicine, DNA Breaks, Double-Stranded, Conserved Sequence, 0303 health sciences, damage response, biology, Reproduction, 10061 Institute of Molecular Cancer Research, rad32(mre11) nuclease, Cell biology, Meiosis, mre11 complex, 030220 oncology & carcinogenesis, DNA end resection, 610 Medicine & health, 03 medical and health sciences, Mre11 complex, 1312 Molecular Biology, DNA double-strand breaks, Amino Acid Sequence, Homologous recombination, gene, Molecular Biology, Gene, 030304 developmental biology, Nuclease, Sequence Homology, Amino Acid, Cell Biology, DNA, Protozoan, human ctip, chemistry, CtIP, end-resection, biology.protein, 570 Life sciences, ctp1, Paramecium tetraurelia, protein, DNA
Abstract: International audience; DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.
Published: 2019

21. Author response: Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium

Author: Mireille Bétermier, Vinciane Régnier, Emeline Dubois, Olivier Arnaiz, Nathalie Mathy, Simran Bhullar, Aditi Singh, Estienne C. Swart, Cyril Denby Wilkes, Julien Bischerour, Linda Sperling, and Mariusz Nowacki
Subjects: Genetics, Carry (arithmetic), Paramecium, Biology, Programmed DNA elimination, biology.organism_classification, Transposase
Published: 2018

22. Improved methods and resources for paramecium genomics: transcription units, gene annotation and gene expression

Author: Erika Sallet, Mireille Bétermier, Linda Sperling, Erwin van Dijk, Augustin de Vanssay, Maoussi Lhuillier-Akakpo, Olivier Arnaiz, Sandra Duharcourt, Jérôme Gouzy, Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Réarrangements programmés du génome (MICMAC), Département Biologie des Génomes (DBG), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Plateforme de séquençage à haut débit (NGS), Département Plateforme (PF I2BC), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Laboratoire des interactions plantes micro-organismes (LIPM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique intramural, Agence Nationale de la Recherche Scientifique 'PIGGYPACK' : ANR-14-CE10-0005-04, INFERNO : ANR-12-BSV6-0017-03, Comite d'lle de France de la Ligue Nationale Contre le Cancer, Equipe FRM : DEQ20160334868, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Commissariat à l'Energie Atomique et aux Energies Alternatives, Université Paris Sud (Paris 11), Université Paris-Saclay, Institut Jacques Monod, UMR 7592, Université Paris Diderot (Paris 7), Centre National de la Recherche Scientifique ( CNRS ), UMR 967, IRCM, Institut National de la Santé et de la Recherche Médicale ( INSERM ), Interactions plantes-microorganismes et santé végétale, Institut National de la Recherche Agronomique ( INRA ) -Université Nice Sophia Antipolis ( UNS ), and Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Centre National de la Recherche Scientifique ( CNRS )
Subjects: 0301 basic medicine, [SDV.SA]Life Sciences [q-bio]/Agricultural sciences, Paramecium, lcsh:QH426-470, autogamy, lcsh:Biotechnology, Genomics, Computational biology, Biology, ciliate, Genome, 03 medical and health sciences, lcsh:TP248.13-248.65, Gene density, Genetics, RNA-Seq, [ SDV.SA ] Life Sciences [q-bio]/Agricultural sciences, Differential gene expression, Paramecium aurelia, amélioration génétique, Sequence Analysis, RNA, Methodology Article, Gene Expression Profiling, Molecular Sequence Annotation, Gene Annotation, biology.organism_classification, Agricultural sciences, TSS, gene annotation, lcsh:Genetics, 030104 developmental biology, Paramecium tetraurelia, cap-Seq, Sciences agricoles, Biotechnology, Reference genome, expression des gènes
Abstract: Background The 15 sibling species of the Paramecium aurelia cryptic species complex emerged after a whole genome duplication that occurred tens of millions of years ago. Given extensive knowledge of the genetics and epigenetics of Paramecium acquired over the last century, this species complex offers a uniquely powerful system to investigate the consequences of whole genome duplication in a unicellular eukaryote as well as the genetic and epigenetic mechanisms that drive speciation. High quality Paramecium gene models are important for research using this system. The major aim of the work reported here was to build an improved gene annotation pipeline for the Paramecium lineage. Results We generated oriented RNA-Seq transcriptome data across the sexual process of autogamy for the model species Paramecium tetraurelia. We determined, for the first time in a ciliate, candidate P. tetraurelia transcription start sites using an adapted Cap-Seq protocol. We developed TrUC, multi-threaded Perl software that in conjunction with TopHat mapping of RNA-Seq data to a reference genome, predicts transcription units for the annotation pipeline. We used EuGene software to combine annotation evidence. The high quality gene structural annotations obtained for P. tetraurelia were used as evidence to improve published annotations for 3 other Paramecium species. The RNA-Seq data were also used for differential gene expression analysis, providing a gene expression atlas that is more sensitive than the previously established microarray resource. Conclusions We have developed a gene annotation pipeline tailored for the compact genomes and tiny introns of Paramecium species. A novel component of this pipeline, TrUC, predicts transcription units using Cap-Seq and oriented RNA-Seq data. TrUC could prove useful beyond Paramecium, especially in the case of high gene density. Accurate predictions of 3′ and 5′ UTR will be particularly valuable for studies of gene expression (e.g. nucleosome positioning, identification of cis regulatory motifs). The P. tetraurelia improved transcriptome resource, gene annotations for P. tetraurelia, P. biaurelia, P. sexaurelia and P. caudatum, and Paramecium-trained EuGene configuration are available through ParameciumDB (http://paramecium.i2bc.paris-saclay.fr). TrUC software is freely distributed under a GNU GPL v3 licence (https://github.com/oarnaiz/TrUC). Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3887-z) contains supplementary material, which is available to authorized users.
Published: 2017

23. International Congress on Transposable elements (ICTE 2016) in Saint Malo: mobile elements under the sun of Brittany

Author: Mireille Bétermier, Jean-Nicolas Volff, Chantal Vaury, Michael Chandler, Antoine Bridier-Nahmias, Pascale Lesage, Gaël Cristofari, Nicolas Gilbert, Hadi Quesneville, Séverine Chambeyron, Pathologie cellulaire : aspects moléculaires et viraux / Pathologie et Virologie Moléculaire, Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Conservatoire National des Arts et Métiers [CNAM] (CNAM), HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM), Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut de génétique humaine (IGH), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche sur le Cancer et le Vieillissement (IRCAN), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Cellules Souches, Plasticité Cellulaire, Médecine Régénératrice et Immunothérapies (IRMB), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Unité de Recherche Génomique Info (URGI), Institut National de la Recherche Agronomique (INRA), Génétique, Reproduction et Développement (GReD), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), Institut de Génomique Fonctionnelle de Lyon (IGFL), École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 ( UPD7 ) -Institut Universitaire d'Hématologie ( IUH ), Université Paris Diderot - Paris 7 ( UPD7 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Département chimie alimentation santé environnement risque-CNAM ( CASER ), Laboratoire de microbiologie et génétique moléculaires ( LMGM ), Université Paul Sabatier - Toulouse 3 ( UPS ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de génétique humaine ( IGH ), Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de Recherche sur le Cancer et le Vieillissement ( IRCAN ), Université Nice Sophia Antipolis ( UNS ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), CHU Saint-Eloi-Centre Hospitalier Régional Universitaire [Montpellier] ( CHRU Montpellier ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université de Montpellier ( UM ), Unité de Recherche Génomique Info ( URGI ), Institut National de la Recherche Agronomique ( INRA ), Génétique, reproduction et développement ( GReD ), Centre National de la Recherche Scientifique ( CNRS ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -IFR79-Université d'Auvergne - Clermont-Ferrand I ( UdA ) -Université Blaise Pascal - Clermont-Ferrand 2 ( UBP ), Institut de Génomique Fonctionnelle de Lyon ( IGFL ), École normale supérieure - Lyon ( ENS Lyon ) -Institut National de la Recherche Agronomique ( INRA ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique ( CNRS ), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire de microbiologie et génétique moléculaires (LMGM), 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 National de la Santé et de la Recherche Médicale (INSERM)-Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA), Génétique, Reproduction et Développement - Clermont Auvergne (GReD), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne (UCA)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), 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 Nice Sophia Antipolis (... - 2019) (UNS), Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Centre National de la Recherche Scientifique (CNRS)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Lyon (ENS Lyon), Lesage, Pascale, and BMC, BMC
Subjects: 0301 basic medicine, Transposable element, lcsh:QH426-470, Evolution of transposable elements, [SDV]Life Sciences [q-bio], education, Control of transposition, Library science, Impact on genomes, Meeting Report, Biology, Mobile DNA, 03 medical and health sciences, Human health, International congress, Molecular Biology, Malo, ComputingMilieux_MISCELLANEOUS, Genetics, [ SDV ] Life Sciences [q-bio], Mechanism of transposition, SAINT, Genome structure, biology.organism_classification, [SDV] Life Sciences [q-bio], lcsh:Genetics, 030104 developmental biology, Transposon-based gene therapy, Transposable elements, Mobile genetic elements
Abstract: International audience; AbstractThe third international conference on Transposable Elements (ICTE) was held 16–19 April 2016 in Saint Malo, France. Organized by the French Transposition Community (Research group of the CNRS: “Mobile genetic elements: from mechanism to populations, an integrative approach”) and the French Society of Genetics, the conference’s goal was to bring together researchers who study transposition in diverse organisms, using multiple experimental approaches. The meeting gathered 180 participants from all around the world. Most of them contributed through poster presentations, invited talks and short talks selected from poster abstracts. The talks were organized into six scientific sessions: “Taming mobile DNA: self and non-self recognition”; “Trans-generational inheritance”; “Mobile DNA genome structure and organization, from molecular mechanisms to applications”; “Remembrance of (retro)transposon past: mobile DNA in genome evolution”; and finally “The yin and the yang of mobile DNA in human health”.
Published: 2016

24. PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia

Author: Mireille Bétermier, Aurélie Kapusta, Eric Meyer, Céline Baudry, Sarah Rosa, Matthieu Restituito, and Sophie Malinsky
Subjects: Genetics, Macronucleus, fungi, Gene rearrangement, Biology, Genome, chemistry.chemical_compound, chemistry, Nuclear dimorphism, Paramecium tetraurelia, Programmed DNA elimination, Transposase, DNA, Developmental Biology
Abstract: Programmed genome rearrangements drive functional gene assembly in ciliates during the development of the somatic macronucleus. The elimination of germline sequences is directed by noncoding RNAs and is initiated by DNA double-strand breaks, but the enzymes responsible for DNA cleavage have not been identified. We show here that PiggyMac (Pgm), a domesticated piggyBac transposase, is required for these rearrangements in Paramecium tetraurelia. A GFP-Pgm fusion localizes in developing macronuclei, where rearrangements take place, and RNAi-mediated silencing of PGM abolishes DNA cleavage. This is the first in vivo evidence suggesting an essential endonucleolytic function of a domesticated piggyBac transposase.
Published: 2009

25. TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements

Author: Olivier Arnaiz, Jacek K. Nowak, Cyril Denby Wilkes, Robert Gromadka, Nathalie Mathy, Kamila Maliszewska-Olejniczak, Julita Gruchota, Sandra Duharcourt, Mireille Bétermier, Institute of Biochemistry and Biophysics PAS, Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Institut Jacques Monod ( IJM ), Université Paris Diderot - Paris 7 ( UPD7 ) -Centre National de la Recherche Scientifique ( CNRS ), Polish National Science Centre (#N N303 808840), Polish Ministry of Science and Higher Education (#IP2010 028470), ARC Foundation for Cancer Research (#SFI20121205487), Centre National de la Recherche Scientifique (CNRS, an ATIP Plus grant), CNRS, Polish Academy of Sciences, CNRS GDRE 'Paramecium Genome Dynamics and Evolution', School of Molecular Biology—Institute of Biochemistry and Biophysics, PAS, European Science Foundation 'Frontiers of Functional Genomics' program), ANR-10-BLAN-1603,GENOMAC,Analyse à grande échelle du positionnement des nucléosomes dans les noyaux de Paramecium tetraurelia ( 2010 ), ANR-12-BSV6-0017,INFERNO,Hérédité épigénétique transgénérationnelle chez les ciliés : mécanismes et conséquences évolutives ( 2012 ), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), ANR-10-BLAN-1603,GENOMAC,Analyse à grande échelle du positionnement des nucléosomes dans les noyaux de Paramecium tetraurelia(2010), ANR-12-BSV6-0017,INFERNO,Hérédité épigénétique transgénérationnelle chez les ciliés : mécanismes et conséquences évolutives(2012), and Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay
Subjects: Cancer Research, Genome evolution, lcsh:QH426-470, Transcription, Genetic, Somatic cell, RNA polymerase II, Genomics, Biology, Genome, 03 medical and health sciences, 0302 clinical medicine, Transcription (biology), Genetics, Nuclear dimorphism, Cell Lineage, Molecular Biology, Genetics (clinical), Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, 0303 health sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, High-Throughput Nucleotide Sequencing, Non-coding RNA, lcsh:Genetics, Germ Cells, biology.protein, RNA, Long Noncoding, RNA Polymerase II, Paramecium tetraurelia, Transcriptional Elongation Factors, [ SDV.GEN ] Life Sciences [q-bio]/Genetics, 030217 neurology & neurosurgery, Research Article
Abstract: Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs). Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium, and establishes for the first time a specific role of TFIIS in non-coding transcription in eukaryotes., Author Summary Paramecium tetraurelia provides an excellent model for studying the mechanisms involved in the production of non-coding transcripts and their mode of action. Different types of non-coding RNAs (ncRNAs) were shown to be implicated in the programmed DNA elimination process that occurs in this organism. At each sexual cycle, during development of the somatic nucleus from the germline nucleus, the genome is massively rearranged through the reproducible elimination of germline-specific sequences including thousands of short, single copy, non-coding Internal Eliminated Sequences (IES). Here, we demonstrate, using RNA interference, that the TFIIS4 gene encoding a development-specific homolog of RNA polymerase II elongation factor TFIIS, is indispensable for ncRNA synthesis in the new somatic nucleus. TFIIS4 depletion impairs the assembly of a functional somatic genome and affects excision of a large fraction of IESs, which leads to strong lethality in the sexual progeny. We propose that TFIIS4-dependent ncRNAs provide an important component of the molecular machinery that is responsible for developmental genome remodeling in Paramecium.
Published: 2015

26. Large-scale genome remodelling by the developmentally programmed elimination of germ line sequences in the ciliate Paramecium

Author: Mireille Bétermier
Subjects: Paramecium, RNA, Untranslated, Somatic cell, Biology, Microbiology, Genome, Germline, Epigenesis, Genetic, Polyploidy, chemistry.chemical_compound, Chromosomal Instability, Animals, Epigenetics, Molecular Biology, Sequence Deletion, Cell Nucleus, Gene Rearrangement, Genetics, Macronucleus, Cell Cycle, Chromosome, DNA, General Medicine, DNA, Protozoan, biology.organism_classification, chemistry, Genome, Protozoan, Gene Deletion
Abstract: In Paramecium, during the development of the somatic macronucleus, precise excision of thousands of single-copy non-coding sequences is initiated by specific DNA double-strand breaks, while imprecise elimination of germ-line-limited repeated sequences leads to internal deletions or chromosome fragmentation. Recent data point to a role of non-coding RNAs in the epigenetic programming of these rearrangements.
Published: 2004

27. Processing of Double-Strand Breaks Is Involved in the Precise Excision of Paramecium Internal Eliminated Sequences

Author: Mireille Bétermier and Ariane Gratias
Subjects: Genetics, Time Factors, Base Sequence, Models, Genetic, Macronucleus, Molecular Sequence Data, Oligonucleotides, Tetrahymena, Chromosome, Cell Biology, Biology, biology.organism_classification, DNA Dynamics and Chromosome Structure, Genome, Chromosomes, DNA Transposable Elements, Nuclear dimorphism, Animals, Paramecium tetraurelia, Paramecium, Programmed DNA elimination, Genome, Protozoan, Molecular Biology, DNA Damage
Abstract: In ciliates, the development of the somatic macronucleus involves the programmed excision of thousands of internal eliminated sequences (IES) scattered throughout the germ line genome. Previous work with Tetrahymena thermophila has suggested that excision is initiated by a staggered double-strand break (DSB) at one IES end. Nucleophilic attack of the other end by the 3'OH group carried by the firstly broken chromosome end leads to macronuclear junction closure. In this study, we mapped the 3'OH and 5'PO(4) groups that are developmentally released at Paramecium IES boundaries, which are marked by two conserved TA dinucleotides, one of which remains in the macronuclear genome after excision. We show that initiating DSBs at both ends generate 4-base 5' overhangs centered on the TA. Based on the observed processing of the 5'-terminal residue of each overhang, we present a new model for the precise closure of macronuclear chromosomes in Paramecium tetraurelia, different from that previously proposed for tetrahymena. In our model, macronucleus-destined broken ends are aligned through the partial pairing of their 5'-nTAn-3' extensions and joined after trimming of the 5' flaps.
Published: 2003

28. Programmed Rearrangement in Ciliates: Paramecium

Author: Sandra Duharcourt, Mireille Bétermier, Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), and Duharcourt, Sandra
Subjects: Microbiology (medical), Transposable element, Genome evolution, RNA, Untranslated, Physiology, Transposases, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Genome, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Genetics, Paramecium, Transposase, ComputingMilieux_MISCELLANEOUS, Gene Rearrangement, Recombination, Genetic, General Immunology and Microbiology, Ecology, Macronucleus, biology, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, DNA, Protozoan, biology.organism_classification, Chromatin, Infectious Diseases, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Paramecium tetraurelia, Programmed DNA elimination
Abstract: Programmed genome rearrangements in the ciliate Paramecium provide a nice illustration of the impact of transposons on genome evolution and plasticity. During the sexual cycle, development of the somatic macronucleus involves elimination of ∼30% of the germline genome, including repeated DNA (e.g., transposons) and ∼45,000 single-copy internal eliminated sequences (IES). IES excision is a precise cut-and-close process, in which double-stranded DNA cleavage at IES ends depends on PiggyMac, a domesticated piggyBac transposase. Genome-wide analysis has revealed that at least a fraction of IESs originate from Tc / mariner transposons unrelated to piggyBac . Moreover, genomic sequences with no transposon origin, such as gene promoters, can be excised reproducibly as IESs, indicating that genome rearrangements contribute to the control of gene expression. How the system has evolved to allow elimination of DNA sequences with no recognizable conserved motif has been the subject of extensive research during the past two decades. Increasing evidence has accumulated for the participation of noncoding RNAs in epigenetic control of elimination for a subset of IESs, and in trans -generational inheritance of alternative rearrangement patterns. This chapter summarizes our current knowledge of the structure of the germline and somatic genomes for the model species Paramecium tetraurelia , and describes the DNA cleavage and repair factors that constitute the IES excision machinery. We present an overview of the role of specialized RNA interference machineries and their associated noncoding RNAs in the control of DNA elimination. Finally, we discuss how RNA-dependent modification and/or remodeling of chromatin may guide PiggyMac to its cognate cleavage sites.
Published: 2014

29. IS911-mediated Transpositional Recombinationin Vitro

Author: Mireille Bétermier, Michael Chandler, Patrice Polard, Laurence Haren, Bao Ton-Hoang, and Robert Walczak
Subjects: DNA, Bacterial, Transposable element, Shigella dysenteriae, Transposases, Peptide, Biology, Cleavage (embryo), Models, Biological, Bacterial cell structure, Plasmid, Bacterial Proteins, Structural Biology, Escherichia coli, Insertion sequence, Molecular Biology, Recombination, Genetic, chemistry.chemical_classification, Base Sequence, Integrases, Escherichia coli Proteins, Molecular biology, Cell biology, Retroviridae, chemistry, DNA Nucleotidyltransferases, Mutation, DNA Transposable Elements, Nucleic Acid Conformation, Recombination, Plasmids
Abstract: A cell-free system is described that accomplishes an unusual type of transposition/recombination involving the bacterial insertion sequence IS911. Using a plasmid substrate carrying a derivative of IS911, we show that bacterial cell extracts enriched for the IS911 transposase, OrfAB, carry out a single-strand cleavage and transfer reaction. This results in the formation of a figure-eight molecule in which a single strand of the element is circularized, faithfully reproducing an event previously detected in vivo. Moreover, when presented with a figure-eight substrate, OrfAB is capable of "reversing" strand transfer. This activity is equivalent to the "disintegration" reaction carried out by retroviral integrases. We demonstrate that the domain of OrfAB responsible for this catalytic activity is located in the carboxy-terminal region of the protein, since a peptide composed of this region retains disintegration activity. The OrfAB-mediated excision-circularization process previously observed in vivo was proposed to proceed via a figure-eight intermediate by circularization of the second transposon strand. The absence of transposon circles in cell-free reaction suggests either that the figure-eight form is not an intermediate or that additional host factors are required that are eliminated from the cell extract. Two types of model, replicative and non-replicative, are discussed to explain how the figure-eight molecule could be processed into the transposon circle.
Published: 1996

30. Mutual stabilisation of bacteriophage Mu repressor and histone-like proteins in a nucleoprotein structure

Author: Robert Alazard, Mireille Bétermier, Philippe Rousseau, and Michael Chandler
Subjects: Integration Host Factors, Protein Conformation, Molecular Sequence Data, HU Protein, Repressor, Biology, Polymerase Chain Reaction, Substrate Specificity, Bacteriophage mu, Histones, chemistry.chemical_compound, Protein structure, Bacterial Proteins, Structural Biology, Consensus Sequence, Escherichia coli, Animals, Deoxyribonuclease I, Binding site, Molecular Biology, DNA Primers, Binding Sites, Base Sequence, High Mobility Group Proteins, Molecular biology, Recombinant Proteins, biological factors, Rats, Nucleoprotein, DNA-Binding Proteins, Models, Structural, Repressor Proteins, Kinetics, Nucleoproteins, chemistry, DNA, Viral, Mutagenesis, Site-Directed, Biophysics, Nucleic Acid Conformation, bacteria, Electrophoresis, Polyacrylamide Gel, Bacteriophage Mu, DNA, Plasmids
Abstract: Integration host factor (IHF) binds in a sequence-specific manner to the bacteriophage Mu early operator. It participates with bound Mu repressor, c, in building stable, large molecular mass nucleoprotein complexes in vitro and enhances repression of early transcription in vivo. We demonstrate that, when the specific IHF binding site with the operator is mutated, the appearance of large molecular mass complexes still depends on IHF and c, but the efficiency of their formation is reduced. Moreover, the IHF-like HU protein, which binds DNA in a non-sequence-specific way, can substitute for IHF and participate in complex formation. Since the complexes require both c and a host factor (IHF or HU), the results imply that these proteins stabilise each other within the nucleoprotein structures. These results suggest that IHF and HU are directed to the repressor-operator complexes, even in the absence of detectable sequence-specific binding. This could be a consequence of their preferential recognition of DNA containing a distortion such as that introduced by repressor binding to the operator. The histone-like proteins could then stabilise the nucleoprotein complexes simply by their capacity to maintain a bend in DNA rather than by specific protein-protein interactions with c. This model is supported by the observation that the unrelated eukaryotic HMG-1 protein, which exhibits a similar marked preference for structurally deformed DNA, is also able to participate in the formation of higher-order complexes with c and the operator DNA.
Published: 1995

31. The Paramecium Germline Genome Provides a Niche for Intragenic Parasitic DNA: Evolutionary Dynamics of Internal Eliminated Sequences

Author: M Prajer, Mariusz Nowacki, Sandra Duharcourt, Benjamin E. Lauderdale, Patrick Wincker, Céline Baudry, Mireille Bétermier, Jean-Marc Aury, Olivier Garnier, Nathalie Mathy, Karine Labadie, Olivier Arnaiz, Antoine Marmignon, Anne Le Mouël, Sophie Malinsky, Cyril Denby Wilkes, Eric Meyer, Julie Poulain, Linda Sperling, Laurent Duret, Centre de génétique moléculaire (CGM), Université Paris-Sud - Paris 11 (UP11)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Génomique d'Evry (IG), Université Paris-Saclay-Institut de Biologie François JACOB (JACOB), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Methodology Institute, London School of Economics, London School of Economics and Political Science (LSE), Universität Bern [Bern] (UNIBE), Department of Experimental Zoology, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Polska Akademia Nauk = Polish Academy of Sciences (PAN), Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Bioinformatique, phylogénie et génomique évolutive (BPGE), Département PEGASE [LBBE] (PEGASE), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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 Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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), CNRS ATIP-Plus grant, FRM, Centre National de Séquençage (Convention GENOSCOPE-CEA number 128/AP 2007_2008/CNRS number 028666), Ministère de l'Enseignement Supérieur et de la Recherche, Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie François JACOB (JACOB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Institute of Cell Biology, University of Bern, University of Bern, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Polska Akademia Nauk (PAN), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Cancer Research, Paramecium, Gene Dosage, Genome, DNA transposons, Molecular cell biology, INDEL Mutation, Gene Order, Nuclear dimorphism, Genome Sequencing, Genome Evolution, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Genetics (clinical), Transposase, Conserved Sequence, Genetics, Gene Rearrangement, 0303 health sciences, Base Composition, Macronucleus, [SDV.BID.EVO]Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], 030302 biochemistry & molecular biology, Genomics, Nucleic acids, Paramecium tetraurelia, Sequence Analysis, Transposons, Research Article, Transposable element, lcsh:QH426-470, DNA recombination, Molecular Sequence Data, DNA repair, Biology, Evolution, Molecular, Molecular Genetics, 03 medical and health sciences, Amino Acid Sequence, Selection, Genetic, Molecular Biology, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, Evolutionary Biology, Models, Genetic, Computational Biology, Genomic Evolution, Gene rearrangement, DNA, DNA, Protozoan, Comparative Genomics, lcsh:Genetics, Germ Cells, DNA Transposable Elements, Programmed DNA elimination, Genome, Protozoan, Sequence Alignment
Abstract: Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of ∼45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a ∼10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated., Author Summary Ciliates are unicellular eukaryotes that rearrange their genomes at every sexual generation when a new somatic macronucleus, responsible for gene expression, develops from a copy of the germline micronucleus. In Paramecium, assembly of a functional somatic genome requires precise excision of interstitial DNA segments, the Internal Eliminated Sequences (IES), involving a domesticated piggyBac transposase, PiggyMac. To study IES origin and evolution, we sequenced germline DNA and identified 45,000 IESs. We found that at least some of these unique-copy elements are decayed Tc1/mariner transposons and that IES insertion is likely an ongoing process. After insertion, elements decay rapidly by accumulation of deletions and substitutions. The 93% of IESs shorter than 150 bp display a remarkable size distribution with a periodicity of 10 bp, the helical repeat of double-stranded DNA, consistent with the idea that evolution has only retained IESs that can form a double-stranded DNA loop during assembly of an excision complex. We propose that the ancient domestication of a piggyBac transposase, which provided a precise excision mechanism, enabled transposons to subsequently invade Paramecium coding sequences, a fraction of the genome that does not usually tolerate parasitic DNA.
Published: 2012

32. Transposon Invasion of the Paramecium Germline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway

Author: Julien Bischerour, Emeline Dubois, Nathalie Mathy, Vinciane Régnier, Mireille Bétermier, Antoine Marmignon, Centre de génétique moléculaire (CGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot, Sorbonne Paris Cité, Paris, France, Université Paris Diderot - Paris 7 (UPD7), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), ANR-08-BLAN-0233,ParaDice,Epissage d'ARN et d'ADN : comment la Paramécie annote son génome(2008), and ANR-10-BLAN-1603,GENOMAC,Analyse à grande échelle du positionnement des nucléosomes dans les noyaux de Paramecium tetraurelia(2010)
Subjects: Genetics, Transposable element, 0303 health sciences, Macronucleus, biology, 030302 biochemistry & molecular biology, Tetrahymena, Review Article, biology.organism_classification, Genome, [SDV.MP.PRO]Life Sciences [q-bio]/Microbiology and Parasitology/Protistology, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Nuclear dimorphism, Paramecium, Programmed DNA elimination, Transposase, 030304 developmental biology
Abstract: Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus), ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliateParamecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists betweenParameciumIESs andTc1/marinertransposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticatedpiggyBactransposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate,Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of apiggyBactransposase might have allowedTc1/marinerelements to spread throughout the germline genome ofParamecium, without strong counterselection against insertion within genes.
Published: 2012

33. Interaction of Fis protein with DNA: Bending and specificity of binding

Author: David J. Galas, Michael Chandler, and Mireille Bétermier
Subjects: Electrophoresis, Integration Host Factors, Conformational change, Chemistry, General Medicine, Plasma protein binding, Factor For Inversion Stimulation Protein, Peptide Mapping, Biochemistry, DNA-binding protein, DNA-Binding Proteins, Crystallography, chemistry.chemical_compound, Consensus sequence, Biophysics, Deoxyribonuclease I, Nucleic Acid Conformation, Binding site, Carrier Proteins, DNA, Plasmids, Protein Binding
Abstract: The Escherichia coli Fis protein is a dimeric DNA-binding protein whose specific binding sites share a weak consensus sequence. Use of the gel retardation technique indicates that binding of Fis on a linear DNA fragment leads to the formation of a ladder of defined retarded complexes, independently of the presence of a specific site. This non-specific binding of Fis is consistent with a model where equivalent low-affinity sites on a given fragment would be bound randomly and independently of each other by consecutive Fis dimers. Evidence is presented that non-specific binding of Fis can, however, induce an apparent site-specific conformational change in the DNA. This observation is discussed in terms of a model in which each Fis:DNA complex detected in gel retardation experiments actually represents a dynamic equilibrium of a fixed number of Fis dimers distributed on the fragment.
Published: 1994

34. Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining

Author: Eric Meyer, Antoine Marmignon, James D. Forney, Aude Silve, Mireille Bétermier, Sophie Malinsky, Atsushi Matsuda, Michael Ku, Aurélie Kapusta, Centre de génétique moléculaire (CGM), Université Paris-Sud - Paris 11 (UP11)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Department of Biochemistry, Purdue University [West Lafayette], Institut de biologie de l'ENS Paris (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), This work was supported by: Centre National de la Recherche Scientifique (CNRS) (ATIP Plus grant to M Bétermier) (http://www.cnrs.fr/), Agence Nationale de la Recherche (grant BLAN08-3_310945 to M Bétermier and E Meyer) (http://www.agence-nationale-recherche.fr/), National Science Foundation (grant MCB-9506009 to JD Forney)(http://www.nsf.gov/), Ministere de l'Enseignement Superieur et de la Recherche (PhD fellowships to A Kapusta and A Marmignon) (http://www.enseignementsup-recherche.gouv.fr/), Fondation pour la Recherche Medicale (PhD fellowship to A Kapusta and Equipe FRM grant to E Meyer) (http://www.frm.org/), and an EMBO short-term fellowship to A Kapusta (http://www.embo.org/programmes/fellowships/short-term.html)., Autard, Delphine, Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Cancer Research, MESH: Sequence Analysis, DNA, Paramecium, DNA Repair, Transcription, Genetic, MESH: DNA Breaks, Double-Stranded, Protozoan Proteins, MESH: DNA Replication, [SDV.GEN] Life Sciences [q-bio]/Genetics, LIG4, Protozoology, Gene Splicing, DNA transposons, DNA Ligase ATP, Molecular cell biology, DNA amplification, DNA Breaks, Double-Stranded, MESH: Phylogeny, MESH: Protozoan Proteins, Genetics (clinical), Transposase, Phylogeny, Genetics, chemistry.chemical_classification, MESH: DNA Repair, 0303 health sciences, Chromosome Biology, 030302 biochemistry & molecular biology, Genomics, MESH: Genome, Protozoan, DNA repair protein XRCC4, Functional Genomics, Non-homologous end joining, Nucleic acids, MESH: DNA Transposable Elements, RNA Interference, Transposons, Research Article, DNA Replication, MESH: DNA Ligases, Chromosome Structure and Function, lcsh:QH426-470, DNA Ligases, DNA repair, DNA recombination, Recombinant Fusion Proteins, MESH: RNA Interference, MESH: DNA, Protozoan, Biology, Microbiology, Molecular Genetics, 03 medical and health sciences, Model Organisms, MESH: Recombinant Fusion Proteins, Gene Regulation, Molecular Biology, Gene, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, DNA ligase, MESH: Paramecium, [SDV.GEN]Life Sciences [q-bio]/Genetics, Protozoan Models, MESH: Transcription, Genetic, DNA replication, DNA, Sequence Analysis, DNA, DNA, Protozoan, lcsh:Genetics, chemistry, DNA Transposable Elements, Gene Function, Genome, Protozoan
Abstract: During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics., Author Summary Double-strand breaks (DSBs) are among the most deleterious lesions that may occur on DNA. Some physiological processes, however, involve the introduction of DSBs and their subsequent repair. In the ciliate Paramecium, programmed DSBs initiate the extensive genome rearrangements that take place at each sexual cycle, during the development of the somatic nucleus. In particular, short intervening germline sequences (one every 1–2 kb along the genome) are spliced out from coding and non-coding regions. In this study, we present evidence that this process is a two-step mechanism and involves DNA cleavage at both ends of each excised sequence, followed by DSB repair. We demonstrate that cellular end-joining proteins, Ligase IV and its partner, Xrcc4p, are essential for the closure of broken excision sites, which has to be precise at the nucleotide level to allow the assembly of functional genes. This precision stands in sharp contrast to the notion that end joining is an error-prone DSB repair pathway. Therefore, Paramecium provides an excellent model for analysis of an intrinsically precise end joining pathway that has been recruited for genome-wide DSB repair.
Published: 2011

35. Functional Study of Genes Essential for Autogamy and Nuclear Reorganization in Paramecium▿§

Author: Mireille Bétermier, Thomas Tang, Jacek K. Nowak, Hervé Delacroix, Olivier Arnaiz, Marek Zagulski, Jean Cohen, Nicolas Agier, Robert Gromadka, C. J. Herbert, Linda Sperling, Marek Juszczuk, Marie-Hélène Mucchielli, Maria Jerka-Dziadosz, Lawrence P. Aggerbeck, Kamila Maliszewska, and Jean-François Gout
Subjects: Regulation of gene expression, Genetics, Macronucleus, Protozoan Proteins, Gene Expression Regulation, Developmental, General Medicine, Articles, Self-Fertilization, Biology, Microbiology, Genome, Karyogamy, Transcriptome, Gene expression, Paramecium tetraurelia, Molecular Biology, Gene, Micronucleus, Germline
Abstract: Like all ciliates, Paramecium tetraurelia is a unicellular eukaryote that harbors two kinds of nuclei within its cytoplasm. At each sexual cycle, a new somatic macronucleus (MAC) develops from the germ line micronucleus (MIC) through a sequence of complex events, which includes meiosis, karyogamy, and assembly of the MAC genome from MIC sequences. The latter process involves developmentally programmed genome rearrangements controlled by noncoding RNAs and a specialized RNA interference machinery. We describe our first attempts to identify genes and biological processes that contribute to the progression of the sexual cycle. Given the high percentage of unknown genes annotated in the P. tetraurelia genome, we applied a global strategy to monitor gene expression profiles during autogamy, a self-fertilization process. We focused this pilot study on the genes carried by the largest somatic chromosome and designed dedicated DNA arrays covering 484 genes from this chromosome (1.2% of all genes annotated in the genome). Transcriptome analysis revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 upregulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old and then to the new MAC, are specifically required during sexual processes. Our work provides a proof of principle that genes essential for meiosis and nuclear reorganization can be uncovered following genome-wide transcriptome analysis.
Published: 2011

36. Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling

Author: Eric Meyer, Mireille Bétermier, Khaled Bouhouche, Aurélie Kapusta, Jean-François Gout, École normale supérieure - Cachan (ENS Cachan), Institut National de la Recherche Agronomique (INRA), Université de Lyon (COMUE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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), Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11), Agence Nationale de la Recherche [ANR-08-BLAN-0233], Fondation pour la Recherche Medicale, Centre National de la Recherche Scientifique, and Ministere de l'Education Superieure et de la Recherche
Subjects: EXPRESSION, [SDV]Life Sciences [q-bio], Protozoan Proteins, Piwi-interacting RNA, DEVELOPMENTALLY PROGRAMMED EXCISION, INTERNAL ELIMINATED SEQUENCES, DIRECTED DNA METHYLATION, RNA-BINDING PROTEIN, TETRAHYMENA-THERMOPHILA, DROSOPHILA-MELANOGASTER, C-ELEGANS, MEMBRANE-FUSION, ARGONAUTE, Biology, 03 medical and health sciences, 0302 clinical medicine, RNA interference, Genetics, RasiRNA, Paramecium, Transgenes, Gene, Molecular Biology, Phylogeny, 030304 developmental biology, RNA, Double-Stranded, 0303 health sciences, Genome, Macronucleus, Argonaute, biology.organism_classification, Paramecium tetraurelia, RNA Interference, 030217 neurology & neurosurgery
Abstract: International audience; Proteins of the Argonaute family are small RNA carriers that guide regulatory complexes to their targets. The family comprises two major subclades. Members of the Ago subclade, which are present in most eukaryotic phyla, bind different classes of small RNAs and regulate gene expression at both transcriptional and post-transcriptional levels. Piwi subclade members appear to have been lost in plants and fungi and were mostly studied in metazoa, where they bind piRNAs and have essential roles in sexual reproduction. Their presence in ciliates, unicellular organisms harbouring both germline micronuclei and somatic macronuclei, offers an interesting perspective on the evolution of their functions. Here, we report phylogenetic and functional analyses of the 15 Piwi genes from Paramecium tetraurelia. We show that four constitutively expressed proteins are involved in siRNA pathways that mediate gene silencing throughout the life cycle. Two other proteins, specifically expressed during meiosis, are required for accumulation of scnRNAs during sexual reproduction and for programmed genome rearrangements during development of the somatic macronucleus. Our results indicate that Paramecium Piwi proteins have evolved to perform both vegetative and sexual functions through mechanisms ranging from post-transcriptional mRNA cleavage to epigenetic regulation of genome rearrangements.
Published: 2011

37. Gene expression in a paleopolyploid: a transcriptome resource for the ciliate Paramecium tetraurelia

Author: Aurélie Kapusta, Jean-François Gout, Eric Meyer, Laurent Duret, Jean Cohen, Khaled Bouhouche, Mireille Bétermier, Olivier Arnaiz, Linda Sperling, Centre de génétique moléculaire (CGM), Université Paris-Sud - Paris 11 (UP11)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Bioinformatique, phylogénie et génomique évolutive (BPGE), Département PEGASE [LBBE] (PEGASE), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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 Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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), Department of biology, Indiana University [Bloomington], Indiana University System-Indiana University System, Institut de biologie de l'ENS Paris (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), The authors acknowledge support from the ANR (Agence National de la Recherche, project ParaDice ANR-08-BLAN-0233), the CNRS (Centre National de la Recherche Scientifique) and an Equipe FRM (Fondation de la Recherche Medicale) grant to EM., BMC, Ed., Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS)
Subjects: Time Factors, MESH: Secretory Vesicles, Genes, Protozoan, Genome, 0302 clinical medicine, Gene Duplication, MESH: Gene Expression Regulation, Developmental, Cluster Analysis, MESH: Animals, Paramecium, MESH: Evolution, Molecular, Oligonucleotide Array Sequence Analysis, Genetics, 0303 health sciences, MESH: Gene Duplication, Gene Expression Regulation, Developmental, MESH: Genes, Protozoan, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Paramecium tetraurelia, DNA microarray, Research Article, Biotechnology, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], MESH: Paramecium tetraurelia, lcsh:QH426-470, lcsh:Biotechnology, Computational biology, Biology, Gene dosage, Evolution, Molecular, Polyploidy, MESH: Gene Expression Profiling, 03 medical and health sciences, MESH: Cilia, lcsh:TP248.13-248.65, MESH: Polyploidy, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Animals, Cilia, Gene, 030304 developmental biology, Gene Expression Profiling, Secretory Vesicles, MESH: Time Factors, biology.organism_classification, MESH: Cluster Analysis, Gene expression profiling, lcsh:Genetics, MESH: Oligonucleotide Array Sequence Analysis, Subfunctionalization, 030217 neurology & neurosurgery
Abstract: Background The genome of Paramecium tetraurelia, a unicellular model that belongs to the ciliate phylum, has been shaped by at least 3 successive whole genome duplications (WGD). These dramatic events, which have also been documented in plants, animals and fungi, are resolved over evolutionary time by the loss of one duplicate for the majority of genes. Thanks to a low rate of large scale genome rearrangement in Paramecium, an unprecedented large number of gene duplicates of different ages have been identified, making this organism an outstanding model to investigate the evolutionary consequences of polyploidization. The most recent WGD, with 51% of pre-duplication genes still in 2 copies, provides a snapshot of a phase of rapid gene loss that is not accessible in more ancient polyploids such as yeast. Results We designed a custom oligonucleotide microarray platform for P. tetraurelia genome-wide expression profiling and used the platform to measure gene expression during 1) the sexual cycle of autogamy, 2) growth of new cilia in response to deciliation and 3) biogenesis of secretory granules after massive exocytosis. Genes that are differentially expressed during these time course experiments have expression patterns consistent with a very low rate of subfunctionalization (partition of ancestral functions between duplicated genes) in particular since the most recent polyploidization event. Conclusions A public transcriptome resource is now available for Paramecium tetraurelia. The resource has been integrated into the ParameciumDB model organism database, providing searchable access to the data. The microarray platform, freely available through NimbleGen Systems, provides a robust, cost-effective approach for genome-wide expression profiling in P. tetraurelia. The expression data support previous studies showing that at short evolutionary times after a whole genome duplication, gene dosage balance constraints and not functional change are the major determinants of gene retention.
Published: 2010

38. Immunocytochemistry of Paramecium cytoskeletal structures

Author: Ching Kung, Janine Beisson, Laurent Duret, Sandra Duharcourt, Eric Meyer, Jean Cohen, John R. Preer, Sophie Malinsky, Marie-Hélène Bré, Linda Sperling, and Mireille Bétermier
Subjects: Paramecium, biology, Immunocytochemistry, Cell Membrane, chemistry.chemical_element, Calcium, biology.organism_classification, Immunohistochemistry, General Biochemistry, Genetics and Molecular Biology, Cell biology, Cell membrane, medicine.anatomical_structure, chemistry, medicine, Homeostasis, Cell shape, Cytoskeleton, Biology, Cell Shape
Abstract: INTRODUCTIONParamecium has a highly complex cellular structure. The cytoskeleton of these organisms interacts with cilia at the cell surface to control cell movement, feeding, and environmental responses. Basal bodies anchored in the cortex organize the cytoskeleton and determine cell shape, and a conspicuous cytoskeletal network, the infraciliary lattice, underlies the cell surface and plays an important role in cellular Ca2+ homeostasis. This protocol describes methods to visualize the cytoskeleton, especially cortical structures. It is not directly applicable for visualization of membrane proteins.
Published: 2010

39. Paramecium tetraurelia: the renaissance of an early unicellular model

Author: Janine Beisson, Ching Kung, John R. Preer, Eric Meyer, Sophie Malinsky, Jean Cohen, Sandra Duharcourt, Mireille Bétermier, Laurent Duret, Linda Sperling, Marie-Hélène Bré, Bioinformatique, phylogénie et génomique évolutive (BPGE), Département PEGASE [LBBE] (PEGASE), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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 Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), and Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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)
Subjects: [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Cellular differentiation, media_common.quotation_subject, Biology, General Biochemistry, Genetics and Molecular Biology, Epigenesis, Genetic, 03 medical and health sciences, 0302 clinical medicine, RNA interference, Macronucleus, Animals, Microscopy, Phase-Contrast, Alleles, 030304 developmental biology, Epigenesis, media_common, 0303 health sciences, Behavior, Animal, Models, Genetic, Reproduction, Homozygote, The Renaissance, Cell Differentiation, Cell biology, Electrophysiology, Fertilization, Models, Animal, Paramecium tetraurelia, RNA Interference, 030217 neurology & neurosurgery
Abstract: INTRODUCTIONParamecium tetraurelia is a widely distributed, free-living unicellular organism that feeds on bacteria and can easily be cultured in the laboratory. Its position within the phylum Ciliophora, remote from the most commonly used models, offers an interesting perspective on the basic cellular and molecular processes of eukaryotic life. Its large size and complex cellular organization facilitate morphogenetic studies of conserved structures, such as cilia and basal bodies, as well as electrophysiological studies of swimming behavior. Like all ciliates, P. tetraurelia contains two distinct types of nuclei, the germline micronucleus (MIC) and the somatic macronucleus (MAC), which differentiate from copies of the zygotic nucleus after fertilization. The sexual cycle can be managed by controlling food uptake, allowing the study of a developmentally regulated differentiation program in synchronous cultures. Spectacular genome rearrangements occur during the development of the somatic macronucleus. Their epigenetic control by RNA-mediated homology-dependent mechanisms, which might underlie long-known cases of non-Mendelian inheritance, provides evolutionary insight into the diversity of small RNA pathways involved in genome regulation. Being endowed with two alternative modes of sexual reproduction (conjugation and autogamy), P. tetraurelia is ideally suited for genetic analyses, and the recent sequencing of its macronuclear genome revealed one of the largest numbers of genes in any eukaryote. Together with the development of new molecular techniques, including complementation cloning and an easily implemented technique for reverse genetics based on RNA interference (RNAi), these features make P. tetraurelia a very attractive unicellular model.
Published: 2010

40. Maintaining clonal Paramecium tetraurelia cell lines of controlled age through daily reisolation

Author: Laurent Duret, Sophie Malinsky, Eric Meyer, Sandra Duharcourt, Janine Beisson, Jean Cohen, John R. Preer, Marie-Hélène Bré, Ching Kung, Linda Sperling, and Mireille Bétermier
Subjects: Time Factors, media_common.quotation_subject, Reproduction, food and beverages, Biology, Immunohistochemistry, Models, Biological, General Biochemistry, Genetics and Molecular Biology, Cell biology, Cell Line, Genetic Techniques, Cell culture, Paramecium tetraurelia, Cell aging, Cellular Senescence, Crosses, Genetic, media_common
Abstract: INTRODUCTIONThe sexual cycle of Paramecium tetraurelia can be managed by controlling food uptake, allowing the study of developmentally regulated differentiation in synchronous cultures. Clonal cell lines can be established easily by single cell isolation and can be grown in bacterized grass infusion medium for a large number of vegetative divisions before entering senescence. Sexual reproduction is triggered by starvation and therefore can be induced or prevented as needed by controlling the availability of food. Given a constant supply of food, P. tetraurelia cells will remain in the vegetative phase of their life cycle and, at 27°C, will divide by binary fission every 6 h. The daily reisolation procedure described here results in populations of P. tetraurelia of known age and produced under reproducible physiological conditions, which can help to standardize experiments. Paramecia produced under these conditions are suitable for immunocytochemical studies or use in genetic cross experiments.
Published: 2010

41. Silencing specific Paramecium tetraurelia genes by feeding double-stranded RNA

Author: John R. Preer, Linda Sperling, Eric Meyer, Jean Cohen, Mireille Bétermier, Sandra Duharcourt, Janine Beisson, Marie-Hélène Bré, Laurent Duret, Ching Kung, and Sophie Malinsky
Subjects: RNA-induced transcriptional silencing, RNA-induced silencing complex, Trans-acting siRNA, RNA, RNA-dependent RNA polymerase, Biology, General Biochemistry, Genetics and Molecular Biology, Cell biology, Antisense RNA, RNA silencing, Phenotype, Genetic Techniques, Paramecium tetraurelia, RNA Interference, Gene Silencing, RNA, Messenger, Promoter Regions, Genetic, Plasmids, RNA, Double-Stranded
Abstract: INTRODUCTIONThis protocol describes an easy and efficient method to knock down gene expression in a homology-dependent manner by feeding Paramecium with bacteria-producing double-stranded (ds) RNA corresponding to a portion of the target gene. The target sequence is cloned in a plasmid between two convergent T7 promoters (the L4440 plasmid) and used to transform an E. coli strain devoid of RNase III activity and expressing the T7 polymerase gene under the control of an inducible Plac promoter (strain HT115-DE3). Inserts corresponding to ~400 bp of the coding sequence to be silenced are recommended; very short inserts (Paramecium tetraurelia cells feed on these bacteria, dsRNA reaches the cytoplasm through unknown mechanisms and triggers a potent RNA interference (RNAi) response, resulting in the degradation of the targeted mRNA. The protocol generally gives a visible phenotype in 24-48 h, depending on the targeted gene, a delay which might correspond to the time necessary for the pre-existing protein stock to be diluted by cell growth. Silencing induced in this manner is only post-transcriptional; when cells are transferred back to normal medium bacterized with Klebsiella pneumoniae, the phenotype usually reverts to wild type within a few cell divisions.
Published: 2010

42. Mass culture of Paramecium tetraurelia

Author: Janine, Beisson, Mireille, Bétermier, Marie-Hélène, Bré, Jean, Cohen, Sandra, Duharcourt, Laurent, Duret, Ching, Kung, Sophie, Malinsky, Eric, Meyer, John R, Preer, and Linda, Sperling
Subjects: Genome, Models, Genetic, Tubulin, Cell Culture Techniques, Temperature, RNA, RNA Interference, Paramecium tetraurelia, Protein Processing, Post-Translational, Epigenesis, Genetic, Mitochondria
Published: 2010

43. Maternal noncoding transcripts antagonize the targeting of DNA elimination by scanRNAs in Paramecium tetraurelia

Author: Mireille Bétermier, Eric Meyer, Gersende Lepère, and Sandra Duharcourt
Subjects: Genetics, Gene Rearrangement, RNA, Untranslated, Macronucleus, Somatic cell, Gene rearrangement, Biology, DNA, Protozoan, biology.organism_classification, Genome, Germline, Cell Line, Nuclear dimorphism, Animals, Paramecium, Programmed DNA elimination, Paramecium tetraurelia, RNA, Small Interfering, Developmental Biology, Research Paper
Abstract: The germline genome of ciliates is extensively rearranged during the development of a new somatic macronucleus from the germline micronucleus, after sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) are precisely excised from coding sequences and intergenic regions. For a subset of IESs, introduction of the IES sequence into the maternal macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus, suggesting that epigenetic regulation of excision involves a global comparison of germline and somatic genomes. ScanRNAs (scnRNAs) produced during micronuclear meiosis by a developmentally regulated RNAi pathway have been proposed to mediate this transnuclear cross-talk. In this study, microinjection experiments provide direct evidence that 25-nucleotide (nt) scnRNAs promote IES excision. We further show that noncoding RNAs are produced from the somatic maternal genome, both during vegetative growth and during sexual events. Maternal inhibition of IES excision is abolished when maternal somatic transcripts containing an IES are targeted for degradation by a distinct RNAi pathway involving 23-nt siRNAs. The results strongly support a scnRNA/macronuclear RNA scanning model in which a natural genomic subtraction, occurring during meiosis between deletion-inducing scnRNAs and antagonistic transcripts from the maternal macronucleus, regulates rearrangements of the zygotic genome.
Published: 2008

44. Developmentally programmed DNA splicing in Paramecium reveals short-distance crosstalk between DNA cleavage sites

Author: Mireille Bétermier, Sophie Malinsky, Eric Meyer, Ariane Gratias, Gersende Lepère, Olivier Garnier, Sarah Rosa, Sandra Duharcourt, Régulation de l'expression génétique (REG), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Duponchelle, Martine
Subjects: Mutant, Cleavage (embryo), Genome, Epigenesis, Genetic, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Macronucleus, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Genetics, Animals, Point Mutation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Paramecium, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, biology, Models, Genetic, Point mutation, Nucleic acid sequence, DNA, Protozoan, biology.organism_classification, AT Rich Sequence, chemistry, RNA splicing, Nucleic Acid Conformation, Paramecium tetraurelia, 030217 neurology & neurosurgery, DNA
Abstract: Somatic genome assembly in the ciliate Paramecium involves the precise excision of thousands of short internal eliminated sequences (IESs) that are scattered throughout the germline genome and often interrupt open reading frames. Excision is initiated by double-strand breaks centered on the TA dinucleotides that are conserved at each IES boundary, but the factors that drive cleavage site recognition remain unknown. A degenerate consensus was identified previously at IES ends and genetic analyses confirmed the participation of their nucleotide sequence in efficient excision. Even for wild-type IESs, however, variant excision patterns (excised or nonexcised) may be inherited maternally through sexual events, in a homology-dependent manner. We show here that this maternal epigenetic control interferes with the targeting of DNA breaks at IES ends. Furthermore, we demonstrate that a mutation in the TA at one end of an IES impairs DNA cleavage not only at the mutant end but also at the wild-type end. We conclude that crosstalk between both ends takes place prior to their cleavage and propose that the ability of an IES to adopt an excision-prone conformation depends on the combination of its nucleotide sequence and of additional determinants.
Published: 2008

45. Translational control of intron splicing in eukaryotes

Author: Khaled Bouhouche, Benjamin Noel, Baptiste Saudemont, Béatrice Segurens, Mariusz Nowacki, Vincent Schächter, Jean-Marc Aury, Olivier Jaillon, Linda Sperling, Patrick Wincker, Jean-François Gout, Betina M. Porcel, Eric Meyer, Jean Cohen, Anne Le Mouël, Mireille Bétermier, Laurent Duret, Gersende Lepère, Vincent Serrano, Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Génomique métabolique (UMR 8030), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'Ecole Normale Supérieure (IBENS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Bioinformatique, phylogénie et génomique évolutive (BPGE), Département PEGASE [LBBE] (PEGASE), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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 Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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), Universität Bern [Bern] (UNIBE), Régulation de l'expression génétique (REG), Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre de génétique moléculaire (CGM), Université Paris-Sud - Paris 11 (UP11)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Savelli, Bruno, Institute of Cell Biology, University of Bern, University of Bern, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris)
Subjects: Paramecium, RNA Stability, Genes, Protozoan, Molecular Sequence Data, Protozoan Proteins, [SDV.GEN] Life Sciences [q-bio]/Genetics, Biology, 03 medical and health sciences, Splicing factor, Exon, 0302 clinical medicine, Minor spliceosome, Animals, Group I catalytic intron, 030304 developmental biology, Expressed Sequence Tags, Genetics, 0303 health sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, Multidisciplinary, Base Sequence, Alternative splicing, Intron, Computational Biology, Group II intron, Introns, Alternative Splicing, Eukaryotic Cells, Protein Biosynthesis, RNA splicing, Codon, Terminator, RNA Interference, RNA, Protozoan, 030217 neurology & neurosurgery
Abstract: Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy.
Published: 2008

46. Analysis of sequence variability in the macronuclear DNA of Paramecium tetraurelia: A somatic view of the germline

Author: Olivier Jaillon, Philippe Dessen, Claire Jubin, Eric Meyer, Jean Cohen, Jean-Marc Aury, Laurent Duret, Olivier Arnaiz, Benjamin Noel, Mireille Bétermier, Patrick Wincker, Linda Sperling, Jean-François Gout, Sylvain Mousset, Bioinformatique, phylogénie et génomique évolutive (BPGE), Département PEGASE [LBBE] (PEGASE), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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 Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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), Sexe et évolution, Department of Psychology (PRINCETON UNIVERSITY), Princeton University, Institut d'Informatique et de Mathématiques Appliquées de Grenoble (IMAG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National Polytechnique de Grenoble (INPG)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Algorithmique Complexité et Logique (LACL), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Centre National de la Recherche Scientifique (CNRS), Institute of Atomic Physics, Université de Roumanie, Architecture, Urbanisme, Sociétés (AUS), Université Paris 8 Vincennes-Saint-Denis (UP8)-Université Paris Nanterre (UPN)-Ministère de la Culture et de la Communication (MCC)-Ecole d'architecture Paris-Belleville-École nationale supérieure d'architecture de Paris-Malaquais (ENSAPM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Coordination nationale des GROG, open Rome, Centre de génétique moléculaire (CGM), Centre National de la Recherche Scientifique (CNRS), Structure et évolution des génomes (SEG), CNS-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Génomes et cancer (GC (FRE2939)), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Génétique Oncologique, Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Enseignement-Formation-Apprentissage (EFA), Université de Mons-Hainaut, Régulation de l'expression génétique (REG), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institute of Physics (NCCR), University of Basel (Unibas), Université Paris 8 Vincennes-Saint-Denis (UP8)-Université Paris Nanterre (UPN)-Ministère de la Culture et de la Communication (MCC)-Ecole d'architecture Paris-Malaquais-Ecole d'architecture Paris-Belleville-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris)
Subjects: MESH: Paramecium tetraurelia, MESH: Sequence Analysis, DNA, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Letter, MESH: DNA, Protozoan, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, MESH: Gene Amplification, Genome, Chromosomes, MESH: Variation (Genetics), 03 medical and health sciences, MESH: Base Composition, Macronucleus, Gene duplication, Genetics, Consensus sequence, Animals, MESH: Animals, MESH: Macronucleus, Gene, Genetics (clinical), Sequence Deletion, 030304 developmental biology, Base Composition, 0303 health sciences, MESH: Micronucleus, Germline, Shotgun sequencing, 030302 biochemistry & molecular biology, Gene Amplification, Genetic Variation, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Sequence Analysis, DNA, Genome project, DNA, Protozoan, MESH: Sequence Deletion, MESH: Chromosomes, Paramecium tetraurelia, Programmed DNA elimination, Micronucleus, Germline
Abstract: Ciliates are the only unicellular eukaryotes known to separate germinal and somatic functions. Diploid but silent micronuclei transmit the genetic information to the next sexual generation. Polyploid macronuclei express the genetic information from a streamlined version of the genome but are replaced at each sexual generation. The macronuclear genome of Paramecium tetraurelia was recently sequenced by a shotgun approach, providing access to the gene repertoire. The 72-Mb assembly represents a consensus sequence for the somatic DNA, which is produced after sexual events by reproducible rearrangements of the zygotic genome involving elimination of repeated sequences, precise excision of unique-copy internal eliminated sequences (IES), and amplification of the cellular genes to high copy number. We report use of the shotgun sequencing data (>106 reads representing 13× coverage of a completely homozygous clone) to evaluate variability in the somatic DNA produced by these developmental genome rearrangements. Although DNA amplification appears uniform, both of the DNA elimination processes produce sequence heterogeneity. The variability that arises from IES excision allowed identification of hundreds of putative new IESs, compared to 42 that were previously known, and revealed cases of erroneous excision of segments of coding sequences. We demonstrate that IESs in coding regions are under selective pressure to introduce premature termination of translation in case of excision failure.
Published: 2008

47. Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia

Author: Claude Scarpelli, Jacek K. Nowak, Jean-Marc Aury, Janine Beisson, Isabelle Blanc, Nathalie Aiach, Vincent Serrano, Anne-Marie Keller, Khaled Bouhouche, Anne Le Mouël, Helmut Plattner, Catherine Klotz, Marek Zagulski, Françoise Ruiz, Vincent Daubin, Jean Weissenbach, Mireille Bétermier, Olivier Arnaiz, Michael Katinka, Julie Poulain, Patrick Wincker, Olivier Jaillon, Delphine Gogendeau, Laurent Duret, Béatrice Ségurens, Véronique Anthouard, Vincent Schächter, Gersende Lepère, Francisco Câmara, Mariusz Nowacki, Roland Kissmehl, Sandra Duharcourt, Philippe Dessen, Claire Jubin, Betina M. Porcel, Eric Meyer, Jean Cohen, Linda Sperling, Alain Billaut, Benjamin Noel, Sophie Malinsky, Roderic Guigó, Structure et évolution des génomes (SEG), CNS-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Bioinformatique, phylogénie et génomique évolutive (BPGE), Département PEGASE [LBBE] (PEGASE), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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 Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-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), Centre de génétique moléculaire (CGM), Centre National de la Recherche Scientifique (CNRS), Régulation de l'expression génétique (REG), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Center for Genomic Regulation (CRG-UPF), CIBER de Epidemiología y Salud Pública (CIBERESP), Department of Biology, Konstanz, University of Konstanz, Institute of Biochemistry and Biophysics, DNA Sequencing Laboratory, Warsaw, Polska Akademia Nauk = Polish Academy of Sciences (PAN), Génomes et cancer (GC (FRE2939)), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Consortium National de Recherche en Génomique, CNRS, the Polish Ministry of Science and the Agence Nationale de la Recherche (ANR), École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris)
Subjects: 0106 biological sciences, MESH: Paramecium tetraurelia, Genes, Protozoan, Molecular Sequence Data, Biology, 010603 evolutionary biology, 01 natural sciences, Genome, Gene dosage, MESH: Eukaryotic Cells, Evolution, Molecular, 03 medical and health sciences, Molecular evolution, Genes, Duplicate, Gene Duplication, ddc:570, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Gene duplication, Animals, MESH: Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Phylogeny, Gene, Phylogeny, MESH: Evolution, Molecular, 030304 developmental biology, Genetics, 0303 health sciences, Multidisciplinary, MESH: Molecular Sequence Data, MESH: Genes, Duplicate, MESH: Genomics, MESH: Gene Duplication, Genomics, MESH: Genome, Protozoan, Paleopolyploidy, Eukaryotic Cells, MESH: Genes, Protozoan, Paramecium tetraurelia, Programmed DNA elimination, Genome, Protozoan
Abstract: The duplication of entire genomes has long been recognized as having great potential for evolutionary novelties, but the mechanisms underlying their resolution through gene loss are poorly understood. Here we show that in the unicellular eukaryote Paramecium tetraurelia, a ciliate, most of the nearly 40,000 genes arose through at least three successive whole-genome duplications. Phylogenetic analysis indicates that the most recent duplication coincides with an explosion of speciation events that gave rise to the P. aurelia complex of 15 sibling species. We observed that gene loss occurs over a long timescale, not as an initial massive event. Genes from the same metabolic pathway or protein complex have common patterns of gene loss, and highly expressed genes are over-retained after all duplications. The conclusion of this analysis is that many genes are maintained after whole-genome duplication not because of functional innovation but because of gene dosage constraints.
Published: 2006

48. Developmentally programmed excision of internal DNA sequences in Paramecium aurelia

Author: Mireille Bétermier and Ariane Gratias
Subjects: Genetics, Cell Nucleus, Gene Rearrangement, Paramecium, biology, Macronucleus, Models, Genetic, Chromosome, General Medicine, Gene rearrangement, DNA Fragmentation, DNA, Protozoan, biology.organism_classification, Biochemistry, Genome, Chromosomes, chemistry.chemical_compound, chemistry, Animals, Site-specific recombination, DNA, Gene Deletion, Paramecium aurelia
Abstract: The development of a new somatic nucleus (macronucleus) during sexual reproduction of the ciliate Paramecium aurelia involves reproducible chromosomal rearrangements that affect the entire germline genome. Macronuclear development can be induced experimentally, which makes P. aurelia an attractive model for the study of the mechanism and the regulation of DNA rearrangements. Two major types of rearrangements have been identified: the fragmentation of the germline chromosomes, followed by the formation of the new macronuclear chromosome ends in association with imprecise DNA elimination, and the precise excision of internal eliminated sequences (IESs). All IESs identified so far are short, A/T rich and non-coding elements. They are flanked by a direct repeat of a 5'-TA-3' dinucleotide, a single copy of which remains at the macronuclear junction after excision. The number of these single-copy sequences has been estimated to be around 60,000 per haploid genome. This review focuses on the current knowledge about the genetic and epigenetic determinants of IES elimination in P. aurelia, the analysis of excision products, and the tightly regulated timing of excision throughout macronuclear development. Several models for the molecular mechanism of IES excision will be discussed in relation to those proposed for DNA elimination in other ciliates.
Published: 2002

49. Is Non-Homologous End-Joining Really an Inherently Error-Prone Process?

Author: Pascale Bertrand, Bernard S. Lopez, and Mireille Bétermier
Subjects: Genome instability, Cancer Research, DNA End-Joining Repair, DNA Ligases, DNA Repair, lcsh:QH426-470, Clinical Research Design, DNA repair, DNA damage, Immunology, Genomics, Review, Computational biology, Biology, Toxicology, Microbiology, Genomic Instability, Model Organisms, Radiation, Ionizing, Molecular Cell Biology, Genetics, DNA Breaks, Double-Stranded, Theoretical Biology, Molecular Biology, Genetics (clinical), Ecology, Evolution, Behavior and Systematics, Recombination, Genetic, Clinical Genetics, chemistry.chemical_classification, Evolutionary Biology, DNA ligase, fungi, Radiobiology, DNA repair protein XRCC4, DNA-Binding Proteins, Non-homologous end joining, lcsh:Genetics, enzymes and coenzymes (carbohydrates), Oncology, chemistry, embryonic structures, Medicine, Clinical Immunology, DNA Damage, Biotechnology
Abstract: DNA double-strand breaks (DSBs) are harmful lesions leading to genomic instability or diversity. Non-homologous end-joining (NHEJ) is a prominent DSB repair pathway, which has long been considered to be error-prone. However, recent data have pointed to the intrinsic precision of NHEJ. Three reasons can account for the apparent fallibility of NHEJ: 1) the existence of a highly error-prone alternative end-joining process; 2) the adaptability of canonical C-NHEJ (Ku- and Xrcc4/ligase IV–dependent) to imperfect complementary ends; and 3) the requirement to first process chemically incompatible DNA ends that cannot be ligated directly. Thus, C-NHEJ is conservative but adaptable, and the accuracy of the repair is dictated by the structure of the DNA ends rather than by the C-NHEJ machinery. We present data from different organisms that describe the conservative/versatile properties of C-NHEJ. The advantages of the adaptability/versatility of C-NHEJ are discussed for the development of the immune repertoire and the resistance to ionizing radiation, especially at low doses, and for targeted genome manipulation.
Published: 2014

50. Timing of Developmentally Programmed Excision and Circularization of Paramecium Internal Eliminated Sequences

Author: Hervé Seitz, Sandra Duharcourt, Eric Meyer, Mireille Bétermier, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-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), Régulation de l'expression génétique (REG), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS 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), École normale supérieure - Paris (ENS-PSL), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL)
Subjects: Paramecium, [SDV]Life Sciences [q-bio], 03 medical and health sciences, chemistry.chemical_compound, Extrachromosomal DNA, Nuclear dimorphism, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Southern blot, 0303 health sciences, Macronucleus, biology, 030302 biochemistry & molecular biology, Cell Biology, DNA, Protozoan, biology.organism_classification, DNA Dynamics and Chromosome Structure, Molecular biology, genomic DNA, Gene Expression Regulation, chemistry, Programmed DNA elimination, DNA
Abstract: Paramecium internal eliminated sequences (IESs) are short AT-rich DNA elements that are precisely eliminated from the germ line genome during development of the somatic macronucleus. They are flanked by one 5'-TA-3' dinucleotide on each side, a single copy of which remains at the donor site after excision. The timing of their excision was examined in synchronized conjugating cells by quantitative PCR. Significant amplification of the germ line genome was observed prior to IES excision, which starts 12 to 14 h after initiation of conjugation and extends over a 2- to 4-h period. Following excision, two IESs were shown to form extrachromosomal circles that can be readily detected on Southern blots of genomic DNA from cells undergoing macronuclear development. On these circular molecules, covalently joined IES ends are separated by one copy of the flanking 5'-TA-3' repeat. The similar structures of the junctions formed on the excised and donor molecules point to a central role for this dinucleotide in IES excision.
Published: 2000

Catalog

Books, media, physical & digital resources

See catalog results

Searchworks

Select search scope, currently: Articles Catalog books, media & more in Jio Institute collections Articles journal articles & other e-resources

Search

Search Constraints

Refine your results

Search Limiters

Topic

Publication Year Range

Language

Publication Type

Journal

Database

Publisher

67 results on '"Mireille Bétermier"'

Search Results

Catalog

Select search scope, currently: Articles

Catalog

books, media & more in Jio Institute collections

Articles

journal articles & other e-resources