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1.
Genome Res ; 32(4): 699-709, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35264448

RESUMO

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.


Assuntos
Nucleossomos , Paramecium , Composição de Bases , Éxons , Íntrons/genética , Nucleossomos/genética , Paramecium/genética , Splicing de RNA/genética
2.
Mol Biol Evol ; 40(5)2023 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-37154524

RESUMO

Whole-genome duplications (WGDs) have shaped the gene repertoire of many eukaryotic lineages. The redundancy created by WGDs typically results in a phase of massive gene loss. However, some WGD-derived paralogs are maintained over long evolutionary periods, and the relative contributions of different selective pressures to their maintenance are still debated. Previous studies have revealed a history of three successive WGDs in the lineage of the ciliate Paramecium tetraurelia and two of its sister species from the Paramecium aurelia complex. Here, we report the genome sequence and analysis of 10 additional P. aurelia species and 1 additional out group, revealing aspects of post-WGD evolution in 13 species sharing a common ancestral WGD. Contrary to the morphological radiation of vertebrates that putatively followed two WGD events, members of the cryptic P. aurelia complex have remained morphologically indistinguishable after hundreds of millions of years. Biases in gene retention compatible with dosage constraints appear to play a major role opposing post-WGD gene loss across all 13 species. In addition, post-WGD gene loss has been slower in Paramecium than in other species having experienced genome duplication, suggesting that the selective pressures against post-WGD gene loss are especially strong in Paramecium. A near complete lack of recent single-gene duplications in Paramecium provides additional evidence for strong selective pressures against gene dosage changes. This exceptional data set of 13 species sharing an ancestral WGD and 2 closely related out group species will be a useful resource for future studies on Paramecium as a major model organism in the evolutionary cell biology.


Assuntos
Duplicação Gênica , Paramecium , Animais , Paramecium/genética , Genoma , Dosagem de Genes , Vertebrados/genética , Evolução Molecular , Filogenia
3.
Trends Genet ; 37(10): 882-889, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34210514

RESUMO

It is generally considered that Polycomb Repressive Complex (PRC)2 deposits the histone mark H3K27me3 on silent protein-coding genes, while transposable elements are repressed by DNA and/or H3K9 methylation. Yet, there is increasing evidence that PRC2 also targets and even silences transposable elements in representatives of several distantly related eukaryotic lineages. In plants and animals, H3K27me3 is present on transposable elements in mutants and specific cell types devoid of DNA methylation. In this Opinion, we summarize the experimental evidence for this phenomenon across the eukaryotic kingdom, and discuss its functional and evolutionary significance. We hypothesize that an ancestral role of Polycomb group (PcG) proteins was to silence transposable elements.


Assuntos
Elementos de DNA Transponíveis/genética , Proteínas do Grupo Polycomb/metabolismo , Animais , Metilação de DNA , Histonas/metabolismo , Humanos , Complexo Repressor Polycomb 2/metabolismo
4.
PLoS Biol ; 19(7): e3001309, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34324490

RESUMO

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression.


Assuntos
Éxons , Genoma de Protozoário , Células Germinativas , Paramecium tetraurellia/genética , Proteínas de Protozoários/genética , Elementos de DNA Transponíveis , Evolução Molecular
5.
PLoS Genet ; 16(7): e1008949, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32702045

RESUMO

In Paramecium tetraurelia, a large proportion of the germline genome is reproducibly removed from the somatic genome after sexual events via a process involving small (s)RNA-directed heterochromatin formation and DNA excision and repair. How germline limited DNA sequences are specifically recognized in the context of chromatin remains elusive. Here, we use a reverse genetics approach to identify factors involved in programmed genome rearrangements. We have identified a P. tetraurelia homolog of the highly conserved histone chaperone Spt16 subunit of the FACT complex, Spt16-1, and show its expression is developmentally regulated. A functional GFP-Spt16-1 fusion protein localized exclusively in the nuclei where genome rearrangements take place. Gene silencing of Spt16-1 showed it is required for the elimination of all germline-limited sequences, for the survival of sexual progeny, and for the accumulation of internal eliminated sequence (ies)RNAs, an sRNA population produced when elimination occurs. Normal accumulation of 25 nt scanRNAs and deposition of silent histone marks H3K9me3 and H3K27me3 indicated that Spt16-1 does not regulate the scanRNA-directed heterochromatin pathway involved in the early steps of DNA elimination. We further show that Spt16-1 is required for the correct nuclear localization of the PiggyMac (Pgm) endonuclease, which generates the DNA double-strand breaks required for DNA elimination. Thus, Spt16-1 is essential for Pgm function during programmed genome rearrangements. We propose a model in which Spt16-1 mediates interactions between the excision machinery and chromatin, facilitating endonuclease access to DNA cleavage sites during genome rearrangements.


Assuntos
Núcleo Celular/genética , Chaperonas de Histonas/genética , Paramecium/genética , Transposases/genética , Sequência de Bases/genética , Quebras de DNA de Cadeia Dupla , Clivagem do DNA , DNA de Protozoário/genética , Endonucleases , Rearranjo Gênico/genética , Genoma de Protozoário/genética , Paramecium/crescimento & desenvolvimento
6.
Bioinformatics ; 37(17): 2738-2740, 2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-33471071

RESUMO

MOTIVATION: Long-read sequencing technologies can be employed to detect and map DNA modifications at the nucleotide resolution on a genome-wide scale. However, published software packages neglect the integration of genomic annotation and comprehensive filtering when analyzing patterns of modified bases detected using Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) data. Here, we present DNA Modification Annotation (DNAModAnnot), a R package designed for the global analysis of DNA modification patterns using adapted filtering and visualization tools. RESULTS: We tested our package using PacBio sequencing data to analyze patterns of the 6-methyladenine (6mA) in the ciliate Paramecium tetraurelia, in which high 6mA amounts were previously reported. We found P. tetraurelia 6mA genome-wide distribution to be similar to other ciliates. We also performed 5-methylcytosine (5mC) analysis in human lymphoblastoid cells using ONT data and confirmed previously known patterns of 5mC. DNAModAnnot provides a toolbox for the genome-wide analysis of different DNA modifications using PacBio and ONT long-read sequencing data. AVAILABILITY AND IMPLEMENTATION: DNAModAnnot is distributed as a R package available via GitHub (https://github.com/AlexisHardy/DNAModAnnot). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

7.
Nucleic Acids Res ; 48(14): e79, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32496553

RESUMO

Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method - coined 'TraPR' for Trans-kingdom, rapid, affordable Purification of RISCs - operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.


Assuntos
Proteínas Argonautas/metabolismo , Cromatografia Líquida/métodos , RNA Interferente Pequeno/isolamento & purificação , Complexo de Inativação Induzido por RNA/química , Animais , Resinas de Troca Aniônica , Proteínas Argonautas/isolamento & purificação , Linhagem Celular Tumoral , Biblioteca Gênica , Camundongos , Camundongos Endogâmicos C57BL , Polinucleotídeo 5'-Hidroxiquinase , RNA Fúngico/isolamento & purificação , RNA de Helmintos/isolamento & purificação , RNA Neoplásico/isolamento & purificação , RNA de Plantas/isolamento & purificação , RNA de Protozoário/isolamento & purificação , RNA Interferente Pequeno/sangue , RNA Interferente Pequeno/metabolismo , Sefarose , Dióxido de Silício , Ultracentrifugação
8.
Nature ; 509(7501): 447-52, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24805235

RESUMO

In the ciliate Paramecium, transposable elements and their single-copy remnants are deleted during the development of somatic macronuclei from germline micronuclei, at each sexual generation. Deletions are targeted by scnRNAs, small RNAs produced from the germ line during meiosis that first scan the maternal macronuclear genome to identify missing sequences, and then allow the zygotic macronucleus to reproduce the same deletions. Here we show that this process accounts for the maternal inheritance of mating types in Paramecium tetraurelia, a long-standing problem in epigenetics. Mating type E depends on expression of the transmembrane protein mtA, and the default type O is determined during development by scnRNA-dependent excision of the mtA promoter. In the sibling species Paramecium septaurelia, mating type O is determined by coding-sequence deletions in a different gene, mtB, which is specifically required for mtA expression. These independently evolved mechanisms suggest frequent exaptation of the scnRNA pathway to regulate cellular genes and mediate transgenerational epigenetic inheritance of essential phenotypic polymorphisms.


Assuntos
Epigênese Genética/genética , Genoma/genética , Padrões de Herança/genética , Paramecium tetraurellia/genética , RNA Interferente Pequeno/genética , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica , Genes/genética , Dados de Sequência Molecular , Paramecium tetraurellia/fisiologia , Regiões Promotoras Genéticas/genética , Reprodução/genética , Reprodução/fisiologia , Deleção de Sequência/genética
9.
Nucleic Acids Res ; 44(4): 1553-65, 2016 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-26503246

RESUMO

A hallmark of active centromeres is the presence of the histone H3 variant CenH3 in the centromeric chromatin, which ensures faithful genome distribution at each cell division. A functional centromere can be inactivated, but the molecular mechanisms underlying the process of centromere inactivation remain largely unknown. Here, we describe the loss of CenH3 protein as part of a developmental program leading to the formation of the somatic nucleus in the eukaryote Paramecium. We identify two proteins whose depletion prevents developmental loss of CenH3: the domesticated transposase Pgm involved in the formation of DNA double strand cleavages and the Polycomb-like lysine methyltransferase Ezl1 necessary for trimethylation of histone H3 on lysine 9 and lysine 27. Taken together, our data support a model in which developmentally programmed centromere loss is caused by the elimination of DNA sequences associated with CenH3.


Assuntos
Autoantígenos/genética , Centrômero/genética , Proteínas Cromossômicas não Histona/genética , DNA/genética , Deleção de Sequência/genética , Divisão Celular/genética , Núcleo Celular/genética , Proteína Centromérica A , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Histonas/genética , Paramecium/genética , Transposases/genética
10.
PLoS Genet ; 11(7): e1005383, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26177014

RESUMO

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.


Assuntos
Genoma , RNA Longo não Codificante/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Linhagem da Célula , Células Germinativas , Sequenciamento de Nucleotídeos em Larga Escala , Paramecium tetraurellia/genética , RNA Polimerase II/genética
11.
BMC Genomics ; 18(1): 327, 2017 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-28446146

RESUMO

BACKGROUND: DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. RESULTS: We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. CONCLUSIONS: We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.


Assuntos
Elementos de DNA Transponíveis/genética , DNA de Protozoário/genética , Citometria de Fluxo , Paramecium/citologia , Paramecium/genética , Genômica
12.
BMC Genomics ; 18(1): 483, 2017 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-28651633

RESUMO

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 ).


Assuntos
Perfilação da Expressão Gênica/métodos , Genômica/métodos , Anotação de Sequência Molecular/métodos , Paramecium/genética , Análise de Sequência de RNA
13.
PLoS Genet ; 10(9): e1004665, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25254958

RESUMO

In the ciliate Paramecium tetraurelia, differentiation of the somatic nucleus from the zygotic nucleus is characterized by massive and reproducible deletion of transposable elements and of 45,000 short, dispersed, single-copy sequences. A specific class of small RNAs produced by the germline during meiosis, the scnRNAs, are involved in the epigenetic regulation of DNA deletion but the underlying mechanisms are poorly understood. Here, we show that trimethylation of histone H3 (H3K27me3 and H3K9me3) displays a dynamic nuclear localization that is altered when the endonuclease required for DNA elimination is depleted. We identified the putative histone methyltransferase Ezl1 necessary for H3K27me3 and H3K9me3 establishment and show that it is required for correct genome rearrangements. Genome-wide analyses show that scnRNA-mediated H3 trimethylation is necessary for the elimination of long, repeated germline DNA, while single copy sequences display differential sensitivity to depletion of proteins involved in the scnRNA pathway, Ezl1- a putative histone methyltransferase and Dcl5- a protein required for iesRNA biogenesis. Our study reveals cis-acting determinants, such as DNA length, also contribute to the definition of germline sequences to delete. We further show that precise excision of single copy DNA elements, as short as 26 bp, requires Ezl1, suggesting that development specific H3K27me3 and H3K9me3 ensure specific demarcation of very short germline sequences from the adjacent somatic sequences.


Assuntos
Epigênese Genética , Rearranjo Gênico , Genoma de Protozoário , Paramecium tetraurellia/genética , Zigoto/metabolismo , Elementos de DNA Transponíveis , Histonas/metabolismo , Macronúcleo , Metilação , Paramecium tetraurellia/classificação , Paramecium tetraurellia/metabolismo , Filogenia , Poliploidia , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Deleção de Sequência
14.
PLoS Genet ; 8(10): e1002984, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23071448

RESUMO

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.


Assuntos
Rearranjo Gênico , Genoma de Protozoário , Células Germinativas , Paramecium/genética , Sequência de Aminoácidos , Composição de Bases , Sequência Conservada , Elementos de DNA Transponíveis , DNA de Protozoário , Evolução Molecular , Dosagem de Genes , Ordem dos Genes , Mutação INDEL , Modelos Genéticos , Dados de Sequência Molecular , Seleção Genética , Alinhamento de Sequência
15.
Wiley Interdiscip Rev RNA ; 15(2): e1848, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38605483

RESUMO

Transposable elements and other repeats are repressed by small-RNA-guided histone modifications in fungi, plants and animals. The specificity of silencing is achieved through base-pairing of small RNAs corresponding to the these genomic loci to nascent noncoding RNAs, which allows the recruitment of histone methyltransferases that methylate histone H3 on lysine 9. Self-reinforcing feedback loops enhance small RNA production and ensure robust and heritable repression. In the unicellular ciliate Paramecium tetraurelia, small-RNA-guided histone modifications lead to the elimination of transposable elements and their remnants, a definitive form of repression. In this organism, germline and somatic functions are separated within two types of nuclei with different genomes. At each sexual cycle, development of the somatic genome is accompanied by the reproducible removal of approximately a third of the germline genome. Instead of recruiting a H3K9 methyltransferase, small RNAs corresponding to eliminated sequences tether Polycomb Repressive Complex 2, which in ciliates has the unique property of catalyzing both lysine 9 and lysine 27 trimethylation of histone H3. These histone modifications that are crucial for the elimination of transposable elements are thought to guide the endonuclease complex, which triggers double-strand breaks at these specific genomic loci. The comparison between ciliates and other eukaryotes underscores the importance of investigating small-RNAs-directed chromatin silencing in a diverse range of organisms. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action.


Assuntos
Histonas , RNA , Animais , Histonas/genética , Histonas/metabolismo , Código das Histonas , Elementos de DNA Transponíveis , Lisina/genética
16.
Biol Cell ; 104(6): 309-25, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22352444

RESUMO

Genomes, like crazy patchwork quilts, are stitched together over evolutionary time from diverse elements, including some unwelcome invaders. To deal with parasitic mobile elements, most eukaryotes employ a genome self-defensive manoeuvre to recognise and silence such elements by homology-dependent interactions with RNA-protein complexes that alter chromatin. Ciliated protozoa employ more 'offensive' tactics by actually unstitching and reassembling their somatic genomes at every sexual generation to eliminate transposons and their remnants, using as patterns the maternal genomes that were rearranged in the previous cycle. Genetic and genomic studies of the distant relatives Paramecium and Tetrahymena have begun to reveal how such events are carried out with remarkable precision. Whole genome, non-coding transcripts from the maternal genome are compared with transcripts from the zygotic genome that are processed through an RNA interference (RNAi)-related process. Sequences found only in the latter are targeted for elimination by the resulting short 'scanRNAs' in many thousand DNA splicing reactions initiated by a domesticated transposase. The involvement of widely conserved mechanisms and protein factors clearly shows the relatedness of these phenomena to RNAi-mediated heterochromatic gene silencing. Such malleability of the genome on a generational time scale also has profound evolutionary implications, possibly including the epigenetic inheritance of acquired adaptive traits.


Assuntos
Cilióforos/genética , DNA de Protozoário/genética , Rearranjo Gênico , RNA de Protozoário/genética , Evolução Molecular , Genoma de Protozoário , Heterocromatina/genética , Pequeno RNA não Traduzido/genética
17.
Methods Mol Biol ; 2624: 87-114, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36723811

RESUMO

Mapping DNA modifications at the base resolution is now possible at the genome level thanks to advances in sequencing technologies. Long-read sequencing data can be used to identify modified base patterns. However, the downstream analysis of Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) data requires the integration of genomic annotation and comprehensive filtering to prevent the accumulation of artifact signals. We present in this chapter, a linear workflow to fully analyze modified base patterns using the DNA Modification Annotation (DNAModAnnot) package. This workflow includes a thorough filtering based on sequencing quality and false discovery rate estimation and provides tools for a global analysis of DNA modifications. Here, we provide an application example of this workflow with PacBio data and guide the user by explaining expected outputs via a fully integrated Rmarkdown script. This protocol is presented with tips showing how to adapt the provided code for annotating epigenomes of any organism according to the user needs.


Assuntos
DNA , Sequenciamento de Nucleotídeos em Larga Escala , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA/genética , Genômica , Análise de Sequência de DNA/métodos , Genoma
18.
Trends Genet ; 25(8): 344-50, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19596481

RESUMO

Several classes of non-protein-coding RNAs have recently been identified as epigenetic regulators of developmental genome rearrangements in ciliates, providing an interesting insight into the role of genome-wide transcription. In these unicellular eukaryotes, extensive rearrangements of the germline genome occur during the development of a new somatic macronucleus from the germline micronucleus. Rearrangement patterns are not dictated by the germline sequence, but reproduce the pre-existing rearrangements of the maternal somatic genome, implying a homology-dependent global comparison of germline and somatic genomes. We review recent evidence showing that this is achieved by a natural genomic subtraction, computed by pairing interactions between meiosis-specific, germline scnRNAs (small RNAs that resemble metazoan piRNAs) and longer non-coding transcripts from the somatic genome. We focus on current models for the RNA-based mechanisms enabling the cell to recognize the germline sequences to be eliminated from the somatic genome and to maintain an epigenetic memory of rearrangement patterns across sexual generations.


Assuntos
Cilióforos/crescimento & desenvolvimento , Cilióforos/genética , Rearranjo Gênico , Genoma/genética , RNA não Traduzido/genética , Animais , Epigênese Genética , Células Germinativas/metabolismo
19.
Nature ; 444(7116): 171-8, 2006 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-17086204

RESUMO

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.


Assuntos
Evolução Molecular , Duplicação Gênica , Genoma de Protozoário/genética , Genômica , Paramecium tetraurellia/genética , Animais , Células Eucarióticas/metabolismo , Genes Duplicados/genética , Genes de Protozoários/genética , Dados de Sequência Molecular , Filogenia
20.
Dev Cell ; 57(8): 1037-1052.e8, 2022 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-35429435

RESUMO

Polycomb repressive complex 2 (PRC2) maintains transcriptionally silent genes in a repressed state via deposition of histone H3K27-trimethyl (me3) marks. PRC2 has also been implicated in silencing transposable elements (TEs), yet how PRC2 is targeted to TEs remains unclear. To address this question, we identified proteins that physically interact with the Paramecium enhancer-of-zeste Ezl1 enzyme, which catalyzes H3K9me3 and H3K27me3 deposition at TEs. We show that the Paramecium PRC2 core complex comprises four subunits, each required in vivo for catalytic activity. We also identify PRC2 cofactors, including the RNA interference (RNAi) effector Ptiwi09, which are necessary to target H3K9me3 and H3K27me3 to TEs. We find that the physical interaction between PRC2 and the RNAi pathway is mediated by a RING finger protein and that small RNA recruitment of PRC2 to TEs is analogous to the small RNA recruitment of H3K9 methylation SU(VAR)3-9 enzymes.


Assuntos
Paramecium , Complexo Repressor Polycomb 2 , Elementos de DNA Transponíveis/genética , Histonas/metabolismo , Paramecium/genética , Paramecium/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , RNA
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