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1.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2022,1869(6):119239
Developmental DNA elimination in Paramecium tetraurelia occurs through a trans-nuclear comparison of the genomes of two distinct types of nuclei: the germline micronucleus (MIC) and the somatic macronucleus (MAC). During sexual reproduction, which starts with meiosis of the germline nuclei, MIC-limited sequences including Internal Eliminated Sequences (IESs) and transposons are eliminated from the developing MAC in a process guided by noncoding RNAs (scnRNAs and iesRNAs). However, our current understanding of this mechanism is still very limited. Therefore, studying both genetic and epigenetic aspects of these processes is a crucial step to understand this phenomenon in more detail. Here, we describe the involvement of homologs of classical meiotic proteins, Spo11, Msh4-1, and Msh5 in this phenomenon. Based on our analyses, we propose that proper functioning of Spo11, Msh4-1, and Msh5 during Paramecium sexual reproduction are necessary for genome reorganization and viable progeny. Also, we show that double-strand breaks (DSBs) in DNA induced during meiosis by Spo11 are crucial for proper IESs excision. In summary, our investigations show that early sexual reproduction processes may significantly influence later somatic genome integrity. 相似文献
2.
Estienne C. Swart Cyril Denby Wilkes Pamela Y. Sandoval Miroslav Arambasic Linda Sperling Mariusz Nowacki 《Nucleic acids research》2014,42(14):8970-8983
During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of ∼45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic. By analyzing the silencing effects of three genes: PGM (responsible for DNA excision), DCL2/3 (scnRNA production) and DCL5 (iesRNA production), we identify key properties required for IES elimination. Based on these results, we propose that, depending on the exact combination of their lengths and end bases, some IESs are less efficiently recognized or excised and have a greater requirement for targeting by scnRNAs and iesRNAs. We suggest that the variation in IES retention following silencing of DCL2/3 is not primarily due to scnRNA density, which is comparatively uniform relative to IES retention, but rather the genetic properties of IESs. Taken together, our analyses demonstrate that in Paramecium the underlying genetic properties of developmentally deleted DNA sequences are essential in determining the sensitivity of these sequences to epigenetic control. 相似文献
3.
Aditi Singh Xyrus X MaurerAlcal Therese Solberg Lilia Hußermann Silvan Gisler Michael Ignarski Estienne C Swart Mariusz Nowacki 《The EMBO journal》2022,41(22)
Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The unicellular ciliate Paramecium is a model to study dynamic genome organization in eukaryotic cells, given its unique feature of nuclear dimorphism. Here, the formation of the somatic macronucleus during sexual reproduction requires eliminating thousands of transposon remnants (IESs) and transposable elements scattered throughout the germline micronuclear genome. The elimination process is guided by Piwi‐associated small RNAs and leads to precise cleavage at IES boundaries. Here we show that IES recognition and precise excision are facilitated by recruiting ISWI1, a Paramecium homolog of the chromatin remodeler ISWI. ISWI1 knockdown substantially inhibits DNA elimination, quantitatively similar to development‐specific sRNA gene knockdowns but with much greater aberrant IES excision at alternative boundaries. We also identify key development‐specific sRNA biogenesis and transport proteins, Ptiwi01 and Ptiwi09, as ISWI1 cofactors in our co‐immunoprecipitation studies. Nucleosome profiling indicates that increased nucleosome density correlates with the requirement for ISWI1 and other proteins necessary for IES excision. We propose that chromatin remodeling together with small RNAs is essential for efficient and precise DNA elimination in Paramecium. 相似文献
4.
Diamantis Sellis Frdric Gurin Olivier Arnaiz Walker Pett Emmanuelle Lerat Nicole Boggetto Sascha Krenek Thomas Berendonk Arnaud Couloux Jean-Marc Aury Karine Labadie Sophie Malinsky Simran Bhullar Eric Meyer Linda Sperling Laurent Duret Sandra Duharcourt 《PLoS biology》2021,19(7)
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.A comparative genomics study of nine Paramecium species reveals successful invasion of genes by transposable elements in their germline genomes, showing that the internal eliminated sequences (IESs) followed an evolutionary trajectory remarkably similar to that of spliceosomal introns. 相似文献
5.
Olivier Arnaiz Nathalie Mathy Céline Baudry Sophie Malinsky Jean-Marc Aury Cyril Denby Wilkes Olivier Garnier Karine Labadie Benjamin E. Lauderdale Anne Le Mou?l Antoine Marmignon Mariusz Nowacki Julie Poulain Malgorzata Prajer Patrick Wincker Eric Meyer Sandra Duharcourt Laurent Duret Mireille Bétermier Linda Sperling 《PLoS genetics》2012,8(10)
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. 相似文献
6.
BACKGROUND: The germline genome of ciliates is extensively rearranged during development of a new somatic macronucleus from the germline micronucleus, a process that follows sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) and multicopy transposons are eliminated, whereas cellular genes are amplified to approximately 800 n. For a subset of IESs, introduction of the IES sequence into the maternal (prezygotic) macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus. This and other homology-dependent maternal effects have suggested that rearrangement patterns are epigenetically determined by an RNA-mediated, trans-nuclear comparison, involving the RNA interference pathway, of germline and somatic genomes. RESULTS: We report the identification of novel developmentally regulated RNA binding proteins, Nowa1p and Nowa2p, which are required for the survival of sexual progeny. Green fluorescent protein (GFP) fusions show that Nowa1p accumulates into the maternal macronucleus shortly before meiosis of germline micronuclei and is later transported to developing macronuclei. Nowa1p/2p depletion impairs the elimination of transposons and of those IESs that are controlled by maternal effects, confirming the existence of distinct IES classes. CONCLUSIONS: The results indicate that Nowa proteins are essential components of the trans-nuclear-crosstalk mechanism that is responsible for epigenetic programming of genome rearrangements. We discuss implications for the current models of genome scanning in ciliates, a process related to the formation of heterochromatin by RNA interference in other eukaryotes. 相似文献
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8.
Maoussi Lhuillier-Akakpo Andrea Frapporti Cyril Denby Wilkes Mélody Matelot Michel Vervoort Linda Sperling Sandra Duharcourt 《PLoS genetics》2014,10(9)
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. 相似文献
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10.
Miroslav Arambasic Pamela Y. Sandoval Cristina Hoehener Aditi Singh Estienne C. Swart Mariusz Nowacki 《PloS one》2014,9(11)
The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization. 相似文献
11.
The somatic genome of the ciliated protist Tetrahymena undergoes DNA elimination of defined sequences called internal eliminated sequences (IESs), which account for ∼30% of the germline genome. During DNA elimination, IES regions are heterochromatinized and assembled into heterochromatin bodies in the developing somatic nucleus. The domesticated piggyBac transposase Tpb2p is essential for the formation of heterochromatin bodies and DNA elimination. In this study, we demonstrate that the activities of Tpb2p involved in forming heterochromatin bodies and executing DNA elimination are genetically separable. The cysteine-rich domain of Tpb2p, which interacts with the heterochromatin-specific histone modifications, is necessary for both heterochromatin body formation and DNA elimination, whereas the endonuclease activity of Tpb2p is only necessary for DNA elimination. Furthermore, we demonstrate that the endonuclease activity of Tpb2p in vitro and the endonuclease activity that executes DNA elimination in vivo have similar substrate sequence preferences. These results strongly indicate that Tpb2p is the endonuclease that directly catalyzes the excision of IESs and that the boundaries of IESs are at least partially determined by the combination of Tpb2p-heterochromatin interaction and relaxed sequence preference of the endonuclease activity of Tpb2p. 相似文献
12.
Antoine Marmignon Julien Bischerour Aude Silve Clémentine Fojcik Emeline Dubois Olivier Arnaiz Aurélie Kapusta Sophie Malinsky Mireille Bétermier 《PLoS genetics》2014,10(8)
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. 相似文献
13.
Paramecium aurelia 《Biochimie》2001,83(11-12)
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. 相似文献
14.
Homology-Dependent Maternal Inhibition of Developmental Excision of Internal Eliminated Sequences in Paramecium tetraurelia 总被引:1,自引:0,他引:1
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Sandra Duharcourt Anne-Marie Keller Eric Meyer 《Molecular and cellular biology》1998,18(12):7075-7085
Thousands of single-copy internal eliminated sequences (IESs) are excised from the germ line genome of ciliates during development of the polygenomic somatic macronucleus, following sexual events. Paramecium IESs are short, noncoding elements that frequently interrupt coding sequences. No absolutely conserved sequence element, other than flanking 5′-TA-3′ direct repeats, has been identified among sequenced IESs; the mechanisms of their specific recognition and precise elimination are unknown. Previous work has revealed the existence of an epigenetic control of excision. It was shown that the presence of one IES in the vegetative macronucleus results in a specific inhibition of the excision of the same element during the development of a new macronucleus, in the following sexual generation. We have assessed the generality and sequence specificity of this transnuclear maternal control by studying the effects of macronuclear transformation with 13 different IESs. We show that at least five of them can be maintained in the new macronuclear genome; sequence specificity is complete both between genes and between different IESs in the same gene. In all cases, the degree of excision inhibition correlates with the copy number of the maternal IES, but each IES shows a characteristic inhibition efficiency. Short internal IES-like segments were found to be excised from two of the IESs when excision between normal boundaries was inhibited. Available data suggest that the sequence specificity of these maternal effects is mediated by pairing interactions between homologous nucleic acids. 相似文献
15.
Piroska Huvos 《Gene》2009
There are over 6000 internally eliminated DNA sequences (IESs) in the Tetrahymena genome that are deleted in a programmed fashion during the development of a polyploid, somatic macronucleus from a diploid germline micronucleus. Recently, based on several results, a homology and small RNA-based mechanism has been proposed for the efficient elimination of IES elements. Since the RNAi machinery is proposed to be intimately involved in silencing potentially harmful repeats such as transposons and viruses, characterization of repeats and the conditions for their developmental elimination from the somatic genome is warranted. Three short (500–600 bp) repeat families, members of which had been experimentally identified in IESs, that is, in micronucleus-specific DNA, are examined here using the Tetrahymena genome database. Members of all three families display varied degrees of truncation and are represented in macronuclear sequences. A 200 bp segment of one of the families can appear in the genome on its own, or as part of a 600 bp repeat detected experimentally, or in association with an unrelated 1 kb sequence to form a 1.2 kb repeat that is also frequently truncated. The 1 kb sequence contains a 300 bp section similar to a repeat associated with a non-long terminal repeat-like element and is often found accompanied by several more copies of this shorter repeat. These observations indicate that transposition may have had a role in the evolution of the short repeat families. 相似文献
16.
17.
James Forney 《BioEssays : news and reviews in molecular, cellular and developmental biology》1997,19(1):5-8
Ciliated protozoa have separate germline and somatic nuclei, yet unlike larger organisms, both nuclei reside in the same cytoplasm. The micronuclei contain the germline and the macronucleus is the somatic nucleus. Thousands of DNA elements are normally removed from the micronuclear genome as it forms a new macronucleus during each sexual cycle. A recent study directly links the excision of these internal eliminated sequences (IESs) to mating type determination by showing that a pleiotropic mutation affecting mating type also prevents the excision of an IES from a surface protein gene(1). Remarkably, once the IES is present in the old macronucleus it prevents excision of that specific IES during formation of the next macronucleus. 相似文献
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19.
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. 相似文献
20.
David M Prescott 《Current opinion in genetics & development》1997,7(6):807-813
Three hypotheses on the evolutionary/molecular origin of internal eliminated segments (IESs) in the germline of hypotrichous ciliates are discussed in the context of the high rate of mutation accumulation in IESs, shifting of IESs during speciation, and evolutionary scrambling of segments within some hypotrich germline genes. Developmental excision of IESs from the germline in Paramecium suggests that the parental macronucleus may provide nucleic acid sequence information to guide excision of IESs and splicing of macronuclear-destined sequences. In ciliates of the oxytrichid/stylonychid group, such a mechanism could explain the precision of excision of IESs and gene unscrambling. Recently initiated molecular/genetic studies may eventually clarify the role of the parental macronucleus in IES excision and gene unscrambling as well as the molecular mechanisms of these events. 相似文献