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Production of artificial piRNAs in flies and mice

ABSTRACT: In animals, a discrete class of small RNAs, the piwi-interacting RNAs (piRNAs), guard germ cell genomes against the activity of mobile genetic elements. piRNAs are generated, via an unknown mechanism, from apparently single-stranded precursors that arise from discrete genomic loci, termed piRNA clusters. The content of piRNA clusters, determines the capacity of the system to respond to a given element, in essence comprising an organism's evolving molecular definition of transposons. Presently, little is known about the signals that distinguish a locus as a source of piRNAs and about how abundant piRNAs are selected. To address these questions, we inserted new sequence information into piRNA clusters in mice and flies. In all cases, this information was incorporated into the piRNA repertoire and in one instance was shown to confer the ability to recognize and silence a corresponding element. Notably, patterns of piRNA abundance suggested that both intrinsic sequence and context with the cluster inform piRNA generation. Though piRNAs themselves are not conserved between species, the genomic location of clusters is often retained. We were able to create artificial piRNA clusters in non-native contexts in both mice and flies, indicating that the signals that define these as generative loci must lie within the clusters themselves rather than being implicit in their genomic position.

ORGANISM(S): Mus musculus Drosophila melanogaster

PROVIDER: GSE32435 | GEO | 2012/01/01

SECONDARY ACCESSION(S): PRJNA147895

REPOSITORIES: GEO

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Production of artificial piRNAs in flies and mice

Project description:In animals, a discrete class of small RNAs, the piwi-interacting RNAs (piRNAs), guard germ cell genomes against the activity of mobile genetic elements. piRNAs are generated, via an unknown mechanism, from apparently single-stranded precursors that arise from discrete genomic loci, termed piRNA clusters. The content of piRNA clusters, determines the capacity of the system to respond to a given element, in essence comprising an organism's evolving molecular definition of transposons. Presently, little is known about the signals that distinguish a locus as a source of piRNAs and about how abundant piRNAs are selected. To address these questions, we inserted new sequence information into piRNA clusters in mice and flies. In all cases, this information was incorporated into the piRNA repertoire and in one instance was shown to confer the ability to recognize and silence a corresponding element. Notably, patterns of piRNA abundance suggested that both intrinsic sequence and context with the cluster inform piRNA generation. Though piRNAs themselves are not conserved between species, the genomic location of clusters is often retained. We were able to create artificial piRNA clusters in non-native contexts in both mice and flies, indicating that the signals that define these as generative loci must lie within the clusters themselves rather than being implicit in their genomic position. Total RNA and RNA associated with Piwi proteins were isolated and size-fractionated by PAGE into 19-33nt. These were processed and sequenced on the Illumina GA2 platform.

2012-01-01 | E-GEOD-32435 | biostudies-arrayexpress

Requirement of Nuclear Pore Complex Subunits for transposon silencing in Drosophila

Project description:The piRNA pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. In Drosophila ovaries, piRNAs are produced from discrete genomic loci, called piRNA clusters, which are composed of inactive transposon copies and fragments and thus constitute a genetically encoded memory of past transposon challenges. Two types of piRNA clusters exist in flies: dual-strand clusters, expressed only in the germline via a highly specialised machinery, and uni-strand cluster, which are predominantly expressed in the somatic follicle cells. Flamenco (flam) is the major uni-strand piRNA cluster in Drosophila, giving rise to the majority of somatic piRNAs. Flam resembles a canonical RNA polymerase II transcriptional unit, nonetheless it can be specifically recognised by the piRNA pathway and directed to the biogenesis machinery. Recent work has implicated the RNA helicase Yb in the licensing of somatic piRNA production, however a detailed understanding of the molecular mechanisms underlying flam export and specification is still lacking. Here, we show that flam export triggers the assembly of peri-nuclear condensates of Yb and provide evidence that piRNA production from flam specifically requires subunits of the Nuclear Pore Complex (NPC). In the absence of some NPC subunits, transposons become de-silenced and piRNA biogenesis is compromised exclusively from flam. We also show that Yb transiently associates with the NPC to promote flam export. Taken together, our data shed light on how the export of uni-strand cluster transcripts is achieved and suggest the evolution of a specialised machinery that couples transcription, nuclear export and piRNA production.

2021-04-15 | PXD019670 | Pride

Requirement of Nuclear Pore Complex subunits for transposon silencing in Drosophila

2021-04-16 | PXD019671 | Pride

Requirement of Nuclear Pore Complex subunits for transposon silencing in Drosophila

2021-07-02 | PXD019674 | Pride

Environmentally-induced epigenetic conversion of a piRNA cluster

Project description:Transposable element (TE) activity is repressed in animal gonads by PIWI-interacting RNAs (piRNAs) produced by piRNA clusters. Current models propose that piRNA clusters are functionally defined by the maternal inheritance of piRNAs produced during the previous generation. Taking advantage of an inactive cluster of P-element derived transgene insertions in Drosophila melanogaster, we show here that raising flies at high temperature (29°C) instead of 25°C results in stable conversion of this locus into an active piRNAs producing one reporting thus the first case of the establishment of an active piRNA cluster by environmental changes and without maternal inheritance of homologous piRNAs.

2019-03-14 | GSE116122 | GEO

Trans-generationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing [Rhino RNA-seq]

Project description:Small non-coding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of Metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that trans-generationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the H3K9me3 mark on genomic piRNA cluster sequences. The HP1 homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that trans-generationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels, by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. total RNA sequencing of RNA from ovaries of shWhite and Rhino knockdown flies

2014-07-26 | E-GEOD-59607 | biostudies-arrayexpress

Trans-generationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing [cuff smallRNA]

Project description:Small non-coding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of Metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that trans-generationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the H3K9me3 mark on genomic piRNA cluster sequences. The HP1 homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that trans-generationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels, by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. small RNA (19-29nt) sequencing of RNA from ovaries of cuff+/- or cuff-/- flies

2014-07-26 | E-GEOD-59608 | biostudies-arrayexpress

Trans-generationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing [cuff RNA-seq]

2014-07-26 | E-GEOD-59609 | biostudies-arrayexpress

Epigenetic requirements for triggering heterochromatinization and Piwi-interacting RNA production from transgenes in the Drosophila germline

Project description:Transgenes containing a fragment of I transposon represent a powerful model of piRNA cluster de novo formation in the Drosophila germline. We revealed that the same transgenes located at different genomic loci form piRNA clusters with various capacity of small RNA production. Transgenic piRNA clusters are not established in piRNA pathway mutants. However, in wild-type context, the endogenous ancestral I-related piRNAs are sufficient to heterochromatinize and convert the I-containing transgenes into piRNA-producing loci. Here, we address how the quantitative level of piRNAs influences the heterochromatinization and piRNA production. We show that neither the piRNAs mediated by active I-element copies nor inheritance of abundant maternal I-derived piRNAs enable to stimulate additional changes of transgenes chromatin state or piRNA production from them. Therefore, chromatin changes and piRNA production are initiated by a minimum threshold level of complementary piRNAs suggesting a selective advantage of prompt cell response to the lowest level of piRNAs. Noteworthy, the weak piRNA clusters do not transform into strong ones after being targeted by a larger amount of I-specific piRNAs, indicating the importance of the genomic context for piRNA cluster establishment. Analysis of ovarian transcription profiles suggests that regions facilitating convergent transcription favor formation of transgenic piRNA clusters .

2020-04-10 | GSE138886 | GEO

Trans-generationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing [ChIP-seq]

Project description:Small non-coding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of Metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that trans-generationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the H3K9me3 mark on genomic piRNA cluster sequences. The HP1 homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that trans-generationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels, by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. ChIPseq of Rhino and Cutoff in Drosophila melanogaster ovaries The Rhino-BioTAP flies were made by fusing the BioTAP tag (Alekseyenko et al. 2014 )to the C-terminal region of the Rhino gene under the UASp promoter. The Cutoff-EGFP fly line (Nanos-GAL4/UASp-Cutoff-GFP), Cuff^wm25 and Cuff^qq37 were a generous gift from T. Schupbach.

2014-07-26 | E-GEOD-59604 | biostudies-arrayexpress

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