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Yb body assembly on the flamenco piRNA precursor transcripts reduces genic piRNA production

ABSTRACT: In Drosophila ovarian somatic cells, piRNAs against transposable elements are mainly produced from the ~180-kb flamenco (flam) locus. flam transcripts are gathered into one-two foci, located close to nuclear envelope, and processed into piRNAs in the cytoplasmic Yb bodies. The mechanism of Yb body formation remains unknown. Using RNA FISH, we found that in the follicle cells of ovaries the 5’-ends of flam transcripts are usually located in close proximity to the nuclear envelope and outside of Yb bodies, while their extended downstream regions mostly overlap with Yb bodies. In flamKG mutant ovaries, flam transcripts containing the 1st and, partially, 2nd exons but lacking downstream regions are gathered into foci at the nuclear envelope, but Yb bodies are not assembled. Strikingly, piRNA production from the protein-coding gene transcripts is enhanced in flamKG ovaries. We propose that normally in ovarian follicle cells flam downstream transcript regions function not only as a substrate for generation of piRNAs but also as a scaffold for Yb body assembly, which is necessary to minimize piRNA production from protein-coding gene transcripts. By contrast, in ovarian somatic cap and escort cells Yb body assembly does not require flam transcription.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE126016 | GEO | 2019/03/29

REPOSITORIES: GEO

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HITS-CLIP analysis of Yb binding sites in Drosophila ovary cell line

Project description:piRNAs direct Piwi to repress transposons to maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. Here, we show that piRNA intermediates arising from the piRNA cluster flamenco (flam) concentrate into perinuclear foci adjacent to Yb bodies, termed Flam bodies. Although flam expression is not required for Yb body formation, Yb, the core component of Yb bodies, is required for Flam body formation. Abolishment of the RNA-binding activity of Yb disrupts both Yb bodies and Flam bodies. Loss of Zucchini, an endoribonuclease necessary for piRNA maturation, enlarges Flam bodies, which now superimpose with Yb bodies. Yb directly binds flam, but not neighboring protein-coding gene, transcripts. Thus, Yb integrates piRNA processing factors and piRNA intermediates into Yb bodies and Flam bodies, respectively, through direct binding to enhance piRNA biogenesis and formation of piRNA-inducing silencing complexes. HITS-CLIP was performed using OSC (Ovarian Somatic Cells). The antibody for Drosophila Yb, which was generated in this study, was used. Obtained CLIP tags were analyzed using illumina HiSeq200.

2014-06-24 | E-GEOD-54875 | biostudies-arrayexpress

HITS-CLIP analysis of Yb binding sites in Drosophila ovary cell line

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Somatic Primary piRNA Biogenesis Driven by Cis-Acting RNA Elements and Trans- Acting Yb

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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.

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