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A heterochromatin-dependent transcription machinery drives piRNA expression

ABSTRACT: Nuclear small RNA pathways safeguard genome integrity by establishing transcription-repressing heterochromatin at transposable elements. This inevitably also targets the transposon-rich source loci of the small RNAs themselves. How small RNA source loci are efficiently transcribed while transposon promoters are potently silenced, is not understood. Here, we show that transcription of Drosophila piRNA clusters—major small RNA source loci in the animal germline—is enforced through formation of the RNA Polymerase II pre-initiation complex within repressive heterochromatin. This is accomplished through the TFIIA-L paralog Moonshiner, which is recruited to piRNA clusters via the Heterochromatin Protein-1 variant Rhino. Moonshiner triggers transcription initiation within piRNA clusters by recruiting the TATA box-binding protein (TBP)-related factor TRF2, a conserved animal TFIID core variant. Our work reveals that transcription of heterochromatic small RNA source loci relies on direct recruitment of the core transcriptional machinery to DNA via histone marks rather than sequence motifs, a concept that we argue is a recurring theme in evolution.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE97719 | GEO | 2017/04/26

SECONDARY ACCESSION(S): PRJNA382719

REPOSITORIES: GEO

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A heterochromatin-dependent transcription machinery drives piRNA expression

Project description:Nuclear small RNA pathways safeguard genome integrity by establishing transcription-repressing heterochromatin at transposable elements. This inevitably also targets the transposon-rich source loci of the small RNAs themselves. How small RNA source loci are efficiently transcribed while transposon promoters are potently silenced, is not understood. Here, we show that transcription of Drosophila piRNA clusters—small RNA source loci in animal gonads—is enforced through RNA Polymerase II pre-initiation complex formation within repressive heterochromatin. This is accomplished through the TFIIA-L paralog Moonshiner, which is recruited to piRNA clusters via the Heterochromatin Protein-1 variant Rhino. Moonshiner triggers transcription initiation within piRNA clusters by recruiting the TATA box-binding protein (TBP)-related factor TRF2, an animal TFIID core variant. Thus, transcription of heterochromatic small RNA source loci relies on direct recruitment of the core transcriptional machinery to DNA via histone marks rather than sequence motifs, a concept that we argue is a recurring theme in evolution.

2017-08-07 | PXD005026 | Pride

A nuclear RNA export factor variant licenses piRNA-guided heterochromatin formation

Project description:Small RNA silencing pathways protect genome integrity in part through establishing heterochromatin at transposon loci. In animals, this process requires piRNA-guided targeting of nuclear PIWI proteins to nascent transcripts. The molecular events contributing to heterochromatin formation upon PIWI binding to nascent RNA, a transient molecule at chromatin, are unknown. Here, we identify SFINX, a protein complex that is required for Piwi-mediated co-transcriptional silencing in Drosophila. It consists of Nxf2—a variant of the nuclear RNA export factor Nxf1/Tap, the mRNA export co-factor Nxt1/p15, and the Piwi-associated protein Panoramix. In the absence of Nxf2, Panoramix is targeted for degradation and piRNA-loaded Piwi is unable to establish heterochromatin. Consequently, nxf2 mutants exhibit severe transposon de-repression and are sterile. We show that within SFINX, Panoramix connects to the heterochromatin machinery while Nxf2 enables target silencing via nascent RNA. Thus, the Nxf2-Nxt1 heterodimer—despite having originated from core mRNA export machinery—has been repurposed for heterochromatin formation. Our data establish an unexpected link between nuclear small RNA biology and NXF-variants, which are widespread in animal lineages, but mostly lack ascribed functions.

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The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters (sRNA-Seq)

Project description:RNA interference systems depend on the synthesis of small RNA precursors whose sequences define the target spectrum of these silencing pathways. The Drosophila Heterochromatin Protein 1 (HP1) variant Rhino permits transcription of PIWI-interacting RNA (piRNA) precursors within transposon-rich heterochromatic loci in germline cells. Current models propose that Rhino’s specific chromatin occupancy at piRNA source loci is determined by histone marks and maternally inherited piRNAs, but also imply the existence of other, undiscovered specificity cues. Here, we identify a member of the diverse family of zinc finger associated domain (ZAD)-C2H2 proteins, Kipferl, as critical Rhino cofactor in ovaries. By binding to guanosine-rich DNA motifs and interacting with the Rhino chromodomain, Kipferl recruits Rhino to specific loci and stabilizes it on chromatin. In kipferl mutant flies, Rhino is lost from most of its target chromatin loci and instead accumulates on pericentromeric satellite arrays, resulting in decreased levels of transposon targeting piRNAs and impaired fertility. Our findings reveal that DNA sequence, in addition to the H3K9me3 mark, determines the identity of piRNA source loci and provide insight into how Rhino might be caught in the crossfire of genetic conflicts.

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The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters (RNA-Seq)

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The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters (ChIP-Seq)

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The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation

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2019-04-17 | GSE120617 | GEO

The Rhino-Deadlock-Cutoff complex licenses non-canonical transcription of dual-strand piRNA clusters in Drosophila

Project description:Argonaute proteins of the PIWI clade are central to transposon silencing in animal gonads. Their target specificity is defined by 22-30nt PIWI interacting RNAs (piRNAs), which mostly originate from discrete genomic loci termed piRNA clusters. Here we show that the RDC complex composed of Rhino, Deadlock and Cutoff defines dual-strand piRNA clusters genome-wide in Drosophila ovaries. The RDC complex is anchored to H3K9me3-marked chromatin in part via Rhino’s chromo-domain. Depletion of Piwi results in loss of the RDC and small RNAs at euchromatic piRNA source loci, demonstrating a feedback loop between Piwi and genomic piRNA sources. Intriguingly, profiles of RNA Polymerase II occupancy, nascent transcription and steady-state RNA levels reveal that the RDC licenses non-canonical transcription of dual-stranded piRNA clusters. Likely, this process involves 5’end protection of nascent RNAs and subsequent suppression of transcription termination. Together, our data provide a comprehensive model for the regulation and evolution of piRNA clusters.

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The Rhino-Deadlock-Cutoff complex licenses non-canonical transcription of dual-strand piRNA clusters in Drosophila

Project description:Argonaute proteins of the PIWI clade are central to transposon silencing in animal gonads. Their target specificity is defined by 22-30nt PIWI interacting RNAs (piRNAs), which mostly originate from discrete genomic loci termed piRNA clusters. Here we show that the RDC complex composed of Rhino, Deadlock and Cutoff defines dual-strand piRNA clusters genome-wide in Drosophila ovaries. The RDC complex is anchored to H3K9me3-marked chromatin in part via RhinoM-bM-^@M-^Ys chromo-domain. Depletion of Piwi results in loss of the RDC and small RNAs at euchromatic piRNA source loci, demonstrating a feedback loop between Piwi and genomic piRNA sources. Intriguingly, profiles of RNA Polymerase II occupancy, nascent transcription and steady-state RNA levels reveal that the RDC licenses non-canonical transcription of dual-stranded piRNA clusters. Likely, this process involves 5M-bM-^@M-^Yend protection of nascent RNAs and subsequent suppression of transcription termination. Together, our data provide a comprehensive model for the regulation and evolution of piRNA clusters. This study aims at indentifying and characterizing genimc sources of piRNA percursour transcripts using genome-wide apporaches such as ChIP-seq, RNA-seq, smallRNA-seq and GRO-seq in adult Drosophila ovaries depleted for several factors implicated in piRNA cluster regulation

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A heterochromatin specific RNA export pathway facilitates piRNA production

Project description:PIWI-interacting RNAs (piRNAs), a class of small RNAs that guide transposon silencing in animals, are processed in the cytoplasm from RNA Polymerase II transcripts. How piRNA precursors, which often lack RNA maturation signatures and thereby violate quality control checkpoints, achieve nuclear export is unknown. Here, we uncover a germline-specific RNA export pathway in Drosophila, that escorts piRNA precursors from their heterochromatic origins to nuage, the cytoplasmic piRNA processing centres. This pathway connects canonical nuclear export factors—the RNA helicase UAP56, the NXF cofactor Nxt1/p15, and the exportin Crm1/Xpo1—with Nxf3, a variant of the mRNA exporter Nxf1/Tap. Nxf3 recruitment to nascent piRNA precursors occurs via the heterochromatin protein 1-variant Rhino and CG13741/Bootlegger, a new piRNA pathway factor, thereby making piRNA precursor export independent of RNA processing events. Thus, similar to retroviral hijacking of cellular export factors, piRNA precursor export evolved to bend canonical gene expression rules through bypassing nuclear RNA surveillance mechanisms.

2019-03-29 | PXD012717 | Pride

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.

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