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Unistrand piRNA clusters are an evolutionarily conserved mechanism to suppress endogenous retroviruses across the Drosophila genus [small RNA-Seq]

ABSTRACT: The PIWI-interacting RNA (piRNA) pathway plays a crucial role in preventing endogenous genomic parasites, transposable elements (TEs), from damaging the genetic material of animal gonadal cells. Specific regions in the genome, called piRNA clusters, define each species’ piRNA repertoire and therefore its capacity to recognize and silence transposons. In the somatic cells of the Drosophila melanogaster ovary, the flamenco (flam) unistrand cluster is the main source of piRNAs and primarily regulates Gypsy family TEs that are able to form virus-like particles and infect neighbouring germ cells. Disruption of the flam locus or failure to process flam precursor transcripts into piRNAs results in sterility, yet it remains unknown whether this silencing mechanism is employed widely across Drosophilidae. Here, using both synteny-based analyses and de novo TE annotation, we identify candidate loci sharing both their organisation and TE targeting repertoire with flam in widely divergent Drosophila species groups. Small RNA-sequencing validated these loci as bona-fide unistrand piRNA clusters and revealed their predominant expression in somatic cells of the ovary, likely to counter TE mobilisation in this tissue. This study provides compelling evidence of co-evolution between virus-like Gypsy family transposons and a host defence mechanism in form of soma-expressed, unistrand piRNA clusters.

ORGANISM(S): Drosophila ananassae Drosophila erecta Drosophila subobscura Drosophila yakuba Drosophila takahashii Drosophila melanogaster Drosophila pseudoobscura Drosophila azteca Drosophila simulans Drosophila suzukii Drosophila ficusphila Drosophila mojavensis Drosophila bifasciata Drosophila persimilis Drosophila virilis Drosophila biarmipes

PROVIDER: GSE225888 | GEO | 2023/09/26

REPOSITORIES: GEO

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Project description:PIWI-clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ~23-30nt piRNAs that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endo-nuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the inter-dependencies between these piRNA biogenesis pathways in the developing Drosophila ovary. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and that defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear, transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. To understand the hierarchical order of primary versus secondary piRNA biogenesis in Drosophila ovaries, we sequenced piRNAs bound to total-Piwi, germline-Piwi, Aubergine and Argonaute3 from ovaries of germline specific knockdowns of control, piwi, aub, ago3 single knockdowns and aub/ago3 double knockdowns. To determine changes in Transposable Element (TE) transcription or TE RNA steady state in perturbed piRNA pathway conditions, we performed Pol2-ChIP-sequencing and polyA bound RNA-sequencing from ovaries of multiple germline knockdown genotypes. We also sequenced genomic DNA from ovaries of control knockdowns to experimentally estimate the TE copy number in our genetic background. Finally, we used CAP-seq from germline specific Piwi depletions to identify the Transcriptional Start Sites (TSS) in TEs in a deregulated background. Replicates are labeled with R1, R2, R3, R4 where indicated.

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Unistrand piRNA clusters are an evolutionarily conserved mechanism to suppress endogenous retroviruses across the Drosophila genus [small RNA-Seq]

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