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Cellular abundance shapes function in piRNA-guided genome defense

ABSTRACT: Abstract: Akin to an adaptive immune system, PIWI-interacting small RNAs (piRNAs) protect the integrity of germline genomes by silencing mobile genetic elements. Precision of piRNA silencing is crucial, as failing to silence a single active transposon or wrongly silencing an essential gene threaten the survival of the species. How piRNAs discriminate between self and nonself and adapt during evolution remains a central question in reproductive biology. Target specificity is determined by basepairing complementarity and places the sequence of piRNAs at the heart of piRNA-guided self/nonself discrimination. Here, we characterize millions of piRNA sequences in flies and mice in search for conserved patterns that determine the specificity of piRNA-guided silencing. We identify two groups of piRNA sequences that differ in abundance, complexity and function. The first group represents the top 1% most abundant sequences and drives piRNA-silencing in a reporter assay. In contrast, the second group of sequences does not function in every cell, every individual or every generation. These rare piRNAs establishes cell-to-cell polymorphism and present opportunity for adaptation to novel genomic invaders during evolution. A model emerges that entrust a selected group of piRNA sequences to establish self/nonself discrimination for future generations, and another group to cultivate polymorphisms.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE156058 | GEO | 2021/07/30

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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Cellular abundance shapes function in piRNA-guided genome defense

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