Cellular abundance shapes function in piRNA-guided genome defense

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. Overall design: Piwi-associated RNAs (piRNAs) were immunoprecipitated from fly ovarian somatic sheet cells (OSC), cloned using ten unique molecular identifiers (UMIs), and sequenced to large depth using next generation sequencing. The sequence identity and sequence abundance characteristics of individual piRNAs accross three biological replicates were then analyzed.