miRNA/siRNA-directed pathway to produce noncoding piRNAs from endogenous protein-coding regions ensures Drosophila spermatogenesis

PIWI-interacting RNA (piRNA) pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the underlying piRNA biogenesis mechanisms remain elusive. Here, we show that endogenous protein coding sequences (CDSs), which are normally used for translation, serve as origins of noncoding piRNA biogenesis in Drosophila melanogaster testes. The product, namely, CDS-piRNAs, formed silencing complexes with Aubergine (Aub) in germ cells. Proximity proteome and functional analyses show that CDS-piRNAs and cluster/TE-piRNAs are distinct species occupying Aub, the former loading selectively relies on chaperone Cyclophilin 40. Moreover, Argonaute 2 (Ago2) and Dicer-2 activities were found critical for CDS-piRNA production. We provide evidence that Ago2-bound short interfering RNAs (siRNAs) and microRNAs (miRNAs) specify precursors to be processed into piRNAs. We further demonstrate that Aub is crucial in spermatid differentiation, regulating chromatins through mRNA cleavage. Collectively, our data illustrate a unique strategy used by male germ line, expanding piRNA repertoire for silencing of endogenous genes during spermatogenesis.

Automated summary

PIWI-interacting RNA (piRNA) pathways are important in animal gamete formation, but the mechanisms of piRNA biogenesis are not fully understood. In this study, we discovered that protein coding sequences (CDSs) in Drosophila melanogaster testes can serve as origins for noncoding piRNA biogenesis. These CDS-piRNAs form silencing complexes with Aubergine (Aub) in germ cells. Our findings also reveal that CDS-piRNAs and cluster/TE-piRNAs are different species and their loading onto Aub depends on chaperone Cyclophilin 40. Furthermore, the activities of Argonaute 2 (Ago2) and Dicer-2 are critical for CDS-piRNA production.