GSE107371
Hierarchical roles of mitochondrial PAPI and Zucchini in Bombyx germline piRNA biogenesis
PIWI-interacting RNAs (piRNAs) are germline-enriched small RNAs that control transposons to maintain genome integrity1,2,3. To achieve this, piRNAs bind PIWI proteins upon being processed from piRNA precursors1,2,3. Bioinformatic studies of piRNA biogenesis in Drosophila showed that the piRNA 5' end is formed by PIWI-Slicer or Zucchini (Zuc) endonucleolytic cleavage, while the 3' end is formed by Zuc or Nibbler (Nbr) 3'-to-5' exonucleolytic activity4,5,6. piRNA 3'-end formation in Bombyx was shown to be mediated by PNLDC1/Trimmer (Trim) 3'-to-5' exonuclease7, while piRNA intermediates are bound with PIWI anchored onto mitochondrial protein PAPI8. However, the requirement for Zuc and Nbr in piRNA biogenesis in Bombyx has not been elucidated. Here, we applied biochemical approaches to understand their involvement in piRNA biogenesis and revealed that Zuc endonuclease, but not Trim and Nbr exonucleases, plays a crucial role in Bombyx piRNA 3'-end formation. Loss of Zuc had little effect on the levels of Trim and Nbr, but led to the aberrant accumulation of piRNA intermediates within the PAPI complex, which were processed to mature piRNAs by recombinant Zuc. Zuc copurified with PAPI, and PAPI exerted RNA-binding activity only when Siwi coexisted with it and PAPI was phosphorylated, suggesting that complex assembly proceeds via a hierarchical process. Bioinformatic analyses of piRNA intermediates within the PAPI complex revealed that both the 5' and the 3' ends showed the hallmark of PIWI-Slicer, yet no phasing pattern was observed in mature piRNAs. These findings strongly support the notion that, in Bombyx piRNA, the 5' end is formed by PIWI-Slicer, but independently of Zuc, while the 3' end is formed by Zuc endonuclease. The Bombyx piRNA biogenesis is simpler than that of Drosophila, which is reasonable considering that Bombyx has no transcriptional silencing machinery relying on phased piRNAs. Overall design: Two RIP-seq libraries: RIP libraries were prepared from RNA associated with PAPI immunoprecipitates (with size selection at 65-100nt). Six smRNA-seq libraries: small RNA libraries were prepared from small RNA associated with Flag-Siwi and Flag-Ago3 immunoprecipitates (with size selection at 23-35nt).