PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that bind to PIWI proteins to control transposons and maintain genome integrity in animal germ lines. piRNA 3′ end formation in the silkworm Bombyx mori has been shown to be mediated by the 3′-to-5′ exonuclease Trimmer (Trim; known as PNLDC1 in mammals), and piRNA intermediates are bound with PIWI anchored onto mitochondrial Tudor domain protein Papi. However, it remains unclear whether the Zucchini (Zuc) endonuclease and Nibbler (Nbr) 3′-to-5′ exonuclease, both of which have pivotal roles in piRNA biogenesis in Drosophila, are required for piRNA processing in other species. Here we show that the loss of Zuc in Bombyx had no effect on the levels of Trim and Nbr, but resulted in the aberrant accumulation of piRNA intermediates within the Papi complex, and that these were processed to form mature piRNAs by recombinant Zuc. Papi exerted its RNA-binding activity only when bound with PIWI and phosphorylated, suggesting that complex assembly involves a hierarchical process. Both the 5′ and 3′ ends of piRNA intermediates within the Papi complex showed hallmarks of PIWI â -slicer' activity, yet no phasing pattern was observed in mature piRNAs. The loss of Zuc did not affect the 5′- A nd 3′-end formation of the intermediates, strongly supporting the idea that the 5′ end of Bombyx piRNA is formed by PIWI slicer activity, but independently of Zuc, whereas the 3′ end is formed by the Zuc endonuclease. The Bombyx piRNA biogenesis machinery is simpler than that of Drosophila, because Bombyx has no transcriptional silencing machinery that relies on phased piRNAs.
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