PIWI-interacting RNAs (piRNAs) silence transposons to maintain genome integrity in animal germ lines1,2,3,4. piRNAs are classified as primary and secondary piRNAs, depending on their biogenesis machinery5,6,7,8,9,10. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters in the genome through the primary processing pathway5,8,9,10. Although the existence of a ribonuclease participating in this pathway has been predicted, its molecular identity remained unknown. Here we show that Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily member11, is an endoribonuclease essential for primary piRNA biogenesis. We solved the crystal structure of Drosophila melanogaster Zuc (DmZuc) at 1.75 Å resolution. The structure revealed that DmZuc has a positively charged, narrow catalytic groove at the dimer interface, which could accommodate a single-stranded, but not a double-stranded, RNA. DmZuc and the mouse homologue MmZuc (also known as Pld6 and MitoPLD)12,13,14 showed endoribonuclease activity for single-stranded RNAs in vitro. The RNA cleavage products bear a 5′-monophosphate group, a hallmark of mature piRNAs. Mutational analyses revealed that the conserved active-site residues of DmZuc are critical for the ribonuclease activity in vitro, and for piRNA maturation and transposon silencing in vivo. We propose a model for piRNA biogenesis in animal germ lines, in which the Zuc endoribonuclease has a key role in primary piRNA maturation.
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We thank A. Kurabayashi, H. Kotani and Y. Tamaki for technical assistance; other members of the Siomi laboratory for discussions and comments on the manuscript; the beamline staff at BL32XU of SPring-8 for assistance in data collection; and T. Suzuki and H. Suga for technical suggestions and comments. This work was supported by a grant from the Japan Society for the Promotion of Science (JSPS) through its ‘Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST) program’ to O.N., by the Core Research for Evolutional Science and Technology (CREST) program ‘The Creation of Basic Medical Technologies to Clarify and Control the Mechanisms Underlying Chronic Inflammation’ of the Japan Science and Technology Agency (JST) to O.N. and M.C.S., and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to H.N., H.I., H.S., M.C.S. and O.N.. K.S. is supported by the JSPS.
The authors declare no competing financial interests.
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Nishimasu, H., Ishizu, H., Saito, K. et al. Structure and function of Zucchini endoribonuclease in piRNA biogenesis. Nature 491, 284–287 (2012). https://doi.org/10.1038/nature11509
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