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Structure and function of Zucchini endoribonuclease in piRNA biogenesis

Nature volume 491, pages 284–287 (2012)Cite this article

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Abstract

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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Figure 1: Crystal structure of DmZuc.
Figure 2: Active site of DmZuc.
Figure 3: Zuc is an endonuclease for single-stranded nucleic acids.
Figure 4: DmZuc endoribonuclease activity is required for transposon silencing.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors are deposited in the Protein Data Bank under accession numbers 4GEL (DmZucWTdimer), 4GEM (DmZuc K171A dimer) and 4GEN (DmZuc WT monomer).

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Acknowledgements

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.

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Author notes

  1. Luc Bonnefond

    Present address: Present address: Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR7104 CNRS-UDS, INSERM U964, 1 rue Laurent Fries, BP 10142, 67404 Illkirch Cedex, France.,

  2. Hiroshi Nishimasu and Hirotsugu Ishizu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo, 113-0032, Japan

    Hiroshi Nishimasu, Hirotsugu Ishizu, Satoshi Fukuhara, Luc Bonnefond, Naoki Matsumoto, Tomohiro Nishizawa, Ryuichiro Ishitani, Mikiko C. Siomi & Osamu Nureki

  2. Department of Molecular Biology, Keio University School of Medicine, Tokyo, 160-8582, Japan

    Hirotsugu Ishizu, Kuniaki Saito, Miharu K. Kamatani, Haruhiko Siomi & Mikiko C. Siomi

  3. Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, 980-8578, Japan

    Keita Nakanaga & Junken Aoki

  4. Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST), Saitama, 332-0012, Japan

    Mikiko C. Siomi & Osamu Nureki

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Contributions

H.N., H.I., K.S., H.S., M.C.S. and O.N. conceived and designed the experiments and wrote the manuscript. H.N., S.F., L.B., N.M., T.N. and R.I. performed the structural analyses. H.I., K.S. and M.K.K. performed biochemical and biological analyses. K.N. and J.A. performed PLD activity assays. All authors discussed the data and the manuscript. M.C.S. and O.N. supervised all the work.

Corresponding authors

Correspondence to Mikiko C. Siomi or Osamu Nureki.

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The authors declare no competing financial interests.

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This file contains Supplementary Table 1, Supplementary Figures 1-10 and an additional reference. (PDF 4268 kb)

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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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