Letter | Published:

Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis

Nature volume 555, pages 260264 (08 March 2018) | Download Citation

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Abstract

PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that bind to PIWI proteins to control transposons and maintain genome integrity in animal germ lines1,2,3. 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)4, and piRNA intermediates are bound with PIWI anchored onto mitochondrial Tudor domain protein Papi5. 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 Drosophila6,7,8, 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 ‘slicer’ activity, yet no phasing pattern was observed in mature piRNAs. The loss of Zuc did not affect the 5′- and 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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Acknowledgements

We are grateful to T. Mannen for preparing materials for mass spectrometry, T. Suzuki for comments on our in vitro Zuc processing assays and Y. Ono for support with the bioinformatics. We also thank S. Ohnishi for technical assistance and other members of the Siomi laboratories for discussions and comments on the manuscript. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan to K.M.N., Y.W.I., Y.M., H.S. and M.C.S. R.M. is supported by CREST, the Japan Science and Technology Agency. T.Ka. was supported by grants from the New Energy and Industrial Technology Development Organization, Japan, and Translational Systems Biology and Medicine Initiative from the Ministry of Education, Culture, Sports, Science and Technology of Japan. T.Ko. is a recipient of Molecular Dynamics for Antibody Drug Development, First Program Grant from the Japan Society of Promotion of Science.

Author information

Author notes

    • Kazumichi M. Nishida
    •  & Kazuhiro Sakakibara

    These authors contributed equally to this work.

Affiliations

  1. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan

    • Kazumichi M. Nishida
    • , Kazuhiro Sakakibara
    • , Hiromi Yamada
    • , Ryo Murakami
    • , Yukiko Murota
    •  & Mikiko C. Siomi
  2. Department of Molecular Biology, Keio University School of Medicine, Tokyo 162-8582, Japan

    • Yuka W. Iwasaki
    •  & Haruhiko Siomi
  3. Proteomics Laboratory, Isotope Science Center, The University of Tokyo, Tokyo 113-0032, Japan

    • Takeshi Kawamura
  4. Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan

    • Takeshi Kawamura
    •  & Tatsuhiko Kodama

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Contributions

K.M.N. generated monoclonal antibodies and performed biochemical analyses of piRNAs, int-piRNAs and piRNA factors. K.S. carried out in vitro experiments with help from R.M. H.Y. performed protein–protein interaction analyses. Y.M. performed immunofluorescence analyses. Y.W.I. performed bioinformatics analyses. T.Ka. and T.Ko. performed LC–MS/MS analysis. M.C.S. designed the experiments with other authors, supervised and discussed the work, and wrote the manuscript. H.S. discussed and supervised the study. All authors commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mikiko C. Siomi.

Reviewer Information Nature thanks J. Brennecke, S. Chameyron and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Supplementary information

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    Life Sciences Reporting Summary

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    Supplementary Figures

    This file contains uncropped gel data for Supplementary Figure 1 and Extended Data Figures.

Excel files

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    Supplementary Table 1

    This file contains all identified Siwi and Ago3 peptides with arginine modifications.

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DOI

https://doi.org/10.1038/nature25788

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