Letter | Published:

Genetic and mechanistic diversity of piRNA 3′-end formation

Nature volume 539, pages 588592 (24 November 2016) | Download Citation



Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing1. Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI-interacting RNAs (piRNAs)—the 22–30-nt-long guides for PIWI-clade Ago proteins that silence transposons in animal gonads—are generated independently of Dicer from single-stranded precursors2,3. piRNA 5′ ends are defined either by Zucchini, the Drosophila homologue of mitoPLD—a mitochondria-anchored endonuclease4,5, or by piRNA-guided target cleavage6,7. Formation of piRNA 3′ ends is poorly understood. Here we report that two genetically and mechanistically distinct pathways generate piRNA 3′ ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub) or Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3′ ends8,9, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3′-to-5′ exoribonuclease Nibbler (Drosophila homologue of Mut-7)10,11,12,13. The relative activity of these two pathways dictates the extent to which piRNAs are directed to cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Notably, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway in which piRNA 5′ and 3′ ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data reveal a coherent model for piRNA biogenesis, and should aid the mechanistic dissection of the processes that govern piRNA 3′-end formation.

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We thank all laboratory members for help and discussions, P. Duchek, J. Gokcezade and K. Meixner for generating fly lines, M. Novatchkova for help on the conservation analysis of nucleases, the VBCF NGS facility for sequencing, and the MFPL monoclonal facility for the Nibbler antibody. This work was supported by the Austrian Academy of Sciences, the European Community’s 7th Framework Program (ERC-StG-260711; ERC-StG-338252), the Austrian Science Fund (Y510-B12; F4303-B09; W12-7-B09; Y733-B22), and an HFSP postdoctoral fellowship to F.M.

Author information

Author notes

    • Rippei Hayashi
    •  & Jakob Schnabl

    These authors contributed equally to this work.

    • Fabio Mohn

    Present address: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.


  1. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Dr. Bohrgasse 3, 1030 Vienna, Austria

    • Rippei Hayashi
    • , Jakob Schnabl
    • , Dominik Handler
    • , Fabio Mohn
    • , Stefan L. Ameres
    •  & Julius Brennecke


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F.M. made the initial observation that Nibbler trims Ago3-bound piRNAs, J.S. and R.H. did all experiments and did the computational analysis with the help of D.H. All authors designed the experiments and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Stefan L. Ameres or Julius Brennecke.

Reviewer Information Nature thanks B. Czech and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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    Supplementary Information

    This file contains the Source Data for Figures 1b, 2a,b, 4f and Extended Data Figures 2i, j, 3d, f as well as a Supplementary Table, which shows the high through datasets analyzed in the study.

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