Letter | Published:

Genetic and mechanistic diversity of piRNA 3′-end formation

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

Subjects

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Small silencing RNAs: an expanding universe. Nat. Rev. Genet. 10, 94–108 (2009)

  2. 2.

    & Small RNAs as guardians of the genome. Cell 136, 656–668 (2009)

  3. 3.

    , & PIWI-interacting RNA: its biogenesis and functions. Annu. Rev. Biochem. 84, 405–433 (2015)

  4. 4.

    et al. Structure and function of Zucchini endoribonuclease in piRNA biogenesis. Nature 491, 284–287 (2012)

  5. 5.

    , , , & The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis. Nature 491, 279–283 (2012)

  6. 6.

    et al. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128, 1089–1103 (2007)

  7. 7.

    et al. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila. Science 315, 1587–1590 (2007)

  8. 8.

    , , , & Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production. Science 348, 817–821 (2015)

  9. 9.

    , & Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis. Science 348, 812–817 (2015)

  10. 10.

    , , , & The 3′-to-5′ exoribonuclease Nibbler shapes the 3′ ends of microRNAs bound to Drosophila Argonaute1. Curr. Biol. 21, 1878–1887 (2011)

  11. 11.

    et al. The exoribonuclease Nibbler controls 3′ end processing of microRNAs in Drosophila. Curr. Biol. 21, 1888–1893 (2011)

  12. 12.

    et al. The exonuclease Nibbler regulates age-associated traits and modulates piRNA length in Drosophila. Aging Cell 14, 443–452 (2015)

  13. 13.

    et al. Antagonistic roles of Nibbler and Hen1 in modulating piRNA 3′ ends in Drosophila. Development 143, 530–539 (2016)

  14. 14.

    et al. Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis. Nat. Struct. Mol. Biol. 19, 773–781 (2012)

  15. 15.

    , , & 3′ end formation of PIWI-interacting RNAs in vitro. Mol. Cell 43, 1015–1022 (2011)

  16. 16.

    , , , & The cochaperone shutdown defines a group of biogenesis factors essential for all piRNA populations in Drosophila. Mol. Cell 47, 954–969 (2012)

  17. 17.

    et al. Identification and functional analysis of the Pre-piRNA 3′ trimmer in silkworms. Cell 164, 962–973 (2016)

  18. 18.

    , , & Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline. EMBO J. 32, 1869–1885 (2013)

  19. 19.

    et al. Krimper enforces an antisense bias on piRNA pools by binding AGO3 in the Drosophila germline. Mol. Cell 59, 553–563 (2015)

  20. 20.

    et al. Aub and Ago3 are recruited to Nuage through two mechanisms to form a ping-pong complex assembled by Krimper. Mol. Cell 59, 564–575 (2015)

  21. 21.

    et al. The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Curr. Biol. 17, 1265–1272 (2007)

  22. 22.

    et al. Pimet, the Drosophila homolog of HEN1, mediates 2′-O-methylation of Piwi- interacting RNAs at their 3′ ends. Genes Dev. 21, 1603–1608 (2007)

  23. 23.

    et al. Early origins and evolution of microRNAs and Piwi-interacting RNAs in animals. Nature 455, 1193–1197 (2008)

  24. 24.

    et al. Ancient and novel small RNA pathways compensate for the loss of piRNAs in multiple independent nematode lineages. PLoS Biol. 13, e1002061 (2015)

  25. 25.

    , , , & The RNase PARN-1 trims piRNA 3′ ends to promote transcriptome surveillance in C. elegans. Cell 164, 974–984 (2016)

  26. 26.

    et al. Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline. Mol. Cell 36, 231–244 (2009)

  27. 27.

    , , & piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire. Genes Dev. 29, 1747–1762 (2015)

  28. 28.

    et al. Slicing and binding by Ago3 or Aub trigger Piwi-bound piRNA production by distinct mechanisms. Mol. Cell 59, 819–830 (2015)

  29. 29.

    , , , & Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat. Genet. 40, 476–483 (2008)

  30. 30.

    , & Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila. G3 (Bethesda) 4, 2279–2282 (2014)

  31. 31.

    et al. A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat. Methods 8, 405–407 (2011)

  32. 32.

    et al. Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster. Nat. Methods 6, 431–434 (2009)

  33. 33.

    et al. Recombination-mediated genetic engineering of large genomic DNA transgenes. Methods Mol. Biol . 772, 445–458 (2011)

  34. 34.

    , , & Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res . 39, e141 (2011)

  35. 35.

    , & Argonaute loading improves the 5′ precision of both MicroRNAs and their miRNA* strand in flies. Curr. Biol. 18, 147–151 (2008)

  36. 36.

    , , & The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157, 1364–1379 (2014)

  37. 37.

    , , , & Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res . 35, e60 (2007)

  38. 38.

    et al. OrthoDB v8: update of the hierarchical catalog of orthologs and the underlying free software. Nucleic Acids Res . 43, D250–D256 (2015)

  39. 39.

    & Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 23, 127–128 (2007)

Download references

Acknowledgements

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.

Affiliations

  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

Authors

  1. Search for Rippei Hayashi in:

  2. Search for Jakob Schnabl in:

  3. Search for Dominik Handler in:

  4. Search for Fabio Mohn in:

  5. Search for Stefan L. Ameres in:

  6. Search for Julius Brennecke in:

Contributions

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.

Extended data

Supplementary information

PDF files

  1. 1.

    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.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature20162

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.