The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements

Journal name:
Nature
Volume:
480,
Pages:
259–263
Date published:
DOI:
doi:10.1038/nature10547
Received
Accepted
Published online
Corrected online

Piwi proteins and Piwi-interacting RNAs (piRNAs) have conserved functions in transposon silencing1. The murine Piwi proteins Mili and Miwi2 (also called Piwil2 and Piwil4, respectively) direct epigenetic LINE1 and intracisternal A particle transposon silencing during genome reprogramming in the embryonic male germ line2, 3, 4. Piwi proteins are proposed to be piRNA-guided endonucleases that initiate secondary piRNA biogenesis5, 6, 7; however, the actual contribution of their endonuclease activities to piRNA biogenesis and transposon silencing remain unknown. To investigate the role of Piwi-catalysed endonucleolytic activity, we engineered point mutations in mice that substitute the second aspartic acid to an alanine in the DDH catalytic triad of Mili and Miwi2, generating the MiliDAH and Miwi2DAH alleles, respectively. Analysis of Mili-bound piRNAs from homozygous MiliDAH fetal gonadocytes revealed a failure of transposon piRNA amplification, resulting in the marked reduction of piRNA bound within Miwi2 ribonuclear particles. We find that Mili-mediated piRNA amplification is selectively required for LINE1, but not intracisternal A particle, silencing. The defective piRNA pathway in MiliDAH mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2DAH mice are fertile, transposon silencing is established normally and no defects in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. We conclude that cycles of intra-Mili secondary piRNA biogenesis fuel piRNA amplification that is absolutely required for LINE1 silencing.

At a glance

Figures

  1. The endonuclease activity of Mili is required for spermatogenesis and L1 silencing.
    Figure 1: The endonuclease activity of Mili is required for spermatogenesis and L1 silencing.

    a, Testicular atrophy in MiliDAH mice. Testicular weights of 3-month-old wild-type (WT) and MiliDAH mice are shown. b, Haematoxylin- and eosin-stained wild-type and MiliDAH testis sections from 3-month-old mice. The percentage of MiliDAH mice with the indicated phenotype is shown. c, Immunofluorescence using anti-L1 Orf1 and anti-IAP Gag antibodies (green) and DAPI-stained DNA (blue) on wild-type and MiliDAH E16.5 fetal testis sections are shown. d, Western blot (WB) using anti-L1 Orf1 and anti-tubulin antibodies on extracts from P10 wild-type and MiliDAH testes is shown. e, The expression levels of L1 and IAP were quantified by qRT–PCR from RNA derived from P10 wild-type and MiliDAH testes. Error bars indicate standard deviation from biological triplicates (n = 3). f, Methylation-sensitive Southern blot on HpaII-digested DNA extracted from P10 wild-type and MiliDAH testis using a L1 promoter probe. The arrowhead indicates the identity of the methylation-sensitive fragment.

  2. piRNA amplification failure in MiliDAH mice.
    Figure 2: piRNA amplification failure in MiliDAH mice.

    a, Mili RNPs were immunoprecipitated from E16.5 fetal testis of the indicated genotypes. Associated piRNAs were visualized by 5′ 32P labelling, resolution on a 15% TBE urea gel and autoradiography. nt, nucleotide. b, Size profiles of cloned Mili-bound piRNAs from biological replicates of wild-type (blue) and MiliDAH (pink) E16.5 fetal gonadocytes. c, Genomic annotation of cloned Mili-bound piRNAs as indicated from pairs of biological replicates of wild-type and MiliDAH E16.5 fetal gonadocytes. LTR, long terminal repeat; ncRNA, non-coding RNA. d, Mapping of Mili-bound piRNAs to the consensus of L1 (left) and IAP (right) elements. Positive and negative values indicate sense and antisense piRNAs, respectively. Schematic representations of the respective elements are also shown (above). e, Percentage of piRNAs from wild-type and MiliDAH Mili RNPs with a U at the first position (1U) without A at position 10 and an A at position 10 (10A) without a U at position 1 are shown for L1 and IAP elements. Error bars indicate standard error of the mean from the biological duplicates (n = 2). f, Ping-pong analysis of Mili-bound piRNAs from biological replicates of wild-type and MiliDAH E16.5 fetal gonadocytes. Relative frequency (y axis) of distances between 5′ ends (x axis) between complementary piRNAs for both L1 and IAP elements is shown. nt, nucleotides.

  3. Marked reduction of Miwi2-bound piRNAs in MiliDAH mice.
    Figure 3: Marked reduction of Miwi2-bound piRNAs in MiliDAH mice.

    a, Miwi2 RNPs were immunoprecipitated from E16.5 fetal testis of the indicated genotypes and piRNA represented as in Fig. 2a. b, Confocal projection images of indirect immunofluorescence with Tdrd9, Dcp1a and Miwi2 antibodies (green) and DAPI-stained DNA (red) from E16.5 fetal testis of the indicated genotypes. c, Annotation of cloned Miwi2-bound piRNAs as indicated from biological replicates of wild-type and MiliDAH E16.5 fetal gonadocytes. d, Mapping of the Miwi2-bound piRNAs to the consensus of L1 (left) and IAP (right) elements. Positive and negative values indicate sense and antisense piRNAs, respectively. Schematic representations of the respective elements are also shown.

  4. Normal spermatogenesis and transposon silencing in Miwi2DAH mice.
    Figure 4: Normal spermatogenesis and transposon silencing in Miwi2DAH mice.

    a, Haematoxylin- and eosin-stained wild-type, Miwi2DAH and Miwi2−/− testis sections from 3-month-old mice. b, Immunofluorescence using anti-L1 Orf1 and anti-IAP Gag antibodies (green) and DAPI-stained DNA (blue) on wild-type, Miwi2DAH and Miwi2−/− E16.5 fetal testis sections are shown. c, Methylation-sensitive Southern blot on HpaII-digested DNA extracted from P10 wild-type, Miwi2DAH and Miwi2−/− testis using a L1 promoter probe is shown. The arrow indicates the identity of the methylation-sensitive fragment. d, Mili (left) and Miwi2 (right) RNPs were immunoprecipitated from E16.5 fetal testis of the indicated genotypes shown as in Fig. 2a. e, Mapping of the Mili-bound (left) and Miwi2-bound (right) piRNAs to the consensus of L1. Positive and negative values indicate sense and antisense piRNAs, respectively. Schematic representation of L1 is shown (above). f, Ping-pong analysis of Mili- and Miwi2-bound piRNAs from biological replicates of wild-type and Miwi2DAH E16.5 fetal gonadocytes are shown. The frequency of the distance between 5′ ends of complementary piRNAs for L1 is presented as in Fig. 2e. g, Model. L1 element silencing is dependent upon Mili’s endonuclease activity for piRNA amplification. IAP silencing is dependent upon Mili and Miwi2 but independent of piRNA amplification. The small blue and green lines represent sense and antisense piRNAs, respectively.

Accession codes

Primary accessions

ArrayExpress

Change history

Corrected online 30 October 2011
The ArrayExpress data accession number was corrected.

References

  1. Ghildiyal, M. & Zamore, P. D. Small silencing RNAs: an expanding universe. Nature Rev. Genet. 10, 94108 (2009)
  2. Aravin, A. A., Sachidanandam, R., Girard, A., Fejes-Toth, K. & Hannon, G. J. Developmentally regulated piRNA clusters implicate MILI in transposon control. Science 316, 744747 (2007)
  3. Carmell, M. A. et al. MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline. Dev. Cell 12, 503514 (2007)
  4. Kuramochi-Miyagawa, S. et al. DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. Genes Dev. 22, 908917 (2008)
  5. Brennecke, J. et al. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128, 10891103 (2007)
  6. Gunawardane, L. S. et al. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila. Science 315, 15871590 (2007)
  7. Aravin, A. A. et al. A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. Mol. Cell 31, 785799 (2008)
  8. Song, J. J., Smith, S. K., Hannon, G. J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 14341437 (2004)
  9. Patel, D. J. et al. Structural biology of RNA silencing and its functional implications. Cold Spring Harb. Symp. Quant. Biol. 71, 8193 (2006)
  10. Saito, K. et al. Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev. 20, 22142222 (2006)
  11. Bestor, T. H. & Bourc’his, D. Transposon silencing and imprint establishment in mammalian germ cells. Cold Spring Harb. Symp. Quant. Biol. 69, 381388 (2004)
  12. Cheloufi, S., Dos Santos, C. O., Chong, M. M. & Hannon, G. J. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 465, 584589 (2010)
  13. Cifuentes, D. et al. A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity. Science 328, 16941698 (2010)
  14. O’Carroll, D. et al. A Slicer-independent role for Argonaute 2 in hematopoiesis and the microRNA pathway. Genes Dev. 21, 19992004 (2007)
  15. Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 14371441 (2004)
  16. Maniataki, E. & Mourelatos, Z. A human, ATP-independent, RISC assembly machine fueled by pre-miRNA. Genes Dev. 19, 29792990 (2005)
  17. Aravin, A. A. et al. Cytoplasmic compartmentalization of the fetal piRNA pathway in mice. PLoS Genet. 5, e1000764 (2009)
  18. Kojima, K. et al. Associations between PIWI proteins and TDRD1/MTR-1 are critical for integrated subcellular localization in murine male germ cells. Genes Cells 14, 11551165 (2009)
  19. Reuter, M. et al. Loss of the Mili-interacting Tudor domain-containing protein-1 activates transposons and alters the Mili-associated small RNA profile. Nature Struct. Mol. Biol. 16, 639646 (2009)
  20. Vagin, V. V. et al. Proteomic analysis of murine Piwi proteins reveals a role for arginine methylation in specifying interaction with Tudor family members. Genes Dev. 23, 17491762 (2009)
  21. Wang, J., Saxe, J. P., Tanaka, T., Chuma, S. & Lin, H. Mili interacts with tudor domain-containing protein 1 in regulating spermatogenesis. Curr. Biol. 19, 640644 (2009)
  22. Zheng, K. et al. Mouse MOV10L1 associates with Piwi proteins and is an essential component of the Piwi-interacting RNA (piRNA) pathway. Proc. Natl Acad. Sci. USA 107, 1184111846 (2010)
  23. Chuma, S. et al. Tdrd1/Mtr-1, a tudor-related gene, is essential for male germ-cell differentiation and nuage/germinal granule formation in mice. Proc. Natl Acad. Sci. USA 103, 1589415899 (2006)
  24. Kuramochi-Miyagawa, S. et al. MVH in piRNA processing and gene silencing of retrotransposons. Genes Dev. 24, 887892 (2010)
  25. Kuramochi-Miyagawa, S. et al. Mili, a mammalian member of piwi family gene, is essential for spermatogenesis. Development 131, 839849 (2004)
  26. Elbashir, S. M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188200 (2001)
  27. Martinez, J. & Tuschl, T. RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes Dev. 18, 975980 (2004)
  28. Mandal, P. K. & Kazazian, H. H., Jr SnapShot: Vertebrate transposons. Cell 135, 192192 (2008)
  29. Wen, J. & Brogna, S. Nonsense-mediated mRNA decay. Biochem. Soc. Trans. 36, 514516 (2008)
  30. Robanus-Maandag, E. et al. p107 is a suppressor of retinoblastoma development in pRb-deficient mice. Genes Dev. 12, 15991609 (1998)
  31. Poueymirou, W. T. et al. F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nature Biotechnol. 25, 9199 (2007)
  32. Farley, F. W., Soriano, P., Steffen, L. S. & Dymecki, S. M. Widespread recombinase expression using FLPeR (flipper) mice. Genesis 28, 106110 (2000)
  33. Schwenk, F., Baron, U. & Rajewsky, K. A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res. 23, 50805081 (1995)
  34. Yoshimizu, T. et al. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev. Growth Differ. 41, 675684 (1999)
  35. Hafner, M. et al. Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods 44, 312 (2008)
  36. Gentleman, R. C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004)
  37. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)
  38. Flicek, P. et al. Ensembl 2011. Nucleic Acids Res. 39, D800D806 (2011)
  39. Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J. & Wheeler, D. L. GenBank. Nucleic Acids Res. 36, D25D30 (2008)
  40. Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 16391645 (2009)

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Author information

  1. These authors contributed equally to this work.

    • Nenad Bartonicek &
    • Monica Di Giacomo

Affiliations

  1. European Molecular Biology Laboratory, Mouse Biology Unit, Via Ramarini 32, Monterotondo Scalo 00015, Italy

    • Serena De Fazio,
    • Monica Di Giacomo,
    • Aditya Sankar,
    • Pedro N. Moreira &
    • Dónal O’Carroll
  2. European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, Cambridge CB10 1SD, UK

    • Nenad Bartonicek,
    • Cei Abreu-Goodger &
    • Anton J. Enright
  3. European Molecular Biology Laboratory, EMBL Meyerhof Str. 1, 69117 Heidelberg, Germany

    • Charlotta Funaya &
    • Claude Antony

Contributions

S.D.F. contributed to the design, execution and analysis of the majority of experiments on MiliDAH and Miwi2DAH mice. N.B. performed the bioinformatic analysis presented in the manuscript with initial assistance from C.A.-G. M.D.G. analysed the spermatogenic defects as well as undertook the co-localization studies in the respective mouse strains. A.S. performed the bisulphite sequencing experiments. C.F. and C.A. performed the electron microscopy experiments. P.N.M. established the 8-cell embryo ES cell injection procedure. A.J.E. supervised the bioinformatic analysis. D.O’C. conceived and supervised this study and wrote the final version of the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

All raw sequencing data are deposited in ArrayExpress (accession number E-MTAB-730) and European Nucleotide Archive (ERP000778). The MiliDAH, Miwi2DAH and Miwi2 null (Miwi2) alleles have been deposited at EMMA (http://www.emmanet.org/) and will be freely available on a non-collaborative basis.

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