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

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Abstract

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.

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Figure 1: The endonuclease activity of Mili is required for spermatogenesis and L1 silencing.
Figure 2: piRNA amplification failure in Mili DAH mice.
Figure 3: Marked reduction of Miwi2-bound piRNAs in Mili DAH mice.
Figure 4: Normal spermatogenesis and transposon silencing in Miwi2 DAH mice.

Accession codes

Primary accessions

ArrayExpress

Data deposits

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.

Change history

  • 30 October 2011

    The ArrayExpress data accession number was corrected.

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Acknowledgements

We are grateful to R. Pillai, B. Cullen, S. Martin, S. Chuma and J. Lykke-Andersen for antibodies used in this study. This study was technically supported by of EMBL’s genomic core facility. We are very grateful to V. Benes, R. Pillai and S. van Dongen for advice. We are grateful to M. Reuter for assistance with the preparation of immunoprecipitations for mass spectroscopy. This study was technically supported by EMBL Monterotondo’s FACS and Microscopy core facilities. We are grateful to A. Wutz for A9 ES cells. We also acknowledge the services of J. Rientjes from Monash University’s Gene Recombineering Facility. We are also very grateful to C. Kutter and D. Odom for advice on small RNA library generation.

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Authors and Affiliations

Authors

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.

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Correspondence to Dónal O’Carroll.

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The authors declare no competing financial interests.

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De Fazio, S., Bartonicek, N., Di Giacomo, M. et al. The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements. Nature 480, 259–263 (2011). https://doi.org/10.1038/nature10547

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