Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing

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Repetitive-element-derived Piwi-interacting RNAs (piRNAs)1, 2 act together with Piwi proteins Mili (also known as Piwil2) and Miwi2 (also known as Piwil4) in a genome defence mechanism that initiates transposon silencing via DNA methylation in the mouse male embryonic germ line. This silencing depends on the participation of the Piwi proteins in a slicer-dependent piRNA amplification pathway and is essential for male fertility3, 4. A third Piwi family member, Miwi (also known as Piwil1), is expressed in specific postnatal germ cells and associates with a unique set of piRNAs of unknown function5, 6, 7. Here we show that Miwi is a small RNA-guided RNase (slicer) that requires extensive complementarity for target cleavage in vitro. Disruption of its catalytic activity in mice by a single point mutation causes male infertility, and mutant germ cells show increased accumulation of LINE1 retrotransposon transcripts. We provide evidence for Miwi slicer activity directly cleaving transposon messenger RNAs, offering an explanation for the continued maintenance of repeat-derived piRNAs long after transposon silencing is established in germline stem cells. Furthermore, our study supports a slicer-dependent silencing mechanism that functions without piRNA amplification. Thus, Piwi proteins seem to act in a two-pronged mammalian transposon silencing strategy: one promotes transcriptional repression in the embryo, the other reinforces silencing at the post-transcriptional level after birth.

At a glance


  1. Miwi is a slicer requiring extensive complementarity for target cleavage.
    Figure 1: Miwi is a slicer requiring extensive complementarity for target cleavage.

    a, Coomassie-stained immunopurified (BVI antibody) Miwi complexes and 5′-end-labelled associated small RNAs (piRNAs). IP, immunoprecipitates. HC and LC are antibody heavy and light chains, respectively. b, Slicer assays with Miwi complexes (purified with BVI or BTO antibodies) or beads alone (contol) and a 5′-end-labelled RNA target bearing complementarity to endogenous piR-A (detected by northern blotting, bottom). Addition of EDTA abolishes Miwi slicer activity. Arrow indicates the 5′ cleavage product. c, d, Slicer assays with target RNAs carrying mismatches at indicated positions relative to the guide piRNA. Protein molecular weight markers in kDa and RNA markers in nucleotides (nt) are shown.

  2. Miwi catalytic activity is essential for spermatogenesis.
    Figure 2: Miwi catalytic activity is essential for spermatogenesis.

    a, Slicer assays with Miwi complexes from the Miwi+/ADH mutant. Total piRNAs are 5′-end labelled, whereas specific piRNAs are detected by northern blotting. b, Haematoxylin and eosin-stained testis sections showing the spermatogenic arrest in different indicated Miwi genotypes. ES, elongating spermatids; RS, round spermatids; PS, pachytene spermatocyes. c, Slicer assay with MiwiADH complex from the Miwi−/ADH mutant. d, Electron micrographs showing morphologically aberrant elongating spermatids in the Miwi+/ADH mutant. e, Electron micrographs of round spermatids showing the chromatoid body (CB) or its absence (residual density marked with a bracket) in Miwi mutants. N, nucleus.

  3. Primary piRNA biogenesis is globally unaffected in the MiwiADH mutants and mRNAs are not targeted by piRNAs.
    Figure 3: Primary piRNA biogenesis is globally unaffected in the MiwiADH mutants and mRNAs are not targeted by piRNAs.

    a, Ethidium bromide staining of total small RNAs. b, Miwi-associated piRNAs detected by 5′-end labelling from indicated Miwi genotypes. c, Normalized density of Miwi piRNA reads over a genomic cluster. d, MA plots showing comparison of gene expression profiles in purified round spermatids from indicated Miwi genotypes. Significantly (P<0.001) changed genes are highlighted in red. The top 100 genes having either exonic sense (red) or exonic antisense (green) piRNAs are highlighted (bottom right). e, Northern blot analyses of select (circled in d) late spermatogenesis genes and loading control (rRNA staining with ethidium bromide) are shown.

  4. Miwi piRNAs target L1 transposons.
    Figure 4: Miwi piRNAs target L1 transposons.

    a, Scatter plot showing fold change of repeat reads in the RNA-seq library from the Miwi−/ADH round spermatids compared to control (+/−). LINE-specific reads are indicated in red and upregulated LINEs are circled. b, Quantitative RT–PCR analysis of L1 transcripts in whole testis and purified round spermatids. Each bar represents individual biological replicate and standard deviation (s.d.) between technical replicates within a biological sample is indicated. c, d, Detection of L1ORF1p in purified round spermatids by western blotting (c) and by immunofluorescence analysis (d) of testis sections. e, Genome-wide 5′-end overlap analysis of global 5′-RACE tags and Miwi piRNAs (top). Similar overlaps over a specific L1 sequence (bottom). f, 5′-end overlap analysis of Miwi piRNAs. The s.d. between three independent biological replicates is indicated.

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Gene Expression Omnibus


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Author information


  1. European Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, 38042 Grenoble, France

    • Michael Reuter,
    • Philipp Berninger &
    • Ramesh S. Pillai
  2. CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell Interactions (UVHCI), 38042 Grenoble, France

    • Michael Reuter,
    • Philipp Berninger &
    • Ramesh S. Pillai
  3. Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan

    • Shinichiro Chuma &
    • Mihoko Hosokawa
  4. Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto 606-8501, Japan

    • Shinichiro Chuma &
    • Mihoko Hosokawa
  5. Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York 10029, USA

    • Hardik Shah &
    • Ravi Sachidanandam
  6. European Molecular Biology Laboratory, EMBL Meyerhof Str. 1, 69117 Heidelberg, Germany

    • Charlotta Funaya &
    • Claude Antony


M.R. performed all biochemical and deep sequencing experiments. S.C., M.R. and M.H. performed imaging. C.F. and C.A. performed electron microscopy analysis. P.B., R.S., R.S.P. and H.S performed bioinformatics analysis. R.S.P., M.R., S.C. and R.S. prepared the manuscript. M.R. and R.S.P. designed research.

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The authors declare no competing financial interests.

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Deep sequencing and microarray data related to this study are deposited with the Gene Expression Omnibus under accession GSE32183.

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