Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts, which are capable of forming long double-stranded RNA structures1,2. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single-cell level and identify key roles for the degradosome components mitochondrial RNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms that have evolved to protect vertebrates against microbial and viral attack.

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We acknowledge V. Bondet for CSF IFNα data. We also thank E. Johnson and A. Pielach from the Dunn School Bioimaging facility for electron microscopy work and M. Alexeyev for sharing plasmids encoding UL12.5M185 and mUNG1 (Addgene #70109 and #70110, respectively). This work was supported by funding to N.J.P. (Wellcome Trust Investigator Award (107928|Z|15|Z), ERC Advanced Grant (339270)) and to M.T. (National Institutes of Health (NIH) (GM073981)). Y.J.C. acknowledges funding from the European Research Council (GA 309449: fellowship), a state subsidy managed by the National Research Agency (ANR, France) under the Investments for the Future (ANR-10-IAHU-01), and an ANR grant CE17001002 to Y.J.C. and D.D. Y.J.C. and D.D. thank ImmunoQure AG for sharing of the antibodies used to assess IFNα protein levels in the Simoa assay. Studies were supported by grant 2014/13/D/NZ2/01114 (to R.J.S.) from the National Science Centre, Poland. Experiments were carried out with the use of CePT infrastructure financed by the European Union through the European Regional Development Fund (Innovative economy 2007–13, Agreement POIG.02.02.00-14-024/08-00).

Reviewer information

Nature thanks C. Reis e Sousa, S. Riis Paludan, G. Shadel and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Somdutta Dhir, Lukasz S. Borowski


  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    • Ashish Dhir
    • , Somdutta Dhir
    • , Takayuki Nojima
    • , Claudia Ribeiro de Almeida
    •  & Nicholas J. Proudfoot
  2. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

    • Lukasz S. Borowski
    • , Andrzej Dziembowski
    •  & Roman J. Szczesny
  3. Faculty of Biology, University of Warsaw, Warsaw, Poland

    • Lukasz S. Borowski
    • , Andrzej Dziembowski
    •  & Roman J. Szczesny
  4. Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    • Laura Jimenez
    •  & Michael Teitell
  5. INSERM UMR1163, Institut Imagine, Paris, France

    • Agnès Rötig
    • , Yanick J. Crow
    • , Christelle Tamby
    • , Arnold Munnich
    •  & Manuel Schiff
  6. Paris Descartes University, Sorbonne-Paris-Cité, Institut Imagine, Paris, France

    • Yanick J. Crow
  7. Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK

    • Yanick J. Crow
  8. Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK

    • Gillian I. Rice
  9. Immunobiology of Dendritic Cells, Institut Pasteur, Paris, France

    • Darragh Duffy
  10. INSERM U1223, Paris, France

    • Darragh Duffy
  11. MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK

    • Jan Rehwinkel


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A.Dh. conceived the study, designed and performed most of the experiments and drafted the paper. S.D. performed the bioinformatics analysis. L.S.B., R.J.S. and A.Dz. performed the experiments in Fig. 1c–f and Extended Data Figs. 1c, d, g, 2c–e, 3, 4. L.J. and M.T. generated the immunofluorescence and gene expression data on PNPase HepKO. A.R., A.M. and M.S. provided patient fibroblasts and the clinical data. C.T. provided the PNPase western blot (Fig. 3a). Y.J.C. and G.I.R. generated the blood ISG expression data. D.D. provided the IFNα CSF data. T.N. provided Extended Data Fig. 1e. C.R.A. performed FACS analysis. J.R. provided the MEF KO cells and agonists. A.Dh. discussed and interpreted results with inputs from N.J.P., R.J.S. and the other authors. N.J.P. and A.Dh. wrote the paper with input from the other authors.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Ashish Dhir or Roman J. Szczesny or Nicholas J. Proudfoot.

Extended data figures and tables

  1. Extended Data Fig. 1 Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation.

    a, RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b, Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 µm. c, Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 µm. d, Quantification of fluorescence signal from HeLa cells treated as in c. Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e, Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly 32P-radiolabelled ssRNA and dsRNA substrates. Signals were visualized and quantified by PhosphorImager. The level of immunoprecipitation signal is shown and expressed as the percentage of input. Images and data are representative of two experiments. For gel source data, see Supplementary Fig. 1. f, Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g, Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 µm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.

  2. Extended Data Fig. 2 RNA degradosome components SUV3 and PNPase are involved in mtdsRNA turnover.

    a, HeLa cells treated with DMSO, DRB (100 μM) and actinomycin-D (0.5 μg ml−1) for 60 min and stained with anti-dsRNA (J2) antibody (green). Mitochondria were stained with MitoTracker Red CMXRos and nuclei with DAPI (blue) (representative of two experiments). b, Flow cytometric analysis of dsRNA levels in HeLa cells treated with the indicated siRNAs. Cells were labelled with J2 antibody or an isotype control. Data are mean ± s.d. from three independent experiments. c, Left, detection of dsRNA with J2 antibody in HeLa cells after depletion of PNPase or SUV3 by On-TARGETplus siRNAs (indicated with an asterisk and listed in Extended Data Table 2). Mitochondria were stained with MitoTracker Deep Red. Nuclei are stained with Hoechst (blue). Scale bars, 10 μm. Right top, western blot showing PNPase or SUV3 depletion. Blots are representative of four experiments. For gel source images, see Supplementary Fig. 1. Far right top, Quantification of dsRNA levels in HeLa cells depleted of PNPase or SUV3. Data are mean ± s.d. from four independent experiments. d, Quantitative analysis of fluorescent signals from dsRNA in HeLa cells with depleted enzymes involved in mitochondrial nucleic acids metabolism. Data are mean ± s.d. from four independent experiments. e, HeLa cells were transfected with siRNA specific for PNPase, SUV3, or non-targeting control. Prior to fixation, cells were treated for indicated times with inhibitors of transcription: actinomycin-D (0.5 μg ml−1) or DRB (100 μM). Immunostaining of dsRNA was performed and cells were imaged using a fluorescent microscope screening station. Data are mean ± s.d. from four independent experiments.

  3. Extended Data Fig. 3 Unwinding activity of SUV3 is required to suppress mtdsRNA accumulation.

    a, Left, confocal images of HEK 293 cells expressing stably integrated wild-type SUV3 (hSUV3_WT) and the catalytically inactive (G207V) dominant negative form (hSUV3_G207V) stained with J2 ab (green). Mitochondria stained with MitoTracker Deep Red (red). Nuclei stained with Hoechst (blue). Right, quantitative analysis of fluorescence signal from HEK 293 cells in the above experiment. Data are mean ± s.e.m. from 16 cells. b, Top, northern blots of J2 immunoprecipitated dsRNA from hSUV3_WT and hSUV3_G207V overexpressing HEK 293 cell lines with four different probes spanning the entire mitochondrial genome. Bottom, diagram depicts positions of probes on mitochondrial genome. Blots are representative of two experiments. For gel source data, see Supplementary Fig. 1.

  4. Extended Data Fig. 4 Exonuclease activity of PNPase is required to suppress mtdsRNA formation.

    a, Diagram of PNPase domain structure showing the position of the R445E/R446E mutation in the RNasePH domain. b, Immunostaining of dsRNA (green) in HeLa stable cell lines transfected with siRNA specific for PNPase or non-targeting control siRNA. Depletion of endogenous PNPase was rescued by expression of siRNA-resistant PNPase-FLAG protein (wild-type or mutated (RNA-degradation deficient version of PNPase (R445E/R446E) was expressed)). Mitochondria are stained with MitoTracker Deep Red. Nuclei are stained with Hoechst (blue). Scale bars, 20 μm. c, Western-blot analysis of PNPase in HeLa cells treated as in b. Exogenous, siRNA-resistant PNPase is expressed as a FLAG fusion. Blots are representative of three experiments. d, Quantitative analysis of fluorescent signals from dsRNA in HeLa treated as in b. Data are mean ± s.d. from three independent experiments. For gel source data, see Supplementary Fig. 1.

  5. Extended Data Fig. 5 dsRNA-seq of HeLa cells following siRNA depletion of SUV3 and PNPase.

    a, dsRNA-seq reads across the mitochondrial genome spanning entire protein coding region (~3.5–16 kb) following siRNA treatment. Data are from two independent experiments. H-strand genes are shown as blue bars and L-strand as red bars. Short tRNA genes are denoted with T as the first letter. b, Correlation plots of J2 immunoprecipitation dsRNA-seq replicates. Pearson correlation coefficients are calculated and shown on each plot.

  6. Extended Data Fig. 6 Upregulation of ISGs in HeLa and murine cells following loss of PNPase accentuated by mitochondrial outer membrane permeabilization.

    a, Heat map of ISGs generated from a subset extracted from a list of significantly expressed genes in siRNA-treated HeLa cells. Gene expression is depicted by colour intensity. Green denotes upregulation and red downregulation. b, RT–qPCR analysis of IFNB1 mRNA in HeLa cells treated with indicated siRNAs and then 8 h of treatment with vehicle or ABT-737. Data are the mean of two independent experiments. c, Western-blot analysis of the cytochrome c (cyt c) release into the cytoplasm of HeLa cells treated with vehicle or ABT-737 for 8 h. Subcellular fractionation purity confirmed by relevant markers. Blots are representative of two experiments. d, log2(fold change) expression levels of ISGs and genes involved in interferon signalling in HepKO versus wild-type female mice. ISG list is based on the Reactome database33. e, Western blot of whole-cell extracts from cells treated with the indicated siRNAs. Blots are representative of two experiments. For gel source data, see Supplementary Fig. 1. Source Data

  7. Extended Data Fig. 7 RNA editing of cytoplasmic mtRNA.

    a, RNA editing sites mapped on the RNA transcriptome of SUV3 and PNPase depleted cells are shown. Each dot represents an editing event. Dots on the upper panel denote editing events on the H-strand and dots on the lower panel denote editing on the L-strand. Triangle denotes single SUV3 editing site. Yellow bars denote the D-loop region. Short red bars denote tRNA genes on the L-strand and green bars denote tRNA genes on the H-strand. b, Frequency of dinucleotide RNA editing sites mapped in the PNPase depleted samples. c, RT–qPCR analysis of IFNB1 mRNA levels in indicated siRNA-treated cells. Data are the mean from two independent experiments. d, Western blot of ADAR1, SUV3 and PNPase in cell treated with the indicated siRNAs. Blots are representative of two experiments. For gel source data, see Supplementary Fig. 1.

  8. Extended Data Fig. 8 EMCV infection results in dsRNA accumulation that partially overlaps with mitochondria.

    a, Left, confocal images of EMCV-infected HeLa cell at MOI 1, 6 h after infection, stained with anti-dsRNA (J2) antibody. Mitochondria are stained with MitoTracker Red CMXRos and nucleus with DAPI. Right, line scan RGB profile for the region of interest (ROI) selected with a white line is shown on the right. Data are representative of two experiments. b, Expanded view of the ROI of an EMCV-infected HeLa cell showing colocalization of dsRNA with mitochondria. Image is representative of two experiments. Scale bars, 10 μm.

  9. Extended Data Table 1 Clinical table of patients carrying PNPT1 mutations
  10. Extended Data Table 2 Oligonucleotide primers and siRNAs used in the study

Supplementary information

  1. Supplementary Figures

    This file contains original source images for gels, western blots and northern blots.

  2. Reporting Summary

  3. Source Data for Figure 2

  4. Source Data for Extended Data Figure 6

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