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Mitochondrial double-stranded RNA triggers antiviral signalling in humans

Abstract

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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Fig. 1: Mitochondria form dsRNA that is suppressed by the RNA degradosome.
Fig. 2: PNPase suppresses a mtdsRNA-mediated type I interferon response.
Fig. 3: Pathological PNPT1 mutations result in mtdsRNA accumulation and activation of ISGs.
Fig. 4: MDA5 is the primary sensor of cytosolic mtdsRNA released in a Bax–Bak dependent fashion.

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References

  1. Aloni, Y. & Attardi, G. Symmetrical in vivo transcription of mitochondrial DNA in HeLa cells. Proc. Natl Acad. Sci. USA 68, 1757–1761 (1971).

    Article  ADS  PubMed  CAS  Google Scholar 

  2. Young, P. G. & Attardi, G. Characterization of double-stranded RNA from HeLa cell mitochondria. Biochem. Biophys. Res. Commun. 65, 1201–1207 (1975).

    Article  PubMed  CAS  Google Scholar 

  3. Borowski, L. S., Dziembowski, A., Hejnowicz, M. S., Stepien, P. P. & Szczesny, R. J. Human mitochondrial RNA decay mediated by PNPase–hSuv3 complex takes place in distinct foci. Nucleic Acids Res. 41, 1223–1240 (2013).

    Article  PubMed  CAS  Google Scholar 

  4. Karikó, K., Buckstein, M., Ni, H. & Weissman, D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23, 165–175 (2005).

    Article  PubMed  CAS  Google Scholar 

  5. Schlee, M. & Hartmann, G. Discriminating self from non-self in nucleic acid sensing. Nat. Rev. Immunol. 16, 566–580 (2016).

    Article  PubMed  CAS  Google Scholar 

  6. McGlasson, S., Jury, A., Jackson, A. & Hunt, D. Type I interferon dysregulation and neurological disease. Nat. Rev. Neurol. 11, 515–523 (2015).

    Article  PubMed  CAS  Google Scholar 

  7. Rongvaux, A. et al. Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA. Cell 159, 1563–1577 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. West, A. P. et al. Mitochondrial DNA stress primes the antiviral innate immune response. Nature 520, 553–557 (2015).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  9. Weber, F., Wagner, V., Rasmussen, S. B., Hartmann, R. & Paludan, S. R. Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J. Virol. 80, 5059–5064 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Spadafora, D., Kozhukhar, N., Chouljenko, V. N., Kousoulas, K. G. & Alexeyev, M. F. Methods for efficient elimination of mitochondrial DNA from cultured cells. PLoS ONE 11, e0154684 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Szczesny, R. J. et al. Human mitochondrial RNA turnover caught in flagranti: involvement of hSuv3p helicase in RNA surveillance. Nucleic Acids Res. 38, 279–298 (2010).

    Article  PubMed  CAS  Google Scholar 

  12. Wang, G. et al. PNPASE regulates RNA import into mitochondria. Cell 142, 456–467 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Hornig-Do, H. T. et al. Isolation of functional pure mitochondria by superparamagnetic microbeads. Anal. Biochem. 389, 1–5 (2009).

    Article  PubMed  CAS  Google Scholar 

  14. Vedrenne, V. et al. Mutation in PNPT1, which encodes a polyribonucleotide nucleotidyltransferase, impairs RNA import into mitochondria and causes respiratory-chain deficiency. Am. J. Hum. Genet. 91, 912–918 (2012). 

    Article  PubMed  CAS  Google Scholar 

  15. Matilainen, S. et al. Defective mitochondrial RNA processing due to PNPT1 variants causes Leigh syndrome. Hum. Mol. Genet. 26, 3352–3361 (2017). 

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Rodero, M. P. et al. Detection of interferon alpha protein reveals differential levels and cellular sources in disease. J. Exp. Med. 214, 1547–1555 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Murr, C., Widner, B., Wirleitner, B. & Fuchs, D. Neopterin as a marker for immune system activation. Curr. Drug Metab. 3, 175–187 (2002).

    Article  PubMed  CAS  Google Scholar 

  18. White, M. J. et al. Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production. Cell 159, 1549–1562 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Mannion, N. M. et al. The RNA-editing enzyme ADAR1 controls innate immune responses to RNA. Cell Rep. 9, 1482–1494 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Hart, T. et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163, 1515–1526 (2015).

    Article  PubMed  CAS  Google Scholar 

  21. West, A. P. & Shadel, G. S. Mitochondrial DNA in innate immune responses and inflammatory pathology. Nat. Rev. Immunol. 17, 363–375 (2017).

    Article  PubMed  CAS  Google Scholar 

  22. Oda, H. et al. Aicardi-Goutières syndrome is caused by IFIH1 mutations. Am. J. Hum. Genet. 95, 121–125 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Rice, G. I. et al. Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling. Nat. Genet. 46, 503–509 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Crow, Y. J. & Manel, N. Aicardi-Goutières syndrome and the type I interferonopathies. Nat. Rev. Immunol. 15, 429–440 (2015).

    Article  PubMed  CAS  Google Scholar 

  25. Rutsch, F. et al. A specific IFIH1 gain-of-function mutation causes Singleton-Merten syndrome. Am. J. Hum. Genet. 96, 275–282 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Lincez, P. J., Shanina, I. & Horwitz, M. S. Reduced expression of the MDA5 gene IFIH1 prevents autoimmune diabetes. Diabetes 64, 2184–2193 (2015).

    Article  PubMed  CAS  Google Scholar 

  27. Gorman, J. A. et al. The A946T variant of the RNA sensor IFIH1 mediates an interferon program that limits viral infection but increases the risk for autoimmunity. Nat. Immunol. 18, 744–752 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Hertzog, J. et al. Infection with a Brazilian isolate of Zika virus generates RIG-I stimulatory RNA and the viral NS5 protein blocks type I IFN induction and signaling. Eur. J. Immunol. https://doi.org/10.1002/eji.201847483 (2018).

  29. Deddouche, S. et al. Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells. eLife 3, e01535 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Meyer, S. et al. AIRE-deficient patients harbor unique high-affinity disease-ameliorating autoantibodies. Cell 166, 582–595 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Nojima, T., Dienstbier, M., Murphy, S., Proudfoot, N. J. & Dye, M. J. Definition of RNA polymerase II CoTC terminator elements in the human genome. Cell Reports 3, 1080–1092 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Nishimura, N. & Yano, M. Separation of the inner and outer mitochondrial membrane in HeLa cells. Bio Protoc. 4, e1299 (2014).

    Article  Google Scholar 

  33. Croft, D. et al. Reactome: a database of reactions, pathways and biological processes. Nucleic Acids Res. 39, D691–D697 (2011).

    Article  PubMed  CAS  Google Scholar 

  34. Johnson, E. et al. Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins. Sci. Rep. 5, 9583 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Kent, W. J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Picardi, E. & Pesole, G. REDItools: high-throughput RNA editing detection made easy. Bioinformatics 29, 1813–1814 (2013).

    Article  PubMed  CAS  Google Scholar 

  40. Hodgkinson, A. et al. High-resolution genomic analysis of human mitochondrial RNA sequence variation. Science 344, 413–415 (2014).

    Article  ADS  PubMed  CAS  Google Scholar 

  41. Blau, N. et al. Cerebrospinal fluid pterins and folates in Aicardi–Goutieres syndrome: a new phenotype. Neurology 61, 642–647 (2003).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

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.

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Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

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

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The authors declare no competing interests.

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Extended data figures and tables

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.

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.

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.

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.

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.

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

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.

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.

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

Supplementary information

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Source Data for Extended Data Figure 6

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Dhir, A., Dhir, S., Borowski, L.S. et al. Mitochondrial double-stranded RNA triggers antiviral signalling in humans. Nature 560, 238–242 (2018). https://doi.org/10.1038/s41586-018-0363-0

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