Abstract

Mitochondria are subcellular organelles that are critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, and on their coordinated translation, import and respiratory complex assembly. Here, we characterize EXD2 (exonuclease 3′–5′ domain-containing 2), a nuclear-encoded gene, and show that it is targeted to the mitochondria and prevents the aberrant association of messenger RNAs with the mitochondrial ribosome. Loss of EXD2 results in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of the Drosophila melanogaster EXD2 orthologue (CG6744) causes developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that is reversed with an antioxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and ageing.

  • Subscribe to Nature Cell Biology for full access:

    $59

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Kauppila, T. E., Kauppila, J. H. & Larsson, N. G. Mammalian mitochondria and aging: an update. Cell Metab. 25, 57–71 (2017).

  2. 2.

    Jourdain, A. A., Boehm, E., Maundrell, K. & Martinou, J. C. Mitochondrial RNA granules: compartmentalizing mitochondrial gene expression. J. Cell Biol. 212, 611–614 (2016).

  3. 3.

    Chinnery, P. F. Mitochondrial disease in adults: what’s old and what’s new? EMBO Mol. Med. 7, 1503–1512 (2015).

  4. 4.

    Yang, W. & Hekimi, S. Two modes of mitochondrial dysfunction lead independently to lifespan extension in Caenorhabditis elegans. Aging Cell 9, 433–447 (2010).

  5. 5.

    Schmeisser, S. et al. Neuronal ROS signaling rather than AMPK/sirtuin-mediated energy sensing links dietary restriction to lifespan extension. Mol. Metab. 2, 92–102 (2013).

  6. 6.

    Dillin, A. et al. Rates of behavior and aging specified by mitochondrial function during development. Science 298, 2398–2401 (2002).

  7. 7.

    Smogorzewska, A. et al. A genetic screen identifies FAN1, a Fanconi anemia-associated nuclease necessary for DNA interstrand crosslink repair. Mol. Cell 39, 36–47 (2010).

  8. 8.

    Broderick, R. et al. EXD2 promotes homologous recombination by facilitating DNA end resection. Nat. Cell Biol. 18, 271–280 (2016).

  9. 9.

    Cox, L. S., Clancy, D. J., Boubriak, I. & Saunders, R. D. Modeling Werner syndrome in Drosophila melanogaster: hyper-recombination in flies lacking WRN-like exonuclease. Ann. NY Acad. Sci. 1119, 274–288 (2007).

  10. 10.

    Biehs, R. et al. DNA double-strand break resection occurs during non-homologous end joining in G1 but is distinct from resection during homologous recombination. Mol. Cell 65, 671–684.e5 (2017).

  11. 11.

    Kudlow, B. A., Kennedy, B. K. & Monnat, R. J. Jr. Werner and Hutchinson–Gilford progeria syndromes: mechanistic basis of human progeroid diseases. Nat. Rev. Mol. Cell Biol. 8, 394–404 (2007).

  12. 12.

    Evans, A. M., DeHaven, C. D., Barrett, T., Mitchell, M. & Milgram, E. Integrated, nontargeted ultrahigh performance liquid chromatography/electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems. Anal. Chem. 81, 6656–6667 (2009).

  13. 13.

    Vinaixa, M. et al. Positional enrichment by proton analysis (PEPA): a one-dimensional 1H-NMR approach for 13C stable isotope tracer studies in metabolomics. Angew. Chem. Int. Ed. Engl. 56, 3531–3535 (2017).

  14. 14.

    Deberardinis, R. J., Sayed, N., Ditsworth, D. & Thompson, C. B. Brick by brick: metabolism and tumor cell growth. Curr. Opin. Genet. Dev. 18, 54–61 (2008).

  15. 15.

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

  16. 16.

    Fukuoh, A. et al. Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase. Mol. Syst. Biol. 10, 734 (2014).

  17. 17.

    West, A. P., Shadel, G. S. & Ghosh, S. Mitochondria in innate immune responses. Nat. Rev. Immunol. 11, 389–402 (2011).

  18. 18.

    Smeitink, J. A., Zeviani, M., Turnbull, D. M. & Jacobs, H. T. Mitochondrial medicine: a metabolic perspective on the pathology of oxidative phosphorylation disorders. Cell Metab. 3, 9–13 (2006).

  19. 19.

    Roux, K. J., Kim, D. I. & Burke, B. BioID: a screen for protein–protein interactions. Curr. Protoc. Protein Sci. 74, 19.23.1–19.23.4 (2013).

  20. 20.

    Sanchez-Caballero, L., Guerrero-Castillo, S. & Nijtmans, L. Unraveling the complexity of mitochondrial complex I assembly: a dynamic process. Biochim. Biophys. Acta 1857, 980–990 (2016).

  21. 21.

    Sasarman, F. & Shoubridge, E. A. Radioactive labeling of mitochondrial translation products in cultured cells. Methods Mol. Biol. 837, 207–217 (2012).

  22. 22.

    Attrill, H. et al. FlyBase: establishing a Gene Group resource for Drosophila melanogaster. Nucleic Acids Res. 44, D786–D792 (2016).

  23. 23.

    Merkey, A. B., Wong, C. K., Hoshizaki, D. K. & Gibbs, A. G. Energetics of metamorphosis in Drosophila melanogaster. J. Insect Physiol. 57, 1437–1445 (2011).

  24. 24.

    Hsu, H. J. & Drummond-Barbosa, D. Insulin levels control female germline stem cell maintenance via the niche in Drosophila. Proc. Natl Acad. Sci. USA 106, 1117–1121 (2009).

  25. 25.

    Hansen, M., Flatt, T. & Aguilaniu, H. Reproduction, fat metabolism, and life span: what is the connection? Cell Metab. 17, 10–19 (2013).

  26. 26.

    Ruzzenente, B. et al. LRPPRC is necessary for polyadenylation and coordination of translation of mitochondrial mRNAs. EMBO J. 31, 443–456 (2012).

  27. 27.

    Bratic, A. et al. The bicoid stability factor controls polyadenylation and expression of specific mitochondrial mRNAs in Drosophila melanogaster. PLoS Genet. 7, e1002324 (2011).

  28. 28.

    Wolf, A. R. & Mootha, V. K. Functional genomic analysis of human mitochondrial RNA processing. Cell Rep. 7, 918–931 (2014).

  29. 29.

    Sessions, O. M. et al. Discovery of insect and human dengue virus host factors. Nature 458, 1047–1050 (2009).

  30. 30.

    Sirbu, B. M. et al. Identification of proteins at active, stalled, and collapsed replication forks using isolation of proteins on nascent DNA (iPOND) coupled with mass spectrometry. J. Biol. Chem. 288, 31458–31467 (2013).

  31. 31.

    Lange, H. et al. Degradation of a polyadenylated rRNA maturation by-product involves one of the three RRP6-like proteins in Arabidopsis thaliana. Mol. Cell Biol. 28, 3038–3044 (2008).

  32. 32.

    Xie, B. et al. Further characterization of human DNA polymerase delta interacting protein 38. J. Biol. Chem. 280, 22375–22384 (2005).

  33. 33.

    Hilton, B. A. et al. ATR plays a direct antiapoptotic role at mitochondria, which is regulated by prolyl isomerase Pin1. Mol. Cell 60, 35–46 (2015).

  34. 34.

    Oberto, J. et al. Qri7/OSGEPL, the mitochondrial version of the universal Kae1/YgjD protein, is essential for mitochondrial genome maintenance. Nucleic Acids Res. 37, 5343–5352 (2009).

  35. 35.

    Ougland, R., Rognes, T., Klungland, A. & Larsen, E. Non-homologous functions of the AlkB homologs. J. Mol. Cell Biol. 7, 494–504 (2015).

  36. 36.

    Liu, P. et al. Removal of oxidative DNA damage via FEN1-dependent long-patch base excision repair in human cell mitochondria. Mol. Cell Biol. 28, 4975–4987 (2008).

  37. 37.

    Zheng, L. et al. Human DNA2 is a mitochondrial nuclease/helicase for efficient processing of DNA replication and repair intermediates. Mol. Cell 32, 325–336 (2008).

  38. 38.

    Copeland, J. M. et al. Extension of Drosophila life span by RNAi of the mitochondrial respiratory chain. Curr. Biol. 19, 1591–1598 (2009).

  39. 39.

    Miwa, S. et al. Low abundance of the matrix arm of complex I in mitochondria predicts longevity in mice. Nat. Commun. 5, 3837 (2014).

  40. 40.

    Scialo, F. et al. Mitochondrial ROS produced via reverse electron transport extend animal lifespan. Cell Metab. 23, 725–734 (2016).

  41. 41.

    Caballero, A. et al. Absence of mitochondrial translation control proteins extends life span by activating sirtuin-dependent silencing. Mol. Cell 42, 390–400 (2011).

  42. 42.

    Guarente, L. & Picard, F. Calorie restriction—the SIR2 connection. Cell 120, 473–482 (2005).

  43. 43.

    Quiros, P. M., Mottis, A. & Auwerx, J. Mitonuclear communication in homeostasis and stress. Nat. Rev. Mol. Cell Biol. 17, 213–226 (2016).

  44. 44.

    Cuykendall, T. N. & Houston, D. W. Identification of germ plasm-associated transcripts by microarray analysis of Xenopus vegetal cortex RNA. Dev. Dyn. 239, 1838–1848 (2010).

  45. 45.

    Oehl-Jaschkowitz, B. et al. Deletions in 14q24.1q24.3 are associated with congenital heart defects, brachydactyly, and mild intellectual disability. Am. J. Med. Genet. A 164A, 620–626 (2014).

  46. 46.

    Gloeckner, C. J., Boldt, K., Schumacher, A., Roepman, R. & Ueffing, M. A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes. Proteomics 7, 4228–4234 (2007).

  47. 47.

    Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).

  48. 48.

    Naviaux, R. K., Costanzi, E., Haas, M. & Verma, I. M. The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J. Virol. 70, 5701–5705 (1996).

  49. 49.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  50. 50.

    Jourdain, A. A. et al. GRSF1 regulates RNA processing in mitochondrial RNA granules. Cell Metab. 17, 399–410 (2013).

  51. 51.

    Gupta, G. D. et al. A dynamic protein interaction landscape of the human centrosome–cilium interface. Cell 163, 1484–1499 (2015).

  52. 52.

    Perry, J. J. et al. WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing. Nat. Struct. Mol. Biol. 13, 414–422 (2006).

  53. 53.

    Graveley, B. R. et al. The developmental transcriptome of Drosophila melanogaster. Nature 471, 473–479 (2011).

  54. 54.

    Gelbart, W. M. & Emmert, D. B. FlyBase High Throughput Expression Pattern Data (2013); http://flybase.org/reports/FBrf0221009.html

  55. 55.

    Guitart, T. et al. New aminoacyl-tRNA synthetase-like protein in insecta with an essential mitochondrial function. J. Biol. Chem. 285, 38157–38166 (2010).

  56. 56.

    Wittig, I., Braun, H. P. & Schagger, H. Blue native PAGE. Nat. Protoc. 1, 418–428 (2006).

  57. 57.

    Reyes, A., Yasukawa, T., Cluett, T. J. & Holt, I. J. Analysis of mitochondrial DNA by two-dimensional agarose gel electrophoresis. Methods Mol. Biol. 554, 15–35 (2009).

  58. 58.

    Yasukawa, T., Yang, M. Y., Jacobs, H. T. & Holt, I. J. A bidirectional origin of replication maps to the major noncoding region of human mitochondrial DNA. Mol. Cell 18, 651–662 (2005).

  59. 59.

    Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

  60. 60.

    Sonnhammer, E. L. & Hollich, V. Scoredist: a simple and robust protein sequence distance estimator. BMC Bioinform. 6, 108 (2005).

  61. 61.

    Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

  62. 62.

    Claros, M. G. & Vincens, P. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem. 241, 779–786 (1996).

  63. 63.

    Ronquist, F. et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542 (2012).

  64. 64.

    Letunic, I. & Bork, P. Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 44, W242–W245 (2016).

  65. 65.

    Krivov, G. G., Shapovalov, M. V. & Dunbrack, R. L. Jr. Improved prediction of protein side-chain conformations with SCWRL4. Proteins 77, 778–795 (2009).

  66. 66.

    Xu, D., Jaroszewski, L., Li, Z. & Godzik, A. FFAS-3D: improving fold recognition by including optimized structural features and template re-ranking. Bioinformatics 30, 660–667 (2014).

  67. 67.

    Zhang, L. et al. Structural and functional characterization of deep-sea thermophilic bacteriophage GVE2 HNH endonuclease. Sci. Rep. 7, 42542 (2017).

  68. 68.

    Sebesta, M., Cooper, C. D. O., Ariza, A., Carnie, C. J. & Ahel, D. Structural insights into the function of ZRANB3 in replication stress response. Nat. Commun. 8, 15847 (2017).

  69. 69.

    Arnoux, P. et al. Structural and redox plasticity in the heterodimeric periplasmic nitrate reductase. Nat. Struct. Biol. 10, 928–934 (2003).

  70. 70.

    Xia, J. & Wishart, D. S. MetPA: a web-based metabolomics tool for pathway analysis and visualization. Bioinformatics 26, 2342–2344 (2010).

Download references

Acknowledgements

We are grateful to A. Zorzano, A. Vaquero, E. Hidalgo, W. M. Keyes, M. Milan, J. Casanova, M. Solà, V. Mootha, J. Guinovart and the Stracker Lab for input and discussions, N. Gallisa for help generating BioID constructs, J. J. P. Perry and J.A. Tainer for the WRN-EXO expression construct, A. Bratic and N.G. Larsson for sharing advice and protocols, and the Advanced Digital Microscopy and Biostatistics/Bioinformatics IRB core facilities. We thank the following bodies for funding: Ministerio de Economía y Competitividad (MINECO) (T.H.S.: BFU2012-39521, BFU2015-68354, Ayudas para incentivar la incorporación estable de doctores (IED) 2015; L.R.d.P.: BIO2015-64572; T.H.S. and L.R.d.P: institutional funding through the Centres of Excellence Severo Ochoa award and from the CERCA Programme of the Catalan Government; and O.Y.: SAF2011-30578 and BFU2014-57466); the Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), an initiative of Instituto de Investigacion Carlos III (ISCIII) to O.Y.; and the Biotechnology and Biological Sciences Research Council (BBSRC: BB/H019723/1 and BB/M008800/1) to A.J.D. and L.B. S.A. was supported by a Finnish Cultural Society Fellowship, J.S. by a fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BD/87025/2012), P.A.K. by an Advanced Postdoc Mobility fellowship from the Swiss National Science Foundation (SNF), I.G.-C. by an Asociación Española Contra el Cáncer (AECC) fellowship, A.A.-F. by a Severo Ochoa FPI fellowship (MINECO; SVP2014068398), and A.A.J. by an EMBO long-term fellowship (ALTF 554-2015).

Author information

Author notes

    • Joana Silva

    Present address: Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain

    • Suvi Aivio

    Present address: Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA

    • Philip A. Knobel

    Present address: Department for Radiation Oncology, University Hospital Zurich, Zurich, Switzerland

    • Pablo Pérez-Ferreros

    Present address: EMBL Australia, University of New South Wales, Lowy Cancer Research Center, Single Molecule Science Node, Sydney and Arc Center of Excellence in Advance Molecular Imaging, Sydney, New South Wales, Australia

Affiliations

  1. Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK

    • Laura J. Bailey
    •  & Aidan J. Doherty
  2. Metabolomics Platform, Department of Electronic Engineering (DEEEA), Universitat Rovira i Virgili, Tarragona, Spain

    • Maria Vinaixa
    • , Sara Samino-Gené
    •  & Oscar Yanes
  3. Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Madrid, Spain

    • Maria Vinaixa
    • , Sara Samino-Gené
    •  & Oscar Yanes
  4. Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada

    • Étienne Coyaud
    •  & Brian Raught
  5. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    • Étienne Coyaud
    •  & Brian Raught
  6. Department of Molecular Biology, Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA, USA

    • Alexis A. Jourdain
  7. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    • Alexis A. Jourdain
  8. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    • Alexis A. Jourdain
  9. Computational Biology and Bioinformatics Group, Institute of Biomedicine of Seville (IBIS/CSIC/US/JA), Campus Hospital Universitario Virgen del Rocio, Seville, Spain

    • Ana M. Rojas
  10. Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide/CSIC/JA, Seville, Spain

    • Acaimo González-Reyes
  11. Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain

    • Lluís Ribas de Pouplana

Authors

  1. Search for Joana Silva in:

  2. Search for Suvi Aivio in:

  3. Search for Philip A. Knobel in:

  4. Search for Laura J. Bailey in:

  5. Search for Andreu Casali in:

  6. Search for Maria Vinaixa in:

  7. Search for Isabel Garcia-Cao in:

  8. Search for Étienne Coyaud in:

  9. Search for Alexis A. Jourdain in:

  10. Search for Pablo Pérez-Ferreros in:

  11. Search for Ana M. Rojas in:

  12. Search for Albert Antolin-Fontes in:

  13. Search for Sara Samino-Gené in:

  14. Search for Brian Raught in:

  15. Search for Acaimo González-Reyes in:

  16. Search for Lluís Ribas de Pouplana in:

  17. Search for Aidan J. Doherty in:

  18. Search for Oscar Yanes in:

  19. Search for Travis H. Stracker in:

Contributions

J.S. and S.A. performed the majority of experiments characterizing cell lines and effects of EXD2 deficiency. J.S. performed sucrose gradient fractionations, and L.J.B. and A.J.D. purified and characterized bacterial EXD2 and WRN, performed mtDNA replication assays and analysed data. J.S., S.A., A.C. and A.G.-R. characterized Drosophila. P.A.K. and P.P.-F. performed BioID assays, and M.V., S.S.-G. and O.Y. carried out metabolomics and mass spectrometry, analysed data and prepared figures. E.C. and B.R. purified biotinylated peptides, performed mass spectrometry and analysed data. A.A.J. performed the MitoString assay, and I.G.-C., J.S., S.A. and T.H.S. performed survival assays and analysed DNA damage responses. T.H.S. and J.S. performed mitochondrial translation assays, and P.A.K. and A.A.F. performed immunofluorescence analysis in Drosophila S2 cells. A.M.R. performed computational and evolutionary analysis, and L.R.d.P. provided critical technical expertise and advice. T.H.S. analysed data, prepared figures, supervised experiments and wrote the manuscript with editorial contributions from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Travis H. Stracker.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–9, Supplementary Table Legends, Supplementary References.

  2. Life Sciences Reporting Checklist

  3. Supplementary Table

    Supplementary Table 1.

  4. Supplementary Table

    Supplementary Table 2.

  5. Supplementary Table

    Supplementary Table 3.

  6. Supplementary Table

    Supplementary Table 4.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41556-017-0016-9

Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.