Abstract

Mitochondria are responsible for energy production through aerobic respiration, and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.

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Data availability

Mass spectrometric data were deposited with the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD010324. Ribo-Seq sequencing data were deposited in the NCBI Gene Expression Omnibus under accession number GSE119655. Cryo-EM data were deposited in EMDataBank under accession number EMDB-4408 for Arabidopsis mitoribosome SSU, and EMDB-4409 for Arabidopsis mitoribosome LSU.

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References

  1. 1.

    Roger, A. J., Muñoz-Gómez, S. A. & Kamikawa, R. The origin and diversification of mitochondria. Curr. Biol. 27, R1177–R1192 (2017).

  2. 2.

    Nunnari, J. & Suomalainen, A. Mitochondria: in sickness and in health. Cell 148, 1145–1159 (2012).

  3. 3.

    De Silva, D., Tu, Y. T., Amunts, A., Fontanesi, F. & Barrientos, A. Mitochondrial ribosome assembly in health and disease. Cell Cycle 14, 226–2250 (2015).

  4. 4.

    Chen, L. & Liu, Y.-G. Male sterility and fertility restoration in crops. Annu. Rev. Plant. Biol. 65, 579–606 (2014).

  5. 5.

    Horn, R., Gupta, K. J. & Colombo, N. Mitochondrion role in molecular basis of cytoplasmic male sterility. Mitochondrion 19, 198–205 (2014).

  6. 6.

    Giegé, P., Sweetlove, L. J., Cognat, V. & Leaver, C. J. Coordination of nuclear and mitochondrial genome expression during mitochondrial biogenesis in Arabidopsis. Plant Cell 17, 1497–1512 (2005).

  7. 7.

    Lightowlers, R. N., Rozanska, A. & Chrzanowska-Lightowlers, Z. M. Mitochondrial protein synthesis: figuring the fundamentals, complexities and complications of mammalian mitochondrial translation. FEBS Lett. 588, 2496–2503 (2014).

  8. 8.

    Hammani, K. & Giegé, P. RNA metabolism in plant mitochondria. Trends Plant Sci. 19, 380–389 (2014).

  9. 9.

    Lopez Sanchez, M. I. G. et al. RNA processing in human mitochondria. Cell Cycle 10, 2904–2916 (2011).

  10. 10.

    Barkan, A. & Small, I. Pentatricopeptide repeat proteins in plants. Annu. Rev. Plant Biol. 65, 415–442 (2014).

  11. 11.

    Chinnery, P. F. et al. The challenges of mitochondrial replacement. PLoS Genet. 10, e1004315 (2014).

  12. 12.

    Giegé, P. Pentatricopeptide repeat proteins. RNA Biol. 10, 1417–1418 (2013).

  13. 13.

    Uyttewaal, M. et al. PPR336 is associated with polysomes in plant mitochondria. J. Mol. Biol. 375, 626–636 (2008).

  14. 14.

    Hammani, K. et al. An Arabidopsis dual-localized pentatricopeptide repeat protein interacts with nuclear proteins involved in gene expression regulation. Plant Cell 23, 730–740 (2011).

  15. 15.

    Haïli, N. et al. The MTL1 pentatricopeptide repeat protein is required for both translation and splicing of the mitochondrial NADH dehydrogenase subunit 7 mRNA in Arabidopsis. Plant Physiol. 170, 354–366 (2016).

  16. 16.

    Mai, N., Chrzanowska-Lightowlers, Z. M. A. & Lightowlers, R. N. The process of mammalian mitochondrial protein synthesis. Cell Tissue Res. 367, 5–20 (2017).

  17. 17.

    Greber, B. J. & Ban, N. Structure and function of the mitochondrial ribosome. Annu. Rev. Biochem. 85, 103–132 . (2016).

  18. 18.

    Planchard, N. et al. The translational landscape of Arabidopsis mitochondria. Nucleic Acids Res. 46, 6218–6228 (2018).

  19. 19.

    Barrell, B. G., Bankier, A. T. & Drouin, J. A different genetic code in human mitochondria. Nature 282, 189–194 (1979).

  20. 20.

    Helm, M. et al. Search for characteristic structural features of mammalian mitochondrial tRNAs. RNA 6, 1356–1379 (2000).

  21. 21.

    Pfeffer, S., Woellhaf, M. W., Herrmann, J. M. & Förster, F. Organization of the mitochondrial translation machinery studied in situ by cryoelectron tomography. Nat. Commun. 6, 6019 (2015).

  22. 22.

    Ott, M., Amunts, A. & Brown, A. Organization and regulation of mitochondrial protein synthesis. Annu. Rev. Biochem. 85, 77–101 (2016).

  23. 23.

    Salinas-Giegé, T. et al. Polycytidylation of mitochondrial mRNAs in Chlamydomonas reinhardtii Thalia Salinas-Gieg e. Nucleic Acids Res. 45, 12963–12973 (2017).

  24. 24.

    Kitakawa, M. et al. Identification and characterization of the genes for mitochondrial ribosomal proteins of Saccharomyces cerevisiae. Eur. J. Biochem. 245, 449–456 (1997).

  25. 25.

    Goldschmidt-Reisin, S. et al. Mammalian mitochondrial ribosomal proteins. N-terminal amino acid sequencing, characterization, and identification of corresponding gene sequences. J. Biol. Chem. 273, 34828–34836 (1998).

  26. 26.

    Koc, E. C. et al. A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins. J. Biol. Chem. 275, 32585–32591 (2000).

  27. 27.

    Koc, E. C. et al. The large subunit of the mammalian mitochondrial ribosome: analysis of the complement of ribosomal proteins present. J. Biol. Chem. 276, 43958–43969 (2001).

  28. 28.

    Bonen, L. & Calixte, S. Comparative analysis of bacterial-origin genes for plant mitochondrial ribosomal proteins. Mol. Biol. Evol. 23, 701–712 (2006).

  29. 29.

    Amunts, A., Brown, A., Toots, J., Scheres, S. H. W. & Ramakrishnan, V. The structure of the human mitochondrial ribosome. Science 348, 95–98 (2015).

  30. 30.

    Greber, B. J. et al. The complete structure of the 55S mammalian mitochondrial ribosome. Science 348, 303–308 (2015).

  31. 31.

    Desai, N., Brown, A., Amunts, A. & Ramakrishnan, V. The structure of the yeast mitochondrial ribosome. Science 355, 528–531 (2017).

  32. 32.

    Chrzanowska-Lightowlers, Z., Rorbach, J. & Minczuk, M. Human mitochondrial ribosomes can switch structural tRNAs – but when and why? RNA Biol. 14, 1668–1671 (2017).

  33. 33.

    Brown, A. et al. Structure of the large ribosomal subunit from human mitochondria. Science 346, 718–722 (2014).

  34. 34.

    Leaver, C. J. & Harmey, M. A. Higher-plant mitochondrial ribosomes contain a 5S ribosomal ribonucleic acid component. Biochem. J. 157, 275–277 (1976).

  35. 35.

    Gold, V. A., Chroscicki, P., Bragoszewski, P. & Chacinska, A. Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo‐tomography. EMBO Rep. 18, 1786–1800 (2017).

  36. 36.

    Lurin, C. et al. Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16, 2089–2103 (2004).

  37. 37.

    Portereiko, M. F. Nuclear fusion defective 1 encodes the Arabidopsis RPL21M protein and is required for karyogamy during female gametophyte development and fertilization. Plant Physiol. 141, 957–965 (2006).

  38. 38.

    Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014).

  39. 39.

    Ramesh, M. & Woolford, J. L. Eukaryote-specific rRNA expansion segments function in ribosome biogenesis. RNA 22, 1153–1162 (2016).

  40. 40.

    Bieri, P., Leibundgut, M., Saurer, M., Boehringer, D. & Ban, N. The complete structure of the chloroplast 70S ribosome in complex with translation factor pY. EMBO J. 36, 475–486 (2017).

  41. 41.

    Boerema, A. P. et al. Structure of the chloroplast ribosome with chl-RRF and hibernation-promoting factor. Nat. Plants 4, 212–217 (2018).

  42. 42.

    Pusnik, M., Small, I., Read, L. K., Fabbro, T. & Schneider, A. Pentatricopeptide repeat proteins in Trypanosoma brucei function in mitochondrial ribosomes. Mol. Cell. Biol. 27, 6876–6888 (2007).

  43. 43.

    Aphasizheva, I., Maslov, D., Wang, X., Huang, L. & Aphasizhev, R. Pentatricopeptide repeat proteins stimulate mRNA adenylation/uridylation to activate mitochondrial translation in trypanosomes. Mol. Cell 42, 106–117 (2011).

  44. 44.

    Aphasizheva, I. et al. Ribosome-associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes. Mol. Microbiol. 99, 1043–1058 (2016).

  45. 45.

    Ramrath, D. et al. Evolutionary shift toward protein-based architecture in trypanosomal mitochondrial ribosomes. Science 7735, 422 (2018).

  46. 46.

    Bieri, P., Greber, B. J. & Ban, N. High-resolution structures of mitochondrial ribosomes and their functional implications. Curr. Opin. Struct. Biol. 49, 44–53 (2018).

  47. 47.

    Kühl, I., Dujeancourt, L., Gaisne, M., Herbert, C. J. & Bonnefoy, N. A genome wide study in fission yeast reveals nine PPR proteins that regulate mitochondrial gene expression. Nucleic Acids Res. 39, 8029–8041 (2011).

  48. 48.

    Tavares-Carreón, F. et al. The pentatricopeptide repeats present in Pet309 are necessary for translation but not for stability of the mitochondrial Cox1 mRNA in yeast. J. Biol. Chem. 283, 1472–1479 (2008).

  49. 49.

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

  50. 50.

    Uyttewaal, M. et al. Characterization of Raphanus sativus pentatricopeptide repeat proteins encoded by the fertility restorer locus for Ogura cytoplasmic male sterility. Plant Cell 20, 3331–3345 (2008).

  51. 51.

    Herbert, C. J. et al. Yeast PPR proteins, watchdogs of mitochondrial gene expression. RNA Biol. 10, 1477–1494 (2013).

  52. 52.

    Wilson, D. N. & Doudna Cate, J. H. The structure and function of the eukaryotic ribosome. Cold Spring Harb. Perspect. Biol. 4, a011536–a011536 (2012).

  53. 53.

    Jobe, A., Liu, Z., Gutierrez-Vargas, C. & Frank, J. New insights into ribosome structure andfunction. Cold Spring Harb. Perspect. Biol. 7, a032615 (2018).

  54. 54.

    Hazle, T. & Bonen, L. Comparative analysis of sequences preceding protein-coding mitochondrial genes in flowering plants. Mol. Biol. Evol. 24, 1101–1112 (2007).

  55. 55.

    Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

  56. 56.

    Gregori, J., Villareal, L., Sanchez, A., Baselga, J. & Villanueva, J. An effect size filter improves the reproducibility in spectral counting-based comparative proteomics. J. Proteom. 16, 55–65 (2013).

  57. 57.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

  58. 58.

    Lamattina, L., Gonzalez, D., Gualberto, J. & Grienenberger, J. M. Higher plant mitochondria encode an homologue of the nuclear-encoded 30-kDa subunit of bovine mitochondrial complex I. Eur. J. Biochem. 217, 831–838 (1993).

  59. 59.

    Elthon, T. E., Nickels, R. L. & McIntosh, L. Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. Plant Physiol. 89, 1311–1317 (1989).

  60. 60.

    Pineau, B., Layoune, O., Danon, A. & De Paepe, R. l-Galactono-1,4-lactone dehydrogenase isrequired for the accumulation of plant respiratory complex I. J. Biol. Chem. 283, 32500–32505 (2008).

  61. 61.

    Carrie, C. et al. Conserved and novel functions for Arabidopsis thaliana MIA40 in assembly of proteins in mitochondria and peroxisomes. J. Biol. Chem. 285, 36138–36148 (2010).

  62. 62.

    Hooper, C. M., Castleden, I. R., Tanz, S. K., Aryamanesh, N. & Millar, A. H. SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations. Nucleic Acids Res. 45, D1064–D1074 (2017).

  63. 63.

    Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

  64. 64.

    Karpenahalli, M. R., Lupas, A. N. & Söding, J. TPRpred: a tool for prediction of TPR-, PPR- and SEL1-like repeats from protein sequences. BMC Bioinformatics 8, 2 (2007).

  65. 65.

    Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845 (2015).

  66. 66.

    Obayashi, T., Aoki, Y., Tadaka, S., Kagaya, Y. & Kinoshita, K. ATTED-II in 2018: a plant coexpression database based on investigation of the statistical property of the mutual rank index. Special Issue – Databases 59, 1–7 (2018).

  67. 67.

    de la Rosa-Trevín, J. M. et al. Scipion: a software framework toward integration, reproducibility and validation in 3D electron microscopy. J. Struct. Biol. 195, 93–99 (2016).

  68. 68.

    de la Rosa-Trevín, J. M. et al. Xmipp 3.0: an improved software suite for image processing in electron microscopy. J. Struct. Biol. 184, 321–328 (2013).

  69. 69.

    Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

  70. 70.

    Abrishami, V. et al. A pattern matching approach to the automatic selection of particles from low-contrast electron micrographs. Bioinformatics 29, 2460–2468 (2013).

  71. 71.

    Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

  72. 72.

    Ban, N. et al. A new system for naming ribosomal proteins. Curr. Opin. Struct. Biol. 24, 165–169 (2014).

  73. 73.

    Stupar, R. M. et al. Complex mtDNA constitutes an approximate 620-kb insertion on Arabidopsis thaliana chromosome 2: Implication of potential sequencing errors caused by large-unit repeats. Proc. Natl Acad. Sci. USA 98, 5099–5103 (2001).

  74. 74.

    Adams, K. L., Daley, D. O., Whelan, J. & Palmer, J. D. Genes for two mitochondrial ribosomal proteins in flowering plants are derived from their chloroplast or cytosolic counterparts. Plant Cell 14, 931–943 (2002).

  75. 75.

    Noeske, J. et al. High-resolution structure of the Escherichia coli ribosome. Nat. Struct. Mol. Biol. 22, 336–341 (2015).

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Acknowledgements

This work was supported by Centre National de la Recherche Scientifique, the University of Strasbourg, by Agence Nationale de la Recherche (ANR) grants (MITRA, no. ANR-16-CE11-0024-02) and (CytoRP, no. ANR-16-CE21-0001-01) to P.G., Y.H. and H.M., and by the LabEx consortium MitoCross within the framework of the French National Program Investissement d’Avenir (no. ANR-11-LABX-0057_MITOCROSS) and LabEx Saclay Plant Sciences-SPS (no. ANR-10-LABX-0040-SPS). Mass spectrometry instruments were funded by the University of Strasbourg, IdEx Equipement mi-lourd 2015 and the LabEx consortium NetRNA within the framework of the French National Program Investissement d’Avenir (no. ANR-10-LABEX-0036_NETRNA). Y.H. was supported by the LabEx consortium NetRNA.

Author information

Affiliations

  1. Institut de biologie de moléculaire des plantes UPR2357 du CNRS, Université de Strasbourg, Strasbourg, France

    • Florent Waltz
    • , Mathilde Arrivé
    •  & Philippe Giegé
  2. Institut Jean-Pierre Bourgin INRA, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France

    • Tan-Trung Nguyen
    • , Martine Quadrado
    •  & Hakim Mireau
  3. Institut Européen de Chimie et Biologie U1212 Inserm, Université de Bordeaux, Pessac, France

    • Anthony Bochler
    •  & Yaser Hashem
  4. Plateforme protéomique Strasbourg Esplanade FRC1589 du CNRS, Université de Strasbourg, Strasbourg, France

    • Johana Chicher
    • , Philippe Hammann
    •  & Lauriane Kuhn

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Contributions

P.G., F.W., H.M. and Y.H. designed and coordinated the experiments. F.W., T.T.N., M.A., A.B., J.C., M.Q., P.H. and L.K. performed the experiments and analysed the results. P.G., F.W., H.M., T.T.N. and Y.H. wrote the manuscript.

Competing interests

The authors declare no competing interest.

Corresponding authors

Correspondence to Hakim Mireau or Yaser Hashem or Philippe Giegé.

Supplementary information

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  3. Supplementary Data

    Supplementary Tables 1–7.

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https://doi.org/10.1038/s41477-018-0339-y