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Structures of the human mitochondrial ribosome in native states of assembly

Nature Structural & Molecular Biology volume 24, pages 866–869 (2017)Cite this article

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Abstract

Mammalian mitochondrial ribosomes (mitoribosomes) have less rRNA content and 36 additional proteins compared with the evolutionarily related bacterial ribosome. These differences make the assembly of mitoribosomes more complex than the assembly of bacterial ribosomes, but the molecular details of mitoribosomal biogenesis remain elusive. Here, we report the structures of two late-stage assembly intermediates of the human mitoribosomal large subunit (mt-LSU) isolated from a native pool within a human cell line and solved by cryo-EM to ∼3-Å resolution. Comparison of the structures reveals insights into the timing of rRNA folding and protein incorporation during the final steps of ribosomal maturation and the evolutionary adaptations that are required to preserve biogenesis after the structural diversification of mitoribosomes. Furthermore, the structures redefine the ribosome silencing factor (RsfS) family as multifunctional biogenesis factors and identify two new assembly factors (L0R8F8 and mt-ACP) not previously implicated in mitoribosomal biogenesis.

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Figure 1: Purification and structural characterization of native mitoribosomal assembly intermediates.
Figure 2: A module of MALSU1–L0R8F8–mt-ACP binds both mt-LSU assembly intermediates.
Figure 3: Models of antiassociation activity.

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References

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Adilakshmi, T., Bellur, D.L. & Woodson, S.A. Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly. Nature 455, 1268–1272 (2008).

    Article  CAS  Google Scholar 

  4. Davis, J.H. et al. Modular assembly of the bacterial large ribosomal subunit. Cell 167, 1610–1622.e15 (2016).

    Article  CAS  Google Scholar 

  5. Bogenhagen, D.F., Martin, D.W. & Koller, A. Initial steps in RNA processing and ribosome assembly occur at mitochondrial DNA nucleoids. Cell Metab. 19, 618–629 (2014).

    Article  CAS  Google Scholar 

  6. De Silva, D., Tu, Y.-T., Amunts, A., Fontanesi, F. & Barrientos, A. Mitochondrial ribosome assembly in health and disease. Cell Cycle 14, 2226–2250 (2015).

    Article  CAS  Google Scholar 

  7. Kim, H.-J., Maiti, P. & Barrientos, A. Mitochondrial ribosomes in cancer. Semin. Cancer Biol. http://dx.doi.org/10.1016/j.semcancer/2017.04.004 (2017).

  8. Stokes, J.M., Davis, J.H., Mangat, C.S., Williamson, J.R. & Brown, E.D. Discovery of a small molecule that inhibits bacterial ribosome biogenesis. eLife 3, e03574 (2014).

    Article  Google Scholar 

  9. Reeves, P.J., Callewaert, N., Contreras, R. & Khorana, H.G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA 99, 13419–13424 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Li, N. et al. Cryo-EM structures of the late-stage assembly intermediates of the bacterial 50S ribosomal subunit. Nucleic Acids Res. 41, 7073–7083 (2013).

    Article  CAS  Google Scholar 

  12. Jomaa, A. et al. Functional domains of the 50S subunit mature late in the assembly process. Nucleic Acids Res. 42, 3419–3435 (2014).

    Article  CAS  Google Scholar 

  13. Maeder, C. & Draper, D.E. A small protein unique to bacteria organizes rRNA tertiary structure over an extensive region of the 50 S ribosomal subunit. J. Mol. Biol. 354, 436–446 (2005).

    Article  CAS  Google Scholar 

  14. Zhang, X. et al. Structural insights into the function of a unique tandem GTPase EngA in bacterial ribosome assembly. Nucleic Acids Res. 42, 13430–13439 (2014).

    Article  CAS  Google Scholar 

  15. Baer, R.J. & Dubin, D.T. Methylated regions of hamster mitochondrial ribosomal RNA: structural and functional correlates. Nucleic Acids Res. 9, 323–337 (1981).

    Article  CAS  Google Scholar 

  16. Bar-Yaacov, D. et al. Mitochondrial 16S rRNA is methylated by tRNA methyltransferase TRMT61B in all vertebrates. PLoS Biol. 14, e1002557 (2016).

    Article  Google Scholar 

  17. Ofengand, J. & Bakin, A. Mapping to nucleotide resolution of pseudouridine residues in large subunit ribosomal RNAs from representative eukaryotes, prokaryotes, archaebacteria, mitochondria and chloroplasts. J. Mol. Biol. 266, 246–268 (1997).

    Article  CAS  Google Scholar 

  18. Häuser, R. et al. RsfA (YbeB) proteins are conserved ribosomal silencing factors. PLoS Genet. 8, e1002815 (2012).

    Article  Google Scholar 

  19. Fung, S., Nishimura, T., Sasarman, F. & Shoubridge, E.A. The conserved interaction of C7orf30 with MRPL14 promotes biogenesis of the mitochondrial large ribosomal subunit and mitochondrial translation. Mol. Biol. Cell 24, 184–193 (2013).

    Article  CAS  Google Scholar 

  20. Li, X. et al. Structure of ribosomal silencing factor bound to mycobacterium tuberculosis ribosome. Structure 23, 1858–1865 (2015).

    Article  CAS  Google Scholar 

  21. Rorbach, J., Gammage, P.A. & Minczuk, M. C7orf30 is necessary for biogenesis of the large subunit of the mitochondrial ribosome. Nucleic Acids Res. 40, 4097–4109 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D Biol. Crystallogr. 71, 136–153 (2015).

    Article  CAS  Google Scholar 

  24. Cronan, J.E., Fearnley, I.M. & Walker, J.E. Mammalian mitochondria contain a soluble acyl carrier protein. FEBS Lett. 579, 4892–4896 (2005).

    Article  CAS  Google Scholar 

  25. Zhu, J. et al. Structure of subcomplex Iβ of mammalian respiratory complex I leads to new supernumerary subunit assignments. Proc. Natl. Acad. Sci. USA 112, 12087–12092 (2015).

    Article  CAS  Google Scholar 

  26. Zhu, J., Vinothkumar, K.R. & Hirst, J. Structure of mammalian respiratory complex I. Nature 536, 354–358 (2016).

    Article  CAS  Google Scholar 

  27. Fiedorczuk, K. et al. Atomic structure of the entire mammalian mitochondrial complex I. Nature 538, 406–410 (2016).

    Article  CAS  Google Scholar 

  28. Andreev, D.E. et al. Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression. eLife 4, e03971 (2015).

    Article  Google Scholar 

  29. Cronan, J.E. The chain-flipping mechanism of ACP (acyl carrier protein)-dependent enzymes appears universal. Biochem. J. 460, 157–163 (2014).

    Article  CAS  Google Scholar 

  30. Van Vranken, J.G. et al. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis. eLife 5, e17828 (2016).

    Article  Google Scholar 

  31. Maio, N. et al. Cochaperone binding to LYR motifs confers specificity of iron sulfur cluster delivery. Cell Metab. 19, 445–457 (2014).

    Article  CAS  Google Scholar 

  32. Klinge, S., Voigts-Hoffmann, F., Leibundgut, M., Arpagaus, S. & Ban, N. Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6. Science 334, 941–948 (2011).

    Article  CAS  Google Scholar 

  33. Weis, F. et al. Mechanism of eIF6 release from the nascent 60S ribosomal subunit. Nat. Struct. Mol. Biol. 22, 914–919 (2015).

    Article  CAS  Google Scholar 

  34. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

    Article  CAS  Google Scholar 

  35. Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    Article  CAS  Google Scholar 

  36. Kimanius, D., Forsberg, B.O., Scheres, S.H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016).

    Article  Google Scholar 

  37. Scheres, S.H. Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014).

    Article  Google Scholar 

  38. Bai, X.-C., Rajendra, E., Yang, G., Shi, Y. & Scheres, S.H. Sampling the conformational space of the catalytic subunit of human α-secretase. eLife 4, e11182 (2015).

    Article  Google Scholar 

  39. Zheng, S.Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  Google Scholar 

  40. Rosenthal, P.B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  Google Scholar 

  41. Kucukelbir, A., Sigworth, F.J. & Tagare, H.D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    Article  CAS  Google Scholar 

  42. Pettersen, E.F. et al. UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

  43. Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics http://dx.doi.org/10.1186/1471-2105-9-40 (2008).

  44. Long, F., Vagin, A.A., Young, P. & Murshudov, G.N. BALBES: a molecular-replacement pipeline. Acta Crystallogr. D Biol. Crystallogr. 64, 125–132 (2008).

    Article  CAS  Google Scholar 

  45. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).

    Article  CAS  Google Scholar 

  46. Cryle, M.J. & Schlichting, I. Structural insights from a P450 Carrier Protein complex reveal how specificity is achieved in the P450(BioI) ACP complex. Proc. Natl. Acad. Sci. USA 105, 15696–15701 (2008).

    Article  CAS  Google Scholar 

  47. Afonine, P.V., Headd, J.J., Terwilliger, T.C. & Adams, P.D. New tool: phenix.real_space_refine. Computational Crystallography Newsletter 4, 43–44 (2013).

    Google Scholar 

  48. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  49. Barad, B.A. et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).

    Article  CAS  Google Scholar 

  50. Marks, D.S. et al. Protein 3D structure computed from evolutionary sequence variation. PLoS One 6, e28766 (2011).

    Article  CAS  Google Scholar 

  51. Weinreb, C. et al. 3D RNA and functional interactions from evolutionary couplings. Cell 165, 963–975 (2016).

    Article  CAS  Google Scholar 

  52. DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific, 2002).

Download references

Acknowledgements

This work was funded by the Swedish Research Council (NT_2015-04107), the Swedish Foundation for Strategic Research (Future Leaders Grant FFL15-0325), and the Ragnar Söderberg Foundation (Fellowship in Medicine M44/16) to A.A., and the UK Medical Research Council (MC_U105184332), the Wellcome Trust (Senior Investigator Award WT096570), and the Agouron Institute and the Louis-Jeantet Foundation to V.R. S.A. was supported by a FEBS Long-Term Fellowship. J.R. and A.A. were supported by Marie Sklodowska Curie Actions (International Career Grant 2015-00579). Funding to M. Minczuk (MC_U105697135) supported the research activities of S.R. and J.R. at the MRC Mitochondrial Biology Unit. Cryo-EM data were collected at the MRC Laboratory of Molecular Biology and the Swedish National Facility. We thank S. Chen, J. Conrad, M. Carroni, and C. Savva for help with data collection; S. Peak-Chew, G. Degliesposti, M. Skehel, and F. Stengel for MS analysis; J. Grimmett, T. Darling, and S. Fleischmann for computing support; D. Marks for help with evolutionary couplings; M. Minczuk for discussions and unpublished data; and C. Tate (MRC Laboratory of Molecular Biology) for providing cells.

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Author notes

  1. Alan Brown

    Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

  2. Xiao-chen Bai

    Present address: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas, USA

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Alan Brown, Xiao-chen Bai, Alexey Amunts & V Ramakrishnan

  2. Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Solna, Sweden

    Sorbhi Rathore, Dari Kimanius, Shintaro Aibara, Joanna Rorbach & Alexey Amunts

  3. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Joanna Rorbach

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Contributions

A.B. processed the data, built and refined the model, and wrote the paper. S.R. processed the data and contributed unpublished data. D.K. processed the data and built the model. S.A. and X.-c.B. collected the data. J.R. contributed unpublished data. A.A. conceived the project and prepared the sample. V.R. initiated the project. All authors contributed to the final version of the manuscript.

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Correspondence to Alexey Amunts or V Ramakrishnan.

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Integrated supplementary information

Supplementary Figure 1 EM data processing pipeline for data set 2.

Initial processing focused on isolating particles with extraneous density by applying a mask over the MALSU1–L0R8F8–mt-ACP module (mask A). The density for this region was further improved by applying a mask over the density for just L0R8F8–mt-ACP (mask B). The particles were separated into two subclasses based on the stability of mt-rRNA using a mask over the interfacial mt-rRNA (mask C). These classes were used for model building and interpretation. Mitoribosomes are viewed as in Fig. 1B, with density corresponding to the MALSU1–L0R8F8–mt-ACP module in yellow. Masks are shown in white. (B) Slices through the 3D projections for both assembly intermediates. Nebulous density at the intersubunit interface for the map with “unstable” interfacial mt-rRNA suggests that the mt-rRNA is present, but adopts multiple conformations.

Supplementary Figure 2 Map quality.

(A) Fourier shell correlation (FSC) curves for the two mt-LSU assembly intermediates, with and without folded interfacial mt-rRNA. Also plotted are the FSC curves calculated between the refined model and final map, and the self and cross-validated correlations. The nominal resolution estimated from the map-to-map correlation at FSC = 0.143 is reported and agrees well with the model-to-map correlation at FSC = 0.5. (B) The cryo-EM map for each assembly intermediate colored by local resolution.

Supplementary Figure 3 Identification of MALSU1.

(A) Superposition of MALSU1 with RsfS from Mycobacterium tuberculosis (PDB ID: 4WCW). (B) Fit of the model for the 5-stranded β-sheet of MALSU1 to the map. (C) Density and model for an α-helix of MALSU1 demonstrating the quality of side-chain density.

Supplementary Figure 4 Identification of mt-ACP.

(A) Schematic of the BALBES-MOLREP pipeline. (B) Table of the top ten hits from BALBES-MOLREP, ranked by the contrast score. The top score belongs to ACP from Escherichia coli. (C) Fit of mammalian mt-ACP to the map. (D) Fit of MALSU1 and humanized mt-ACP to the additional density leaves 3 helices unassigned. These helices were subsequently assigned to L0R8F8.

Supplementary Figure 5 Identification of L0R8F8.

(A) There are two copies of mt-ACP in mammalian complex I (SDAP-α and SDAP-β), each of which contacts a different LYR-motif protein (NDUFA6 and NDUFB9, respectively). (B) Fit of NDUFA6 to the map. (C) Fit of NDUFB9 to the map. (D) Fit of L0R8F8 to the map. (E) Aromatic residues guided the de novo modeling of L0R8F8. (F) Density present in the core of L0R8F8 corresponds to the 4′-phosphopantetheine (4′-PP) modification of serine 112 of mt-ACP. (G) The top seven highest-ranking evolutionary couplings for L0R8F8 plotted as colored dots. The predicted secondary structure closely matches the secondary structure extracted from the model. (H) Table of the top seven highest-ranking evolutionary couplings colored according to G. (I) Evolutionary couplings mapped onto the structure of L0R8F8. Evolutionary coupled residues are typically in close spatial proximity, as would be expected for a correctly built fold.

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Brown, A., Rathore, S., Kimanius, D. et al. Structures of the human mitochondrial ribosome in native states of assembly. Nat Struct Mol Biol 24, 866–869 (2017). https://doi.org/10.1038/nsmb.3464

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