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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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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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