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The structure of the human 80S ribosome at 1.9 Å resolution reveals the molecular role of chemical modifications and ions in RNA

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

The ribosomal RNA of the human protein synthesis machinery comprises numerous chemical modifications that are introduced during ribosome biogenesis. Here we present the 1.9 Å resolution cryo electron microscopy structure of the 80S human ribosome resolving numerous new ribosomal RNA modifications and functionally important ions such as Zn2+, K+ and Mg2+, including their associated individual water molecules. The 2′-O-methylation, pseudo-uridine and base modifications were confirmed by mass spectrometry, resulting in a complete investigation of the >230 sites, many of which could not be addressed previously. They choreograph key interactions within the RNA and at the interface with proteins, including at the ribosomal subunit interfaces of the fully assembled 80S ribosome. Uridine isomerization turns out to be a key mechanism for U–A base pair stabilization in RNA in general. The structural environment of chemical modifications and ions is primordial for the RNA architecture of the mature human ribosome, hence providing a structural framework to address their role in healthy states and in human diseases.

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Fig. 1: High-resolution features, ions and water molecules in the human 80S ribosome.
Fig. 2: Assignment of ion coordinations in the human 80S ribosome.
Fig. 3: 2′-O-methylation sites in the human 80S ribosome.
Fig. 4: Pseudo-uridines in the human 80S ribosome.
Fig. 5: Nucleotide base modifications in the human 80S ribosome.
Fig. 6: Complete annotation of chemical modification in the human 80S ribosome through MS and structural analysis.
Fig. 7: Comparison of chemical modifications and conserved ion sites in the human 80S and E. coli 70S ribosome.

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Data availability

Atomic coordinates and cryo-EM maps have been deposited in the PDB and Electron Microscopy Data Bank (EMDB) under accession codes 8QOI and EMD-18539 (full 80S ribosome); EMD-18812, EMD-18813 and EMD-18814 for the focused refinements of the 60S ribosomal subunit, the 40S ribosomal subunit body and head regions, respectively; and EMD-18815 for the last global refinement of the 80S ribosome before the focused refinements. MS data have been deposited in the ProteomeXchange Consortium available via the PRIDE database at http://www.ebi.ac.uk/pride (ref. 109) with the dataset identifiers PXD046739, PXD046743, PXD046744 and PXD046747 for the 28S, 18S, 5.8S and 5S rRNAs, respectively. All other materials such as cells are available on reasonable request.

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Acknowledgements

We thank the TFS and Jeol companies for kindly making their microscope setups available for test data collection, which was performed by Evgeniya Pechnikova (TFS, Eindhoven, NL) and Fumiaki Makino (on behalf of Jeol, Osaka, Japan). We acknowledge J. Michalon, M. Schaeffer and S. Ballet for IT support and the IGBMC cell culture facilities for HeLa cell production, members of the integrated structural biology platform at CBI for support and the late J.-F. Ménétret for his constant interest. A.L. and P.W. thank B. Chane-Woon-Ming for the implementation of the MassSpec-Toolkit for RNAs software. This work was supported by CNRS (Centre national de la recherche scientifique), Association pour la Recherche sur le Cancer, Institut National du Cancer (INCa_16099), the Fondation pour la Recherche Médicale (FRM and FDT202304016898), Ligue nationale contre le cancer (Ligue), Agence Nationale pour la Recherche and the University of Strasbourg Institute for Advanced Study (USIAS-2018-012). This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021-2028 program of the University of Strasbourg, CNRS and Inserm, was supported by IdEx Unistra (ANR-10-IDEX-0002) and by SFRI-STRAT’US project (ANR 20-SFRI-0012), EUR IMCBio (ANR-17-EURE-0023) under the framework of the France 2030 program and LabexNetRNA (ANR-10-LABX-0036_NETRNA) administered by Agence Nationale pour la Recherche and by the epiRNA funding from the Region Grand Est. The electron microscope facility was supported by the Region Grand Est, FEDER, the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01/France 2030 program, Instruct-ERIC and iNEXT-Discovery.

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Author notes

  1. These authors contributed equally: Samuel Holvec, Charles Barchet, Antony Lechner, Léo Fréchin.

Authors and Affiliations

  1. Centre for Integrative Biology, Department of Integrated Structural Biology, Institute of Genetics and of Molecular and Cellular Biology, Illkirch, France

    Samuel Holvec, Charles Barchet, Antony Lechner, Léo Fréchin, S. Nimali T. De Silva, Isabelle Hazemann, Ottilie von Loeffelholz & Bruno P. Klaholz

  2. Centre National de la Recherche Scientifique UMR, Illkirch, France

    Samuel Holvec, Charles Barchet, Antony Lechner, Léo Fréchin, S. Nimali T. De Silva, Isabelle Hazemann, Ottilie von Loeffelholz & Bruno P. Klaholz

  3. Institut National de la Santé et de la Recherche Médicale, Illkirch, France

    Samuel Holvec, Charles Barchet, Antony Lechner, Léo Fréchin, S. Nimali T. De Silva, Isabelle Hazemann, Ottilie von Loeffelholz & Bruno P. Klaholz

  4. Université de Strasbourg, Strasbourg, France

    Samuel Holvec, Charles Barchet, Antony Lechner, Léo Fréchin, S. Nimali T. De Silva, Isabelle Hazemann, Ottilie von Loeffelholz & Bruno P. Klaholz

  5. Architecture et Réactivité de l’ARN, CNRS UPR9002, Institute of Molecular and Cellular Biology, Université de Strasbourg, Strasbourg, France

    Antony Lechner & Philippe Wolff

Authors
  1. Samuel Holvec
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Contributions

I.H., L.F. and S.H. performed sample preparation. L.F., S.H. and O.v.L. carried out the image processing. A.L. and P.W. carried out the MS analysis and annotations. S.N.T.D.S. and O.v.L. performed the rRNA sequencing, and C.B., L.F., S.H., O.v.L. and B.P.K. performed the structure refinement and model building, structural analysis and annotations. All authors analyzed the data. B.P.K. supervised the project and wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Bruno P. Klaholz.

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The authors declare no competing financial interests.

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Extended data

Extended Data Fig. 1

Particle sorting scheme.

Extended Data Fig. 2

Resolution improvement of features in the cryo-EM map (present work compared to our previous study38).

Extended Data Fig. 3 Post-translational modifications of ribosomal proteins in the human ribosome.

Iso-Asp138 (uS11), methyl-arginine (ribosomal protein eS19), hydroxy-histidine (uL15) and methyl-histidine (uL3).

Extended Data Fig. 4

Visualization of endogenous polyamines (2 spermidines) in the human ribosome.

Extended Data Fig. 5 Assignment of octahedral Mg2+ coordinations with water molecules at N7, O6 and O4 positions of nucleotide bases.

Confirmation of Mg2+-coordinated rRNA bases that were identified previously by electrostatic potential map calculations40; respective σ-levels are indicated.

Extended Data Fig. 6 MS analysis using chimeric oligo nucleotides.

Deconvoluted MS/MS spectrum of 1770AUCUCAACC[Ψ]A[Ψ][Ψ]1783C from HeLa 28S rRNA with fragmentation assignments. x-axis: mass (Da); y-axis: Relative abundance.

Extended Data Fig. 7 RNA & DNA oligos used for mass spectrometry analysis.

Deoxyribonucleotides are in parenthesis; Nm corresponds to 2′-O-methylated nucleotides.

Extended Data Fig. 8 Correction of the rRNA reference sequence as found by sequencing and confirmed from the structure and MS analysis.

A4910 is labeled in red in the 28S rRNA sequence (see Supplementary Fig. 4).

Extended Data Fig. 9 Primer sequences used for human 18S, 28S, 5.8S and 5S rRNA sequencing.

Oligonucleotide primers used for PCR.

Supplementary information

Supplementary Information

Supplementary Fig. 1: Chemical modifications seen in the cryo-EM map (40S ribosomal subunit).

Supplementary Fig. 2: Chemical modifications seen in the cryo-EM map (60S ribosomal subunit).

Supplementary Fig. 3: MS analysis of 18S, 28S, 5.8S and 5S rRNA fragments with 2′-O-Me’s, Ψs and nucleotide base modifications.

Supplementary Fig. 4: The 18S, 28S, 5.8S and 5S rRNA sequencing with annotations of chemically modified nucleotides.

Reporting Summary

Supplementary Table 1

Legend to Supplementary File 1. Complete list of sites analyzed by MS and cryo-EM and the corresponding annotations, including previously reported rRNA modifications38,88,89. RNAses that were used to identify the respective modifications are indicated. Chemical modifications from E. coli, rabbit and plant ribosome structures58,59,60,84 are included for comparison.

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Holvec, S., Barchet, C., Lechner, A. et al. The structure of the human 80S ribosome at 1.9 Å resolution reveals the molecular role of chemical modifications and ions in RNA. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01274-x

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