Letter | Published:

Modular assembly of the nucleolar pre-60S ribosomal subunit

Nature volume 556, pages 126129 (05 April 2018) | Download Citation

Abstract

Early co-transcriptional events during eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits1. A multitude of transient assembly factors regulate and chaperone the systematic folding of pre-ribosomal RNA subdomains. However, owing to a lack of structural information, the role of these factors during early nucleolar 60S assembly is not fully understood. Here we report cryo-electron microscopy (cryo-EM) reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions of up to 3.4 Å. These reconstructions reveal how steric hindrance and molecular mimicry are used to prevent both premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Among these factors, three Brix-domain proteins and their binding partners form a ring-like structure at ribosomal RNA (rRNA) domain boundaries to support the architecture of the maturing particle. The existence of mutually exclusive conformations of these pre-60S particles suggests that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly. These structures rationalize previous genetic and biochemical data and highlight the mechanisms that drive eukaryotic ribosome assembly in a unidirectional manner.

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Acknowledgements

We thank M. Ebrahim and J. Sotiris for their support with data collection at the Evelyn Gruss Lipper Cryo-EM resource center, M. Tesic-Mark for analysis of mass spectrometry data, C. Cheng for help with the initial manual curation and analysis of the nucleolar pre-60S particles and members of the Walz laboratory for helpful discussions. L.M. is supported in part by NIH T32 GM115327-Tan. J.B. is supported by an EMBO long-term fellowship (ALTF 51-2014) and a Swiss National Science Foundation fellowship (155515). M.C.-M. is supported by a postgraduate scholarship from NSERC. S.K. is supported by the Robertson Foundation, the Irma T. Hirschl Trust, the Alexandrine and Alexander L. Sinsheimer Fund, the Rita Allen Foundation and an NIH New Innovator Award (1DP2GM123459). B.T.C. is supported by National Institute of Health Grant Nos. P41GM103314 and P41GM109824.

Author information

Author notes

    • Zahra Assur Sanghai
    •  & Linamarie Miller

    These authors contributed equally to this work.

Affiliations

  1. Laboratory of Protein and Nucleic Acid Chemistry, The Rockefeller University, New York, New York 10065, USA

    • Zahra Assur Sanghai
    • , Linamarie Miller
    • , Jonas Barandun
    • , Mirjam Hunziker
    • , Malik Chaker-Margot
    •  & Sebastian Klinge
  2. Tri-Institutional Training Program in Chemical Biology, The Rockefeller University, New York, New York 10065, USA

    • Linamarie Miller
    •  & Malik Chaker-Margot
  3. Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10065, USA

    • Kelly R. Molloy
    • , Junjie Wang
    •  & Brian T. Chait

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Contributions

S.K. and Z.A.S. established purification conditions. Z.A.S., L.M. and S.K. determined the cryo-EM structure of the yeast nucleolar pre-60S particle. K.R.M., J.W. and B.T.C. processed and analysed DSS cross-linking data. Z.A.S., L.M., J.B., M.C.-M., M.H. and S.K. built the model. L.M. performed all RNA work, and Z.A.S., L.M., J.B., M.H., M.C.-M. and S.K. interpreted the results and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Sebastian Klinge.

Reviewer Information Nature thanks A. Amunts, D. Tollervey and J. Woolford for their contribution to the peer review of this work.

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

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

  2. 2.

    Supplementary Figure 1

    Source data for the RNA gel and northern blots showing the uncropped images for Extended Data Figure 1d and e.

CSV files

  1. 1.

    Supplementary Data 1

    DSS cross-links for the nucleolar pre-60S ribosomal subunit.

Zip files

  1. 1.

    Supplementary Data 2

    PyMOL session file for the structural analysis of the nucleolar pre-60S ribosomal subunit.

Videos

  1. 1.

    360° view of the cryo-EM reconstruction of the S. cerevisiae nucleolar pre-60S particle

    A 360° rotation of a composite cryo-EM map consisting of the 3.4 Å state 1 and the 3.7 Å state 2 maps. Densities for nucleolar pre-60S assembly factors and rRNA domains are colour-coded as in Figure 1 and ribosomal proteins are colored in grey. The rotation is paused at front and back view as shown in Figure 1.

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DOI

https://doi.org/10.1038/nature26156

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