Letter | Published:

Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes

Nature volume 534, pages 133137 (02 June 2016) | Download Citation

Abstract

Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm1,2. Hundreds of assembly factors, organized into sequential functional groups3,4, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.

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Accessions

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The cryo-EM density maps of state 1 and state 2 have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-6615 and EMD-6616, respectively. The atomic model of state 1 has been deposited in the Protein Data Bank (PDB) under accession number 3JCT. The XL–MS data have been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org/) with the dataset identifier PXD003736.

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Acknowledgements

We thank the National Center for Protein Sciences (Beijing, China) for providing resource for cryo-EM data collection and computation. We also thank members of Woolford laboratory for reading the manuscript. This work was supported by the Ministry of Science and Technology of China (2013CB910404 to N.G. and 2014CB849800 to M.-Q.D.), the National Natural Science Foundation of China (31422016 and 31470722 to N.G., and 21375010 to M.-Q.D.) and National Institutes of Health grant R01GM028301 (to J.L.W.).

Author information

Affiliations

  1. Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China

    • Shan Wu
    • , Kaige Yan
    • , Yixiao Zhang
    • , Yi Yuan
    • , Zhifei Li
    • , Chengying Ma
    • , Jianlin Lei
    •  & Ning Gao
  2. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA

    • Beril Tutuncuoglu
    • , Hailey Brown
    • , Michael Gamalinda
    • , Jelena Jakovljevic
    •  & John L. Woolford
  3. Graduate Program in Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China

    • Dan Tan
    •  & Meng-Qiu Dong
  4. National Institute of Biological Sciences, Beijing 102206, China

    • Dan Tan
    •  & Meng-Qiu Dong

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Contributions

N.G. and J.L.W. designed and directed experiments; B.K., H.B., M.G. and J.J. purified samples; D.T. and M.-Q.D. performed XL–MS; S.W. collected cryo-EM data (with J.L., Y.Y., Z.L., and C.M.), performed image processing (with Y.Z.), and analysed structures (with Y.K.). N.G., S.W. and K.Y. performed structural modelling. S.W., J.L.W. and N.G. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to John L. Woolford or Ning Gao.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    This file contains the original gel image for Extended Data Fig. 1a.

Excel files

  1. 1.

    Supplementary Table 1

    This file shows cross-linked peptides identified in CL-MS data.

  2. 2.

    Supplementary Table 2

    This file shows the analysis results of CL-MS data.

Videos

  1. 1.

    Overall structure of Nog2-particles in State 1

    The cryo-EM density (sharpened) is first shown in surface representative, with densities of factors individually colored (as in Fig. 1). The rRNA and r-proteins are colored grey and beige, respectively. Groups of factors are then highlighted in zoom-in views.

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DOI

https://doi.org/10.1038/nature17942

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