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The Cbf5–Nop10 complex is a molecular bracket that organizes box H/ACA RNPs

Nature Structural & Molecular Biology volume 12, pages 11011107 (2005) | Download Citation

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Abstract

Box H/ACA ribonucleoprotein particles (RNPs) catalyze RNA pseudouridylation and direct processing of ribosomal RNA, and are essential architectural components of vertebrate telomerases. H/ACA RNPs comprise four proteins and a multihelical RNA. Two proteins, Cbf5 and Nop10, suffice for basal enzymatic activity in an archaeal in vitro system. We now report their cocrystal structure at 1.95-Å resolution. We find that archaeal Cbf5 can assemble with yeast Nop10 and with human telomerase RNA, consistent with the high sequence identity of the RNP components between archaea and eukarya. Thus, the Cbf5–Nop10 architecture is phylogenetically conserved. The structure shows how Nop10 buttresses the active site of Cbf5, and it reveals two basic troughs that bidirectionally extend the active site cleft. Mutagenesis results implicate an adjacent basic patch in RNA binding. This tripartite RNA-binding surface may function as a molecular bracket that organizes the multihelical H/ACA and telomerase RNAs.

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Acknowledgements

We thank C. Hoang for experimental contributions to initial stages of this project, A. Roll-Mecak for advice on protein coexpression, J. Bolduc for in-house X-ray support, the staff of Advanced Light Source beamline 5.0.2. for synchrotron data collection support, N. Isern at Pacific Northwest National Laboratories and the staff at the National Magnetic Resonance Facility at Madison for support with NMR data collection, N. Leuliott (IBBMC, Université Paris-Sud) for providing yNop10 expression vector and T. Edwards, K. Godin, D. Klein, J. Pitt, B. Shen, B. Stoddard and H. Xiao for discussions. This work was supported by the US National Institutes of Health (grants to A.R.F. and G.V. and Viral Oncology training grant to T.H.). T.H. is a Leukemia & Lymphoma Society Special Fellow. A.R.F. is a Distinguished Young Scholar in Medical Research of the W.M. Keck Foundation.

Author information

Affiliations

  1. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109-1024, USA.

    • Tomoko Hamma
    •  & Adrian R Ferré-D'Amaré
  2. Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195-1700, USA.

    • Steve L Reichow
    •  & Gabriele Varani
  3. Department of Biochemistry, University of Washington, Box 357350, Seattle, Washington 98195-7350, USA.

    • Gabriele Varani

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Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Gabriele Varani or Adrian R Ferré-D'Amaré.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Multiple sequence alignment of Nop10.

  2. 2.

    Supplementary Fig. 2

    Zinc-ribbon domain of free aNop10.

  3. 3.

    Supplementary Fig. 3

    In vitro reconstitution of an archaeal H/ACA RNP analyzed by the electrophoretic mobility shift assay.

  4. 4.

    Supplementary Fig. 4

    Multiple sequence alignment of Cbf5/Dyskerin

  5. 5.

    Supplementary Fig. 5

    Multiple sequence alignment of the PUA domain.

  6. 6.

    Supplementary Fig. 6

    Molecular surface of the aCbf5–aNop10 complex cocrystal structure and a hypothetical RNA double helix.

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    Supplementary Fig. 7

    Portion of the 1.95-Å resolution solvent-flattened MAD experimental electron density map contoured at 1.1 standard deviations above mean peak height.

  8. 8.

    Supplementary Methods

Word documents

  1. 1.

    Supplementary Table 1

    NMR and refinement statistics for aNop10 and yNop10.

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DOI

https://doi.org/10.1038/nsmb1036

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