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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 1101–1107 (2005)Cite this article

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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Figure 1: Sequences and secondary structures of a box H/ACA snoRNA bound to its substrate RNA and of the box H/ACA snoRNA–like region of human telomerase RNA.
Figure 2: Functional conservation of Nop10 and the Cbf5–Nop10 complex between archaea and eukarya.
Figure 3: Structure of the Cbf5–Nop10 complex.
Figure 4: Structural comparison of the Cbf5–Nop10 complex and TruB.
Figure 5: Conserved surfaces of the Cbf5–Nop10 complex interact with H/ACA guide RNA.

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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.

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Authors and Affiliations

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

    Tomoko Hamma & Adrian R Ferré-D'Amaré

  2. Department of Chemistry, University of Washington, Box 351700, Seattle, 98195-1700, Washington, USA

    Steve L Reichow & Gabriele Varani

  3. Department of Biochemistry, University of Washington, Box 357350, Seattle, 98195-7350, Washington, USA

    Gabriele Varani

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

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Supplementary information

Supplementary Fig. 1

Multiple sequence alignment of Nop10. (PDF 183 kb)

Supplementary Fig. 2

Zinc-ribbon domain of free aNop10. (PDF 334 kb)

Supplementary Fig. 3

In vitro reconstitution of an archaeal H/ACA RNP analyzed by the electrophoretic mobility shift assay. (PDF 878 kb)

Supplementary Fig. 4

Multiple sequence alignment of Cbf5/Dyskerin (PDF 384 kb)

Supplementary Fig. 5

Multiple sequence alignment of the PUA domain. (PDF 495 kb)

Supplementary Fig. 6

Molecular surface of the aCbf5–aNop10 complex cocrystal structure and a hypothetical RNA double helix. (PDF 730 kb)

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. (PDF 1675 kb)

Supplementary Table 1

NMR and refinement statistics for aNop10 and yNop10. (DOC 48 kb)

Supplementary Methods (PDF 148 kb)

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Hamma, T., Reichow, S., Varani, G. et al. The Cbf5–Nop10 complex is a molecular bracket that organizes box H/ACA RNPs. Nat Struct Mol Biol 12, 1101–1107 (2005). https://doi.org/10.1038/nsmb1036

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