Article

Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution

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Accepted:
Published online:

Abstract

U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5′-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre.

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Change history

  • Corrected online 17 February 2016

    The Electron Microscopy Data Bank accession codes were listed in full, rather than as a range.

Accessions

Data deposits

The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with accession codes EMD-8006, EMD-8007, EMD-8008, EMD-8009, EMD-8010, EMD-8011, EMD-8012, EMD-8013 and EMD-8014. The coordinates of the atomic models have been deposited in the Protein Data Bank under accession codes 5GAN (overall), 5GAP (body domain), 5GAO (head domain) and 5GAM (foot domain).

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Acknowledgements

We thank C. Savva, S. Chen, G. McMullan, J. Grimmett and T. Darling for running the electron microscopy and computing facilities, A. Brown, P. Emsley, G. Murshudov for advice and help with model building and refinement, R. O’Keefe for the ∆Snu114 yeast strain, the members of the spliceosome group for help and advice throughout the project and R. Leiro for help with data processing. We thank S. Fica for critical reading of the manuscript and J. Löwe, V. Ramakrishnan, and R. Henderson for their continuing support and encouragements. The project was supported by the Medical Research Council (MC_U105184330 to K.N. and MC_UP_A025_1013 to S.H.W.S.).

Author information

Author notes

    • Thi Hoang Duong Nguyen
    •  & Wojciech P. Galej

    These authors contributed equally to this work.

Affiliations

  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK

    • Thi Hoang Duong Nguyen
    • , Wojciech P. Galej
    • , Xiao-chen Bai
    • , Chris Oubridge
    • , Andrew J. Newman
    • , Sjors H. W. Scheres
    •  & Kiyoshi Nagai

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Contributions

T.H.D.N. purified yeast tri-snRNP and prepared EM grids, T.H.D.N., W.P.G. and X.-C.B collected all EM images. T.H.D.N. processed data and calculated the maps with the help of X.-C.B and S.H.W.S.; T.H.D.N., W.P.G. and C.O. built a model into the map and refined the structure. T.H.D.N. performed mutagenesis experiments with the help of A.J.N.; T.H.D.N. and W.P.G. prepared all illustrations. K.N. initiated and orchestrated the project. T.H.D.N., W.P.G. and K.N. analysed the structure and wrote the paper with invaluable contributions from all other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Thi Hoang Duong Nguyen or Wojciech P. Galej or Kiyoshi Nagai.

Extended data

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  1. 1.

    Supplementary Data

    This zipped file contains the pymol session file of the PDB coordinate.

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