Abstract
The active centre of the spliceosome consists of an intricate network formed by U5, U2 and U6 small nuclear RNAs, and a pre-messenger-RNA substrate. Prp8, a component of the U5 small nuclear ribonucleoprotein particle, crosslinks extensively with this RNA catalytic core. Here we present the crystal structure of yeast Prp8 (residues 885–2413) in complex with Aar2, a U5 small nuclear ribonucleoprotein particle assembly factor. The structure reveals tightly associated domains of Prp8 resembling a bacterial group II intron reverse transcriptase and a type II restriction endonuclease. Suppressors of splice-site mutations, and an intron branch-point crosslink, map to a large cavity formed by the reverse transcriptase thumb, and the endonuclease-like and RNaseH-like domains. This cavity is large enough to accommodate the catalytic core of group II intron RNA. The structure provides crucial insights into the architecture of the spliceosome active site, and reinforces the notion that nuclear pre-mRNA splicing and group II intron splicing have a common origin.
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Acknowledgements
We thank G. Murshudov for his help and guidance with crystallography; T.H.D. Nguyen, Y. Kondo, M. van Roon, J. Hardin, J. Li, C. Norman, A. Andreeva, A. Murzin and M. Yu for discussion and help; T. Ignjatovic and H. Oshikane for their contributions at the early stage of the project; L. Passmore and L. Jovine for reading of the manuscript; and M. Ikura for the gift of a calmodulin clone. We are grateful to the beamline staff at Diamond Light Source and European Synchrotron Radiation Facility for their help, and to E. Stephens and the LMB mass spectrometry facility for their help. W.P.G. thanks the Cambridge European Trust and Downing College for scholarships. This project was funded by the UK Medical Research Council.
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A.J.N. and K.N. initiated the project and worked on protein expression and purification for many years. Co-expression of Prp8 and Aar2 by A.J.N. was a crucial step of the project. W.P.G. successfully identified and expressed a stable large fragment of Prp8, crystallized the Prp8–Aar2 complex and solved and refined the structure almost single-handedly with practical support from K.N. and A.J.N. C.O. analysed the mercury derivative data and refined the structure of the P212121 crystal form. W.P.G. and K.N. analysed the structure and wrote the paper with important input from A.J.N. and C.O.
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Galej, W., Oubridge, C., Newman, A. et al. Crystal structure of Prp8 reveals active site cavity of the spliceosome. Nature 493, 638–643 (2013). https://doi.org/10.1038/nature11843
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DOI: https://doi.org/10.1038/nature11843
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