Crystal structure of Prp8 reveals active site cavity of the spliceosome

Journal name:
Nature
Volume:
493,
Pages:
638–643
Date published:
DOI:
doi:10.1038/nature11843
Received
Accepted
Published online

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 groupII 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 groupII intron splicing have a common origin.

At a glance

Figures

  1. Structure of the large domain in yeast Prp8 (residues 885-1824).
    Figure 1: Structure of the large domain in yeast Prp8 (residues 885–1824).

    a, The large domain consists of a group II intron reverse transcriptase-like domain and a type II restriction endonuclease-like domain. b, The palm subdomain of hepatitis C virus RNA-dependent RNA polymerase (1NB6). Asp residues (Asp220 in motif A, and Asp318 and Asp319 motif C) coordinate two catalytic Mg2+ ions. c, The corresponding residues in the palm subdomain of the group II intron reverse transcriptase-like domain of Prp8. d, The catalytic centre of the endonuclease domain of the influenza virus polymerase acidic subunit (2W69). His41, Glu80, Asp108 and Glu119 coordinate two catalytic divalent ions. e, The corresponding residues in the endonuclease domain of Prp8.

  2. Overall structure of yeast Prp8885-2413 in complex with Aar2.
    Figure 2: Overall structure of yeast Prp8885–2413 in complex with Aar2.

    a, Domain architecture of Prp8885–2413 and Aar2. CTD, C-terminal domain; NTD, N-terminal domain; RT, reverse transcriptase. b, Aar2 organizes the arrangement of the RT/En, RNaseH-like and Jab1/MPN domains. c, Orthogonal view of the complex. d, A view (as in c) without Aar2 and the Jab1/MPN domain. The RNaseH-like domain has no direct contact with the RT/En domain.

  3. Overview of the Prp8 active site cavity in an /`open book/' view.
    Figure 3: Overview of the Prp8 active site cavity in an ‘open book’ view.

    a, Overview with the suppressors of splice site (5′-SS, 3′-SS and branch point) mutations (red spheres). Green spheres indicate the sequence (1966-Ser-Ala-Ala-Met-Ser-1970) corresponding to the crosslinking site of hPRP8 to the 5′-SS (ref. 18). b, Stereo view of the RNaseH-like domain surface making up the active site cavity. c, Stereo view of the RT/En domain surface making up the active site cavity. Crosslink of the pre-mRNA branch point (BP+2) nucleotide is located between residues 1585 and 1598 in sequence (C. M. Norman and A.J.N., unpublished observations). This site is found within the disordered loop (blue dotted line) between residues 1575 and 1598 (blue spheres).

  4. Suppressors of U4-cs1 and brr2-1 alleles mapped on the Prp8 structure.
    Figure 4: Suppressors of U4-cs1 and brr2-1 alleles mapped on the Prp8 structure.

    a, U4-cs-1 (blue spheres) and brr2-1 (green spheres) suppressor mutants map on one face of the RT/En domain of Prp8. b, A view rotated by 120° along the y axis. c, Both types of suppressor mutant map to the same region of the Prp8 reverse transcriptase domain. Residues that suppress both alleles are marked with an asterisk.

  5. Comparison between the active site of group II intron and the spliceosome (Prp8).
    Figure 5: Comparison between the active site of group II intron and the spliceosome (Prp8).

    a, Group II intron from O. iheyensis (PDB accession 3IGI). Domain V, red; exon-binding loop helix, blue; spliced exons, green; catalytic Mg2+ ions, yellow sphere; scaffolding RNA, grey. b, The RT/En domain with the RNaseH-like domain of Prp8 with the active RNA elements of group II intron modelled on its surface for size comparison. At present there are insufficient experimental constraints for the precise position or orientation of the RNA. c, Electrostatic potential (±5kTe−1) plotted on the solvent-accessible surface of the Prp8 (calculated with adaptive Poisson–Boltzmann solver, see Methods).

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Primary accessions

Protein Data Bank

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Author information

Affiliations

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

    • Wojciech P. Galej,
    • Chris Oubridge,
    • Andrew J. Newman &
    • Kiyoshi Nagai

Contributions

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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The authors declare no competing financial interests.

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Atomic coordinates and structure factors for the Prp8–Aar2 complex have been deposited in the Protein Data Bank under accession codes 4I43 (C2221) and 3ZEF (P212121).

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