Crystal structure of Prp8 reveals active site cavity of the spliceosome

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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


  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).

Accession codes

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Protein Data Bank


  1. Wahl, M. C., Will, C. L. & Lührmann, R. The spliceosome: design principles of a dynamic RNP machine. Cell 136, 701718 (2009)
  2. Wassarman, D. A. & Steitz, J. A. Interactions of small nuclear RNA’s with precursor messenger RNA during in vitro splicing. Science 257, 19181925 (1992)
  3. Madhani, H. D. & Guthrie, C. A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome. Cell 71, 803817 (1992)
  4. Kandels-Lewis, S. & Seraphin, B. Involvement of U6 snRNA in 5′ splice site selection. Science 262, 20352039 (1993)
  5. Lesser, C. F. & Guthrie, C. Mutations in U6 snRNA that alter splice site specificity: Implications for the active site. Science 262, 19821988 (1993)
  6. Sun, J. S. & Manley, J. L. A novel U2–U6 snRNA structure is necessary for mammalian mRNA splicing. Genes Dev. 9, 843854 (1995)
  7. Yean, S.-L., Wuenschell, G., Termini, J. & Lin, R. J. Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome. Nature 408, 881884 (2000)
  8. Steitz, T. A. & Steitz, J. A. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl Acad. Sci. USA 90, 64986502 (1993)
  9. Newman, A. J. & Norman, C. U5 snRNA interacts with exon sequences at 5′ and 3′ splice sites. Cell 68, 743754 (1992)
  10. Sontheimer, E. J. & Steitz, J. A. The U5 and U6 small nuclear RNAs as active site components of the spliceosome. Science 262, 19891996 (1993)
  11. O'Keefe, R. T., Norman, C. & Newman, A. J. The invariant U5 snRNA loop 1 sequence is dispensable for the first catalytic step of pre-mRNA splicing in yeast. Cell 86, 679689 (1996)
  12. Achsel, T., Ahrens, K., Brahms, H., Teigelkamp, S. & Lührmann, R. The human U5-220kD protein (hPrp8) forms a stable RNA-free complex with several U5-specific proteins, including an unwindase, a homologue of ribosomal elongation factor EF-2, and a novel WD-40 protein. Mol. Cell. Biol. 18, 67566766 (1998)
  13. Bartels, C., Urlaub, H., Lührmann, R. & Fabrizio, P. Mutagenesis suggests several roles of Snu114p in pre-mRNA splicing. J. Biol. Chem. 278, 2832428334 (2003)
  14. Small, E. C., Leggett, S. R., Winans, A. A. & Staley, J. P. The EF-G-like GTPase Snu114p regulates spliceosome dynamics mediated by Brr2p, a DExD/H box ATPase. Mol. Cell 23, 389399 (2006)
  15. Teigelkamp, S., Newman, A. J. & Beggs, J. D. Extensive interactions of PRP8 protein with the 5′ and 3′ splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA. EMBO J. 14, 26022612 (1995)
  16. Dix, I., Russell, C. S., O’Keefe, R. T., Newman, A. J. & Beggs, J. D. Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae. RNA 4, 12391250 (1998)
  17. Vidal, V. P., Verdone, L., Mayes, A. E. & Beggs, J. D. Characterization of U6 snRNA-protein interactions. RNA 5, 14701481 (1999)
  18. Reyes, J. L., Gustafson, E. H., Luo, H. R., Moore, M. J. & Konarska, M. M. The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5′ splice site. RNA 5, 167179 (1999)
  19. MacMillan, A. M. et al. Dynamic association of proteins with the pre-mRNA branch region. Genes Dev. 8, 30083020 (1994)
  20. Turner, I. A., Norman, C. M., Churcher, M. J. & Newman, A. J. Dissection of Prp8 protein defines multiple interactions with crucial RNA sequences in the catalytic core of the spliceosome. RNA 12, 375386 (2006)
  21. Grainger, R. J. & Beggs, J. D. Prp8 protein: at the heart of the spliceosome. RNA 11, 533557 (2005)
  22. Pena, V., Rozov, A., Fabrizio, P., Lührmann, R. & Wahl, M. C. Structure and function of an RNase H domain at the heart of the spliceosome. EMBO J. 27, 29292940 (2008)
  23. Ritchie, D. B. et al. Structural elucidation of a PRP8 core domain from the heart of the spliceosome. Nature Struct. Mol. Biol. 15, 11991205 (2008)
  24. Yang, K., Zhang, L., Xu, T., Heroux, A. & Zhao, R. Crystal structure of the β-finger domain of Prp8 reveals analogy to ribosomal proteins. Proc. Natl Acad. Sci. USA 105, 1381713822 (2008)
  25. Pena, V., Liu, S., Bujnicki, J. M., Lührmann, R. & Wahl, M. C. Structure of a multipartite protein-protein interaction domain in splicing factor Prp8 and its link to Retinitis pigmentosa. Mol. Cell 25, 615624 (2007)
  26. Zhang, L. et al. Crystal structure of the C-terminal domain of splicing factor Prp8 carrying retinitis pigmentosa mutants. Protein Sci. 16, 10241031 (2007)
  27. Dlakić, M. & Mushegian, A. Prp8, the pivotal protein of the spliceosomal catalytic center, evolved from a retroelement-encoded reverse transcriptase. RNA 17, 799808 (2011)
  28. Boon, K. L. et al. Prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast. Nature Struct. Mol. Biol. 14, 10771083 (2007)
  29. Weber, G. et al. Mechanism for Aar2p function as a U5 snRNP assembly factor. Genes Dev. 25, 16011612 (2011)
  30. Joyce, C. M. & Steitz, T. A. Function and structure relationships in DNA polymerase. Annu. Rev. Biochem. 63, 777822 (1994)
  31. Dias, A. et al. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458, 914918 (2009)
  32. Yuan, P. et al. Crystal structure of an avian influenza polymerase PAN reveals an endonuclease active site. Nature 458, 909913 (2009)
  33. Wyatt, J. R., Sontheimer, E. J. & Steitz, J. A. Site-specific cross-linking of mammalian U5 snRNP to the 5′ splice site before the first step of pre-mRNA splicing. Genes Dev. 6, 25422553 (1992)
  34. Urlaub, H., Hartmuth, K., Kostka, S., Grelle, G. & Lührmann, R. A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). J. Biol. Chem. 275, 4145841468 (2000)
  35. Query, C. C. & Konarska, M. M. Suppression of multiple substrate mutations by spliceosomal prp8 alleles suggests functional correlations with ribosomal ambiguity mutants. Mol. Cell 14, 343354 (2004)
  36. Umen, J. G. & Guthrie, C. Mutagenesis of the yeast gene PRP8 reveals domains governing the specificity and fidelity of 3′ splice site selection. Genetics 143, 723739 (1996)
  37. Kuhn, A. N. & Brow, D. A. Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in the splicing factor Prp8 that influence spliceosome activation. Genetics 155, 16671682 (2000)
  38. Kuhn, A. N., Reichl, E. M. & Brow, D. A. Distinct domains of splicing factor Prp8 mediate different aspects of spliceosome activation. Proc. Natl Acad. Sci. USA 99, 91459149 (2002)
  39. Sharp, P. A. On the origin of RNA splicing and introns. Cell 42, 397400 (1985)
  40. Cech, T. R. The generality of self-splicing RNA: relationship to nuclear mRNA splicing. Cell 44, 207210 (1986)
  41. Michel, F., Umesono, K. & Ozeki, H. Comparative and functional anatomy of group II catalytic introns—a review. Gene 82, 530 (1989)
  42. Sharp, P. A. Five easy pieces. Science 254, 663 (1991)
  43. Lambowitz, A. M. & Zimmerly, S. Mobile group II introns. Annu. Rev. Genet. 38, 135 (2004)
  44. Pyle, A. M. & Lambowitz, A. M. in The RNA World 3rd edn (eds Gesteland, R. F., Cech, T. R. & Atkins, J. F.) 469505 (Cold Spring Harbor Laboratory Press, 2006)
  45. Qui, Y.-L. & Palmer, J. D. Many different origins of trans splicing in a plant mitochondrial group II intron. J. Mol. Evol. 59, 8089 (2004)
  46. Toor, N. et al. Tertiary architecture of the Oceanobacillus iheyensis group II intron. RNA 16, 5769 (2010)
  47. Marcia, M. & Pyle, A. M. Visualizing group II intron catalysis through the stages of splicing. Cell 151, 497507 (2012)
  48. Matsuura, M., Noah, J. W. & Lambowitz, A. M. Mechanism of maturase-promoted group II intron splicing. EMBO J. 20, 72597270 (2001)
  49. Rambo, R. P. & Doudna, J. A. Assembly of an active group II intron-maturase complex by protein dimerization. Biochemistry 43, 64866497 (2004)
  50. Gu, S. Q. et al. Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase. RNA 16, 732747 (2010)
  51. Wagenbach, M. et al. Synthesis of wild type and mutant human hemoglobins in Saccharomyces cerevisiae. Biotechnology (N Y) 9, 5761 (1991)
  52. Christianson, T. W., Sikorski, R. S., Dante, M., Shero, J. H. & Hieter, P. Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119122 (1992)
  53. Leslie, A. G. W. & Powell, H. R. Processing diffraction data with Mosflm. Evolv. Methods Macromol. Crystallograph. 245, 4151 (2007)
  54. Kabsch, W. XDS. Acta Crystallogr. D 66, 125132 (2010)
  55. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 7282 (2006)
  56. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658674 (2007)
  57. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D 66, 2225 (2010)
  58. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240255 (1997)
  59. Sheldrick, G. M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479485 (2010)
  60. Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nature Protocols 3, 11711179 (2008)
  61. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 21262132 (2004)
  62. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 1221 (2010)
  63. Krissinel, E. & Henrick, K. Secondary-structure matching (PDBeFold), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D 60, 22562268 (2004)
  64. Holm, L. & Park, J. DaliLite workbench for protein structure comparison. Bioinformatics 16, 566567 (2000)
  65. Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 1003710041 (2001)
  66. Ashkenazy, H., Erez, E., Martz, E., Pupko, T. & Ben-Tal, N. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, 529533 (2010)
  67. Larkin, M. A. et al. ClustalW and ClustalX version 2. Bioinformatics 23, 29472948 (2007)
  68. Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 25772637 (1983)
  69. Heinig, M. & Frishman, D. STRIDE: a Web server for secondary structure assignment from known atomic coordinates of proteins. Nucleic Acids Res. 32, W500W502 (2004)
  70. Sikorski, R. S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 1927 (1989)

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

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


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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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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