Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A conformational switch in PRP8 mediates metal ion coordination that promotes pre-mRNA exon ligation

Nature Structural & Molecular Biology volume 20, pages 728–734 (2013)Cite this article

Subjects

Abstract

Splicing of pre-mRNAs in eukaryotes is catalyzed by the spliceosome, a large RNA-protein metalloenzyme. The catalytic center of the spliceosome involves a structure comprising the U2 and U6 snRNAs and includes a metal bound by U6 snRNA. The precise architecture of the splicesome active site, however, and the question of whether it includes protein components, remains unresolved. A wealth of evidence places the protein PRP8 at the heart of the spliceosome through assembly and catalysis. Here we provide evidence that the RNase H domain of PRP8 undergoes a conformational switch between the two steps of splicing, rationalizing yeast prp8 alleles that promote either the first or second step. We also show that this switch unmasks a metal-binding site involved in the second step. Together, these data establish that PRP8 is a metalloprotein that promotes exon ligation within the spliceosome.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Conformational switch in the PRP8 RH domain unmasks a Mg2+-binding site.
Figure 2: Characterization of PRP8 RH-domain alleles.
Figure 3: PRP8 mutant alleles favor distinct conformations within the PRP8 RH domain.
Figure 4: PRP8 Asp1853 mutations selectively impair the second step of splicing.
Figure 5: Bimolecular exon ligation assay implicates PRP8 Asp1853 in the second transesterification step of pre-mRNA splicing.
Figure 6: PRP8 RH undergoes a conformational switch to present a functionally important metal ion to the spliceosome.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Jurica, M.S. & Moore, M.J. Pre-mRNA splicing: awash in a sea of proteins. Mol. Cell 12, 5–14 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Rappsilber, J., Ryder, U., Lamond, A.I. & Mann, M. Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231–1245 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Zhou, Z., Licklider, L.J., Gygi, S.P. & Reed, R. Comprehensive proteomic analysis of the human spliceosome. Nature 419, 182–185 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. 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, 803–817 (1992).

    Article  CAS  PubMed  Google Scholar 

  5. 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, 167–179 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 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, 375–386 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Vidal, V.P., Verdone, L., Mayes, A.E. & Beggs, J.D. Characterization of U6 snRNA-protein interactions. RNA 5, 1470–1481 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liu, L., Query, C.C. & Konarska, M.M. Opposing classes of prp8 alleles modulate the transition between the catalytic steps of pre-mRNA splicing. Nat. Struct. Mol. Biol. 14, 519–526 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. 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, 9145–9149 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 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, 1667–1682 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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, 723–739 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Grainger, R.J. & Beggs, J.D. Prp8 protein: at the heart of the spliceosome. RNA 11, 533–557 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Abelson, J. Is the spliceosome a ribonucleoprotein enzyme? Nat. Struct. Mol. Biol. 15, 1235–1237 (2008).

    Article  CAS  PubMed  Google Scholar 

  14. Ritchie, D.B. et al. Structural elucidation of a PRP8 core domain from the heart of the spliceosome. Nat. Struct. Mol. Biol. 15, 1199–1205 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. 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, 13817–13822 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Pena, V., Rozov, A., Fabrizio, P., Luhrmann, R. & Wahl, M.C. Structure and function of an RNase H domain at the heart of the spliceosome. EMBO J. 27, 2929–2940 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Galej, W.P., Oubridge, C., Newman, A.J. & Nagai, K. Crystal structure of Prp8 reveals active site cavity of the spliceosome. Nature 493, 638–643 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Nowotny, M., Gaidamakov, S.A., Crouch, R.J. & Yang, W. Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis. Cell 121, 1005–1016 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. 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, 343–354 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Fouser, L.A. & Friesen, J.D. Mutations in a yeast intron demonstrate the importance of specific conserved nucleotides for the two stages of nuclear mRNA splicing. Cell 45, 81–93 (1986).

    Article  CAS  PubMed  Google Scholar 

  21. Vijayraghavan, U. et al. Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome. EMBO J. 5, 1683–1695 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Anderson, K. & Moore, M.J. Bimolecular exon ligation by the human spliceosome. Science 276, 1712–1716 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Anderson, K. & Moore, M.J. Bimolecular exon ligation by the human spliceosome bypasses early 3′ splice site AG recognition and requires NTP hydrolysis. RNA 6, 16–25 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gordon, P.M., Sontheimer, E.J. & Piccirilli, J.A. Metal ion catalysis during the exon-ligation step of nuclear pre-mRNA splicing: extending the parallels between the spliceosome and group II introns. RNA 6, 199–205 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Collins, C.A. & Guthrie, C. Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome. Genes Dev. 13, 1970–1982 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kuhn, A.N., Li, Z. & Brow, D.A. Splicing factor Prp8 governs U4/U6 RNA unwinding during activation of the spliceosome. Mol. Cell 3, 65–75 (1999).

    Article  CAS  PubMed  Google Scholar 

  27. Siatecka, M., Reyes, J.L. & Konarska, M.M. Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center. Genes Dev. 13, 1983–1993 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Adams, P.L., Stahley, M.R., Kosek, A.B., Wang, J. & Strobel, S.A. Crystal structure of a self-splicing group I intron with both exons. Nature 430, 45–50 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. 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, 881–884 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Koodathingal, P., Novak, T., Piccirilli, J.A. & Staley, J.P. The DEAH box ATPases Prp16 and Prp43 cooperate to proofread 5′ splice site cleavage during pre-mRNA splicing. Mol. Cell 39, 385–395 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sontheimer, E.J., Sun, S. & Piccirilli, J.A. Metal ion catalysis during splicing of premessenger RNA. Nature 388, 801–805 (1997).

    Article  CAS  PubMed  Google Scholar 

  32. Sashital, D.G., Cornilescu, G., McManus, C.J., Brow, D.A. & Butcher, S.E. U2–U6 RNA folding reveals a group II intron-like domain and a four-helix junction. Nat. Struct. Mol. Biol. 11, 1237–1242 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. Toor, N., Keating, K.S., Taylor, S.D. & Pyle, A.M. Crystal structure of a self-spliced group II intron. Science 320, 77–82 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Steitz, T.A. & Steitz, J.A. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl. Acad. Sci. USA 90, 6498–6502 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Schmidt, B.H., Burgin, A.B., Deweese, J.E., Osheroff, N. & Berger, J.M. A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases. Nature 465, 641–644 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Nakamura, T., Zhao, Y., Yamagata, Y., Hua, Y.J. & Yang, W. Watching DNA polymerase η make a phosphodiester bond. Nature 487, 196–201 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Le Du, M.H. et al. Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase. J. Mol. Biol. 316, 941–953 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Kim, E.E. & Wyckoff, H.W. Reaction mechanism of alkaline phosphatase based on crystal structures: two-metal ion catalysis. J. Mol. Biol. 218, 449–464 (1991).

    Article  CAS  PubMed  Google Scholar 

  39. Romier, C., Dominguez, R., Lahm, A., Dahl, O. & Suck, D. Recognition of single-stranded DNA by nuclease P1: high resolution crystal structures of complexes with substrate analogs. Proteins 32, 414–424 (1998).

    Article  CAS  PubMed  Google Scholar 

  40. Garcin, E.D. et al. DNA apurinic-apyrimidinic site binding and excision by endonuclease IV. Nat. Struct. Mol. Biol. 15, 515–522 (2008).

    Article  CAS  PubMed  Google Scholar 

  41. Shan, S., Yoshida, A., Sun, S., Piccirilli, J.A. & Herschlag, D. Three metal ions at the active site of the Tetrahymena group I ribozyme. Proc. Natl. Acad. Sci. USA 96, 12299–12304 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Tanaka, N. & Schwer, B. Mutations in PRP43 that uncouple RNA-dependent NTPase activity and pre-mRNA splicing function. Biochemistry 45, 6510–6521 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Mayas, R.M., Maita, H., Semlow, D.R. & Staley, J.P. Spliceosome discards intermediates via the DEAH box ATPase Prp43p. Proc. Natl. Acad. Sci. USA 107, 10020–10025 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Otwinowski, Z.M. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    CAS  PubMed  Google Scholar 

  45. Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. A graphical user interface to the CCP4 program suite. Acta Crystallogr. D Biol. Crystallogr. 59, 1131–1137 (2003).

    Article  PubMed  Google Scholar 

  46. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  PubMed  Google Scholar 

  47. Terwilliger, T.C. Automated main-chain model building by template matching and iterative fragment extension. Acta Crystallogr. D Biol. Crystallogr. 59, 38–44 (2003).

    Article  PubMed  CAS  Google Scholar 

  48. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  49. Umen, J.G. & Guthrie, C. A novel role for a U5 snRNP protein in 3′ splice site selection. Genes Dev. 9, 855–868 (1995).

    Article  CAS  PubMed  Google Scholar 

  50. Brown, J.D. & Beggs, J.D. Roles of PRP8 protein in the assembly of splicing complexes. EMBO J. 11, 3721–3729 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Boeke, J.D., Trueheart, J., Natsoulis, G. & Fink, G.R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154, 164–175 (1987).

    Article  CAS  PubMed  Google Scholar 

  52. Lesser, C.F. & Guthrie, C. Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1. Genetics 133, 851–863 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Köhrer, K. & Domdey, H. Preparation of high molecular weight RNA. Methods Enzymol. 194, 398–405 (1991).

    Article  PubMed  Google Scholar 

  54. Lin, R.J., Newman, A.J., Cheng, S.C. & Abelson, J. Yeast mRNA splicing in vitro. J. Biol. Chem. 260, 14780–14792 (1985).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by an Operating grant to A.M.M. from the Canadian Institutes of Health Research (CIHR) and a US National Institutes of Health grant to J.P.S. and J. Piccirilli (R01GM088656). We would like to thank M. Friis and M. Schultz for helpful advice, J. Beggs (University of Edinburgh, Edinburgh, UK) and C. Guthrie (University of California, San Francisco, San Francisco, California, USA) for providing yeast strains and plasmids and D. Brow (University of Wisconsin–Madison, Madison, Wisconsin, USA) for providing the PRP8 V1862Y plasmid. Research described in this paper was performed at the Advanced Light Source (Berkeley, California, USA; supported by the US Department of Energy under contract no. DE-AC02-05CH11231) and the Canadian Light Source (supported by the Natural Sciences and Engineering Research Council of Canada, the National Research Council Canada, CIHR, the Province of Saskatchewan, Western Economic Diversification Canada and the University of Saskatchewan).

Author information

Author notes

  1. Matthew J Schellenberg & Dustin B Ritchie

    Present address: Present addresses: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, US Department of Health and Human Services, Research Triangle Park, North Carolina, USA (M.J.S.) and Department of Physics, University of Alberta, Edmonton, Alberta, Canada (D.B.R.).,

  2. Matthew J Schellenberg and Tao Wu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada

    Matthew J Schellenberg, Tao Wu, Dustin B Ritchie, Karim A Atta & Andrew M MacMillan

  2. Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA

    Sebastian Fica & Jonathan P Staley

  3. Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada

    Paul LaPointe

Authors
  1. Matthew J Schellenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tao Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dustin B Ritchie
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sebastian Fica
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jonathan P Staley
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Karim A Atta
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Paul LaPointe
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Andrew M MacMillan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.J.S., T.W., D.B.R. and A.M.M. designed the study; M.J.S. created mutant yeast strains, crystallized protein, collected X-ray diffraction data, solved the structures and performed in vivo and in vitro assays including development of the bimolecular exon ligation; T.W. created mutant PRP8 yeast strains, crystallized protein, collected X-ray diffraction data and carried out in vivo and in vitro assays; K.A.A. purified and crystallized proteins; S.F. and J.P.S. independently designed and tested mutants and critically analyzed data; D.B.R. designed, crystallized and analyzed mutants; P.L. provided technical expertise and helped design experiments in yeast; M.J.S., T.W. and A.M.M. wrote the manuscript.

Corresponding author

Correspondence to Andrew M MacMillan.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1 and 2 and Supplementary Note (PDF 2088 kb)

Supplementary Movie 1

Representation of the rearrangement of the PRP8 RH domain from the closed form to the metal-binding open conformation. Highlighted are the displacement of Thr1783 and Mg2+ coordination including the inner sphere ligand Asp1781. The Mg2+ ion is shown in purple and inner-sphere waters are shown in red. (MOV 5205 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schellenberg, M., Wu, T., Ritchie, D. et al. A conformational switch in PRP8 mediates metal ion coordination that promotes pre-mRNA exon ligation. Nat Struct Mol Biol 20, 728–734 (2013). https://doi.org/10.1038/nsmb.2556

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2556

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing