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Three-dimensional structure of C complex spliceosomes by electron microscopy

Nature Structural & Molecular Biology volume 11, pages 265–269 (2004)Cite this article

Abstract

The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs. Recruitment of the spliceosome to splice sites and subsequent splicing require a series of dynamic interactions among the spliceosome's component U snRNPs and many additional protein factors. These dynamics present several challenges for structural analyses, including purification of stable complexes to compositional homogeneity and assessment of conformational heterogeneity. We have isolated spliceosomes arrested before the second chemical step of splicing (C complex) in which U2, U5 and U6 snRNAs are stably associated. Using electron microscopy, we obtained images of C complex spliceosomes under cryogenic conditions and determined a three-dimensional structure of a core complex to a resolution of 30 Å. The structure reveals a particle of dimensions 27 × 22 × 24 nm with a relatively open arrangement of three primary domains.

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Figure 1: Purification and imaging of spliceosomes.
Figure 2: Predominant view of C complex spliceosomes.
Figure 3: Class averages from untilted images (top) and the structures derived from the corresponding tilted images (bottom).
Figure 4
Figure 5: Stereo views of the surface representation of the refined three-dimensional structure.

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References

  1. Nilsen, T.W. The spliceosome: no assembly required? Mol. Cell 9, 8–9 (2002).

    Article  CAS  Google Scholar 

  2. Jiang, J., Horowitz, D.S. & Xu, R.M. Crystal structure of the functional domain of the splicing factor Prp18. Proc. Natl. Acad. Sci. USA 97, 3022–3027 (2000).

    Article  CAS  Google Scholar 

  3. Kambach, C. et al. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell 96, 375–387 (1999).

    Article  CAS  Google Scholar 

  4. Kielkopf, C.L., Rodionova, N.A., Green, M.R. & Burley, S.K. A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer. Cell 106, 595–605 (2001).

    Article  CAS  Google Scholar 

  5. Oubridge, C., Ito, N., Evans, P.R., Teo, C.H. & Nagai, K. Crystal structure at 1.92 Å resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin. Nature 372, 432–438 (1994).

    Article  CAS  Google Scholar 

  6. Price, S.R., Evans, P.R. & Nagai, K. Crystal structure of the spliceosomal U2B″-U2A′ protein complex bound to a fragment of U2 small nuclear RNA. Nature 394, 645–650 (1998).

    Article  CAS  Google Scholar 

  7. Vidovic, I., Nottrott, S., Hartmuth, K., Luhrmann, R. & Ficner, R. Crystal structure of the spliceosomal 15.5kD protein bound to a U4 snRNA fragment. Mol. Cell 6, 1331–2342 (2000).

    Article  CAS  Google Scholar 

  8. Reuter, K., Nottrott, S., Fabrizio, P., Luhrmann, R. & Ficner, R. Identification, characterization and crystal structure analysis of the human spliceosomal U5 snRNP-specific 15 kD protein. J. Mol. Biol. 294, 515–525 (1999).

    Article  CAS  Google Scholar 

  9. Stark, H., Dube, P., Luhrmann, R. & Kastner, B. Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle. Nature 409, 539–542 (2001).

    Article  CAS  Google Scholar 

  10. Golas, M.M., Sander, B., Will, C.L., Luhrmann, R. & Stark, H. Molecular architecture of the multiprotein splicing factor SF3b. Science 300, 980–984 (2003).

    Article  CAS  Google Scholar 

  11. Jurica, M., Licklider, L., Gygi, S., Grigorieff, N. & Moore, M. Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. RNA 8, 426–439 (2002).

    Article  CAS  Google Scholar 

  12. Stevens, S.W. et al. Composition and functional characterization of the yeast spliceosomal penta-snRNP. Mol. Cell 9, 31–44. (2002).

    Article  CAS  Google Scholar 

  13. Van Heel, M. Angular reconstitution: a posteriori assignment of projection directions for 3D reconstruction. Ultramicroscopy 21, 111–123 (1987).

    Article  CAS  Google Scholar 

  14. Radermacher, M., Wagenknecht, T., Verschoor, A. & Frank, J. Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J. Microsc. 146 (Pt 2), 113–136 (1987).

    Article  CAS  Google Scholar 

  15. Frank, J. & Radermacher, M. Three-dimensional reconstruction of single particles negatively stained or in vitreous ice. Ultramicroscopy 46, 241–262 (1992).

    Article  CAS  Google Scholar 

  16. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).

    Article  CAS  Google Scholar 

  17. Grigorieff, N. Structure of the respiratory NADH:ubiquinone oxidoreductase (complex I). Curr. Opin. Struct. Biol. 9, 476–483 (1999).

    Article  CAS  Google Scholar 

  18. Ajuh, P. et al. Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry. EMBO J. 19, 6569–6581. (2000).

    Article  CAS  Google Scholar 

  19. Neubauer, G. et al. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat. Genet. 20, 46–50. (1998).

    Article  CAS  Google Scholar 

  20. Ohi, M.D. et al. Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs. Mol. Cell. Biol. 22, 2011–2024. (2002).

    Article  CAS  Google Scholar 

  21. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. Staley, J.P. & Guthrie, C. Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 92, 315–326 (1998).

    Article  CAS  Google Scholar 

  24. Kastner, B., Bach, M. & Luhrmann, R. Electron microscopy of small nuclear ribonucleoprotein (snRNP) particles U2 and U5: evidence for a common structure-determining principle in the major U snRNP family. Proc. Natl. Acad. Sci. USA 87, 1710–1714 (1990).

    Article  CAS  Google Scholar 

  25. Makarov, E.M. et al. Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome. Science 298, 2205–2208 (2002).

    Article  CAS  Google Scholar 

  26. Behrens, S.E., Tyc, K., Kastner, B., Reichelt, J. & Luhrmann, R. Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions. Mol. Cell. Biol. 13, 307–319 (1993).

    Article  CAS  Google Scholar 

  27. Mindell, J.A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  Google Scholar 

  28. Crowther, R.A., Henderson, R. & Smith, J.M. MRC image processing programs. J. Struct. Biol. 116, 9–16 (1996).

    Article  CAS  Google Scholar 

  29. Matthews, B.W. Solvent content of protein crystals. J. Mol. Biol. 33, 491–497 (1968).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank T. Walz and M. Ohi for discussion and H. Stark for advice on sample preparation. N.G. is an assistant investigator and M.J.M is an associate investigator with the Howard Hughes Medical Institute; M.S.J. is a Paul Sigler/Agouron Institute fellow of the Helen Hay Whitney Foundation; D.R.S. is supported by a US National Science Foundation integrative graduate education and research training grant. This work was supported by US National Institutes of Health grant 1 P01 GM-62580 (N.G.) and GM 53007 (M.J.M.) and funding from the Keck Foundation.

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  1. Melissa S Jurica and Duncan Sousa: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, Howard Hughes Medical Institute, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street, Waltham, 02454, Massachusetts, USA

    Melissa S Jurica, Duncan Sousa, Melissa J Moore & Nikolaus Grigorieff

  2. Department of Molecular, Center for the Molecular Biology of RNA, Cell and Developmental Biology, University of California Santa Cruz, 1156 High Street, Santa Cruz, 95064, California, USA

    Melissa S Jurica

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  1. Melissa S Jurica
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Correspondence to Melissa S Jurica.

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Jurica, M., Sousa, D., Moore, M. et al. Three-dimensional structure of C complex spliceosomes by electron microscopy. Nat Struct Mol Biol 11, 265–269 (2004). https://doi.org/10.1038/nsmb728

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