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.

  • Technical Report
  • Published:

A protein-based EM label for RNA identifies the location of exons in spliceosomes

Nature Structural & Molecular Biology volume 15, pages 213–215 (2008)Cite this article

Abstract

To locate key RNA features in the structure of the spliceosome by EM, we fused a sequence-specific RNA binding protein to a protein with a distinct donut-shaped structure. We used this fusion to label spliceosomes assembled on a pre-mRNA that contained the target sequence in the exons. The label is clearly visible in EM images of the spliceosome, and subsequent image processing with averaging shows that the exons sit close to each other in the complex. This labeling strategy will serve as a general tool for analyzing the structures of RNA-containing macromolecular complexes.

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: Beta-PP7 label design, RNA binding and imaging.
Figure 2: Difference maps of two-dimensional averages of labeled and unlabeled C complexes.

Similar content being viewed by others

References

  1. Boehringer, D. et al. Three-dimensional structure of a pre-catalytic human spliceosomal complex B. Nat. Struct. Mol. Biol. 11, 463–468 (2004).

    Article  CAS  Google Scholar 

  2. Jurica, M.S., Sousa, D., Moore, M.J. & Grigorieff, N. Three-dimensional structure of C complex spliceosomes by electron microscopy. Nat. Struct. Mol. Biol. 11, 265–269 (2004).

    Article  CAS  Google Scholar 

  3. Deckert, J. et al. Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions. Mol. Cell. Biol. 26, 5528–5543 (2006).

    Article  CAS  Google Scholar 

  4. Sander, B. et al. Organization of core spliceosomal components U5 snRNA loop I and U4/U6 Di-snRNP within U4/U6.U5 Tri-snRNP as revealed by electron cryomicroscopy. Mol. Cell 24, 267–278 (2006).

    Article  CAS  Google Scholar 

  5. Behzadnia, N. et al. Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes. EMBO J. 26, 1737–1748 (2007).

    Article  CAS  Google Scholar 

  6. Ohi, M.D., Ren, L., Wall, J.S., Gould, K.L. & Walz, T. Structural characterization of the fission yeast U5.U2/U6 spliceosome complex. Proc. Natl. Acad. Sci. USA 104, 3195–3200 (2007).

    Article  CAS  Google Scholar 

  7. Lim, F. & Peabody, D.S. RNA recognition site of PP7 coat protein. Nucleic Acids Res. 30, 4138–4144 (2002).

    Article  CAS  Google Scholar 

  8. Kong, X.P., Onrust, R., O'Donnell, M. & Kuriyan, J. Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell 69, 425–437 (1992).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Chiara, M.D. et al. Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly. Mol. Cell. Biol. 16, 3317–3326 (1996).

    Article  CAS  Google Scholar 

  11. Chiara, M.D., Palandjian, L., Feld Kramer, R. & Reed, R. Evidence that U5 snRNP recognizes the 3′ splice site for catalytic step II in mammals. EMBO J. 16, 4746–4759 (1997).

    Article  CAS  Google Scholar 

  12. Reichert, V.L., Le Hir, H., Jurica, M.S. & Moore, M.J. 5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly. Genes Dev. 16, 2778–2791 (2002).

    Article  CAS  Google Scholar 

  13. Ludtke, S.J., Baldwin, P.R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).

    Article  CAS  Google Scholar 

  14. 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 

  15. van Heel, M., Harauz, G., Orlova, E.V., Schmidt, R. & Schatz, M. A new generation of IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996).

    Article  CAS  Google Scholar 

  16. Tars, K., Fridborg, K., Bundule, M. & Liljas, L. The three-dimensional structure of bacteriophage PP7 from Pseudomonas aeruginosa at 3.7- Å resolution. Virology 272, 331–337 (2000).

    Article  CAS  Google Scholar 

  17. Frank, J. Three-Dimensional Electron Microscopy of Macromolecular Assemblies (Academic, New York, 1996).

Download references

Acknowledgements

J. Ilagan provided support for EM data collection and image processing. M. O' Donnell (Rockefeller University, New York, USA) provided a plasmid encoding full-length DnaN and K. Collins (University of California, Berkeley, USA) gave us pET28ZZTPP7H including the PP7 sequence. We thank N. Grigorieff (Brandeis University, Waltham, Massachusetts, USA) and members of the Jurica laboratory for advice and discussion. This work was funded by US National Institutes of Health grant 5R01GM72649 to M.S.J., which included a Diversity Supplement for E.A.A.

Author information

Authors and Affiliations

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

    Eric A Alcid & Melissa S Jurica

Authors
  1. Eric A Alcid
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Melissa S Jurica
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.A.A. and M.S.J. designed the experiments; E.A.A. performed the experiments; E.A.A. and M.S.J. analyzed the data. E.A.A. prepared figures and M.S.J. wrote the paper.

Corresponding author

Correspondence to Melissa S Jurica.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Alcid, E., Jurica, M. A protein-based EM label for RNA identifies the location of exons in spliceosomes. Nat Struct Mol Biol 15, 213–215 (2008). https://doi.org/10.1038/nsmb.1378

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1378

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