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Localization of Prp8, Brr2, Snu114 and U4/U6 proteins in the yeast tri-snRNP by electron microscopy

Nature Structural & Molecular Biology volume 15, pages 1206–1212 (2008)Cite this article

Abstract

The U4/U6-U5 tri–small nuclear ribonucleoprotein (snRNP) is a major, evolutionarily highly conserved spliceosome subunit. Unwinding of its U4/U6 snRNA duplex is a central event of spliceosome activation that requires several components of the U5 portion of the tri-snRNP, including the RNA helicase Brr2, Prp8 and the GTPase Snu114. Here we report the EM projection structure of the Saccharomyces cerevisiae tri-snRNP. It shows a modular organization comprising three extruding domains that contact one another in its central portion. We have visualized genetically tagged tri-snRNP proteins by EM and show here that U4/U6 snRNP forms a domain termed the arm. Conversely, a separate head domain adjacent to the arm harbors Brr2, whereas Prp8 and the GTPase Snu114 are located centrally. The head and arm adopt variable relative positions. This molecular organization and dynamics suggest possible scenarios for structural events during catalytic activation.

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Figure 1: Structure of the S. cerevisiae U4/U6-U5 tri-snRNP as visualized by EM.
Figure 2: Localization of U5 proteins by EM.
Figure 3: EM localization of U4/U6 proteins.
Figure 4: EM localization of the bridging proteins.
Figure 5: Location of functionally important yeast U4/U6-U5 tri-snRNP proteins.

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Acknowledgements

We thank H. Urlaub for MS analysis and M. Raabe for excellent technical assistance. We are grateful to C.L. Will for helpful comments on the manuscript and to R. Karaduman for tagging Prp8 and Snu114 with the TAP tag. This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie and the Ernst Jung Stiftung to R.L. Research in the laboratory of H.S. was supported by grants from the Federal Ministry of Education and Research, Germany (0311899) and the Sixth Framework Program of the European Union via the Integrated Project '3D Repertoire'. I.H. was supported by the Stiftung Stipendien-Fonds des Verbandes der Chemischen Industrie.

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Author notes

  1. Irina Häcker, Bjoern Sander and Monika M Golas: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany

    Irina Häcker, Elmar Wolf, Elif Karagöz, Berthold Kastner, Patrizia Fabrizio & Reinhard Lührmann

  2. Three-dimensional Electron Cryo-Microscopy Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077, Göttingen, Germany

    Bjoern Sander, Monika M Golas & Holger Stark

  3. Department of Chemistry, Utrecht University, Padualaan 8, NL-3584 CH, Utrecht, The Netherlands

    Elif Karagöz

  4. Three-dimensional Cryo-Electron Microscopy, Göttinger Zentrum für Molekulare Biologie, Universität Göttingen, Justus-von-Liebig-Weg 11, D-37077, Göttingen, Germany

    Holger Stark

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Contributions

I.H., B.S., M.M.G., P.F. and R.L. designed the experiments. P.F., H.S. and R.L. supervised the project. I.H. performed most of the biochemical and genetic research. B.S. and M.M.G. performed most of the EM research. E.W. performed the PA-gold labeling of the immunocomplexes and the EM of the PA-gold–labeled complexes. I.H. and E.W. analyzed the data of the PA-gold–labeled complexes. E.K. contributed to the biochemical and genetic experiments in the initial phase of the project. B.K. contributed to the EM research in the initial phase of the project. I.H., B.S., M.M.G., P.F., H.S. and R.L. analyzed the data and wrote the paper.

Corresponding author

Correspondence to Reinhard Lührmann.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Methods (PDF 4223 kb)

Supplementary Video 1

Here, the two views show a repositioning of the head, which kinks to the left, accompanied by a small additional bending of the foot to the left, while the arm remains in the closed position. (GIF 54 kb)

Supplementary Video 2

In addition to the kinking of the head that is also evident in Supplementary Video 1, the arm changes between the closed and open position. No changes are observed in the foot region, while a slight bending of the central portion accompanies the changes in head and arm. (GIF 49 kb)

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Häcker, I., Sander, B., Golas, M. et al. Localization of Prp8, Brr2, Snu114 and U4/U6 proteins in the yeast tri-snRNP by electron microscopy. Nat Struct Mol Biol 15, 1206–1212 (2008). https://doi.org/10.1038/nsmb.1506

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