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Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components

Nature Structural & Molecular Biology volume 16, pages 1237–1243 (2009)Cite this article

Abstract

The spliceosome is a ribonucleoprotein machine that removes introns from pre-mRNA in a two-step reaction. To investigate the catalytic steps of splicing, we established an in vitro splicing complementation system. Spliceosomes stalled before step 1 of this process were purified to near-homogeneity from a temperature-sensitive mutant of the RNA helicase Prp2, compositionally defined, and shown to catalyze efficient step 1 when supplemented with recombinant Prp2, Spp2 and Cwc25, thereby demonstrating that Cwc25 has a previously unknown role in promoting step 1. Step 2 catalysis additionally required Prp16, Slu7, Prp18 and Prp22. Our data further suggest that Prp2 facilitates catalytic activation by remodeling the spliceosome, including destabilizing the SF3a and SF3b proteins, likely exposing the branch site before step 1. Remodeling by Prp2 was confirmed by negative stain EM and image processing. This system allows future mechanistic analyses of spliceosome activation and catalysis.

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Figure 1: Affinity purification of precatalytic Bact Δprp2 spliceosomes.
Figure 2: Reconstitution of both steps of S. cerevisiae pre-mRNA splicing with native proteins.
Figure 3: Reconstitution of both steps of S. cerevisiae pre-mRNA splicing with recombinant proteins.
Figure 4: Catalytic activation of the spliceosome by Prp2.
Figure 5: EM images of yeast spliceosomes before and after catalytic activation by Prp2.
Figure 6: Model for the catalytic activation of the spliceosome by Prp2 before Cwc25-promoted step 1.

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Acknowledgements

We thank M. Raabe, U. Plessmann and J. Lehne for their help in MS analysis; R.-J. Lin (Beckman Research Institute) for providing the yeast mutant prp2-1; and R. Rauhut and C.L. Will for helpful comments on the manuscript. This work was supported by grants from the European Commission (EURASNET-518238) and the Ernst Jung Stiftung to R.L., a fellowship provided by the Stiftung Stipendien–Fonds des Verbandes der Chemischen Industrie to Z.W., by the Stiftung der deutschen Wirtschaft to P.O., and a Young Investigator Programme grant from EURASNET to H.U.

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Authors and Affiliations

  1. Department of Cellular Biochemistry, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany

    Zbigniew Warkocki, Peter Odenwälder, Jana Schmitzová, Patrizia Fabrizio & Reinhard Lührmann

  2. 3D Electron Cryomicroscopy Group, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany

    Florian Platzmann & Holger Stark

  3. Göttingen Center of Molecular Biology, Institute for Microbiology and Genetics, Georg August University Göttingen, Göttingen, Germany

    Holger Stark

  4. Bioanalytical Mass Spectrometry Group, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany

    Henning Urlaub

  5. Department for Molecular Structural Biology, Institute for Microbiology and Genetics, Georg August University Göttingen, Göttingen, Germany

    Ralf Ficner

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  1. Zbigniew Warkocki
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Contributions

Z.W., P.O., J.S., F.P., H.S., R.F., P.F. and R.L. designed experiments; Z.W., P.O., J.S. and F.P. performed the experiments; Z.W., P.F. and R.L. analyzed the data; F.P. and H.S. analyzed the EM data; H.U. analyzed the MS data; Z.W., J.S., H.S., P.F. and R.L. wrote the paper.

Corresponding authors

Correspondence to Patrizia Fabrizio or Reinhard Lührmann.

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Warkocki, Z., Odenwälder, P., Schmitzová, J. et al. Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components. Nat Struct Mol Biol 16, 1237–1243 (2009). https://doi.org/10.1038/nsmb.1729

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