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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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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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.
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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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DOI: https://doi.org/10.1038/nsmb.1729
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