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Isolation of an active step I spliceosome and composition of its RNP core

Nature volume 452, pages 846–850 (2008)Cite this article

Abstract

Formation of catalytically active RNA structures within the spliceosome requires the assistance of proteins. However, little is known about the number and nature of proteins needed to establish and maintain the spliceosome’s active site. Here we affinity-purified human spliceosomal C complexes and show that they catalyse exon ligation in the absence of added factors. Comparisons of the composition of the precatalytic versus the catalytic spliceosome revealed a marked exchange of proteins during the transition from the B to the C complex, with apparent stabilization of Prp19–CDC5 complex proteins and destabilization of SF3a/b proteins. Disruption of purified C complexes led to the isolation of a salt-stable ribonucleoprotein (RNP) core that contained both splicing intermediates and U2, U5 and U6 small nuclear RNA plus predominantly U5 and human Prp19–CDC5 proteins and Prp19-related factors. Our data provide insights into the spliceosome’s catalytic RNP domain and indicate a central role for the aforementioned proteins in sustaining its catalytically active structure.

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Figure 1: Splicing of PM5 pre-mRNA lacking a 3′ splice site and 3′ exon.
Figure 2: Affinity-purified C complexes catalyse bimolecular exon ligation on their own.
Figure 3: Characterization of MS2 affinity-purified B and C complexes.
Figure 4: U5– and Prp19–CDC5-proteins remain associated with C-complex RNP cores.

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Acknowledgements

We thank T. Conrad, P. Kemkes, H. Kohansal and I. Öchsner for technical assistance; M. Raabe and U. Plessmann for their help in MS analysis; E. Makarov for raising anti-peptide antibodies; and C. Smith for providing the PYP pre-mRNA. This work was supported by grants from the Deutsche Forschungsgemeinschaft, EURASNET, the Fonds der Chemischen Industrie and the Ernst Jung Stiftung to R.L., and a Young Investigator Programme grant from EURASNET to H.U.

Author Contributions S.B., M.A., H.U. and R.L. designed experiments; S.B., M.A. and H.U. performed the experiments; S.B., M.A., C. L.W., H.U. and R.L. analysed the data; and S.B., C. L.W., H.U. and R.L. wrote the paper.

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Authors and Affiliations

  1. Department of Cellular Biochemistry, and,

    Sergey Bessonov, Maria Anokhina, Cindy L. Will & Reinhard Lührmann

  2. Bioanalytical Mass Spectrometry Group, Max Planck Institute of Biophysical Chemistry, D-37077 Göttingen, Germany,

    Henning Urlaub

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  1. Sergey Bessonov
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  2. Maria Anokhina
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Correspondence to Reinhard Lührmann.

Supplementary information

Supplementary Information

The file contains Supplementary Tables S1 and S2. Table S1 summarizes those proteins reproducibly identified by mass spectrometry in affinity-purified human spliceosomal B and C complexes, plus those in salt-resistant C complex RNP cores. Table S2 summarizes proteins not reproducibly detected by mass spectrometry in B and C complexes, or in salt-resistant C complex RNP cores. (PDF 415 kb)

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Bessonov, S., Anokhina, M., Will, C. et al. Isolation of an active step I spliceosome and composition of its RNP core. Nature 452, 846–850 (2008). https://doi.org/10.1038/nature06842

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