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Phosphorylation of human PRP28 by SRPK2 is required for integration of the U4/U6-U5 tri-snRNP into the spliceosome

Nature Structural & Molecular Biology volume 15, pages 435–443 (2008)Cite this article

Abstract

Several protein kinases, including SRPK1 and SRPK2, have been implicated in spliceosome assembly and catalytic activation. However, little is known about their targets. Here we show that SRPK1 is predominantly associated with U1 small nuclear ribonucleoprotein (snRNP), whereas SRPK2 associates with the U4/U6-U5 tri-snRNP. RNAi-mediated depletion in HeLa cells showed that SRPK2 is essential for cell viability, and it is required for spliceosomal B complex formation. SRPK2 knock down results in hypophosphorylation of the arginine-serine (RS) domain–containing human PRP28 protein (PRP28, also known as DDX23), and destabilizes PRP28 association with the tri-snRNP. Immunodepletion of PRP28 from HeLa cell nuclear extract and complementation studies revealed that PRP28 phosphorylation is required for its stable association with the tri-snRNP and for tri-snRNP integration into the B complex. Our results demonstrate a role for SRPK2 in splicing and reveal a previously unknown function for PRP28 in spliceosome assembly.

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Figure 1: SRPK1 and SRPK2 are associated with the U1 snRNP and the tri-snRNP, respectively.
Figure 2: RNAi-mediated depletion of SRPK2, but not of SRPK1, blocks splicing.
Figure 3: PRP28 dissociates from the tri-snRNP upon SRPK2 knockdown.
Figure 4: Differential loss of PRP28 isoelectric variants upon SRPK1 or SRPK2 knockdown.
Figure 5: PRP28 dissociates from the tri-snRNP upon phosphatase (PPase) treatment.
Figure 6: Immunodepletion of PRP28 blocks spliceosome assembly.
Figure 7: Phosphorylation of PRP28 is required for B complex formation.
Figure 8: Restoration of splicing activity to SRPK2 knockdown extracts.

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Acknowledgements

We thank I. Lemm and M. Hoßbach for help with RNAi experiments, X.-D. Fu (University of California, San Diego, USA) for the plasmid encoding SRPK2, H. Manniga for synthesis of siRNAs, and S. Trowitzsch and G. Weber for the gift of purified tri-snRNP. We thank C.L. Will for critically reading the manuscript. We acknowledge the expert technical assistance of H. Kohansal, P. Kempkes and T. Conrad with HeLa cell extract and snRNP preparation, and M. Raabe with MS. This work was funded by grants from the DFG, the Fonds der Chemischen Industrie, the Volkswagen Stiftung and the EURASNET (within the 6th EU framework; LSHG-CT-2005-518238).

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Authors and Affiliations

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

    Rebecca Mathew, Klaus Hartmuth & Reinhard Lührmann

  2. Department of Molecular Structural Biology, Institute for Microbiology and Genetics, University of Göttingen, Justus-von-Liebig Weg 11,, Göttingen, D-37077, Germany

    Sina Möhlmann & Ralf Ficner

  3. Bioanalytical Mass Spectrometry Group, MPI for Biophysical Chemistry, Am Fassberg 11, Göttingen, D-37077, Germany

    Henning Urlaub

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Contributions

R.M., K.H. and R.L. designed the research; R.M. performed the research; S.M. and R.F. provided recombinant PRP28; H.U. performed MS; R.M., K.H. and R.L. analyzed data and wrote the paper.

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Correspondence to Reinhard Lührmann.

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Mathew, R., Hartmuth, K., Möhlmann, S. et al. Phosphorylation of human PRP28 by SRPK2 is required for integration of the U4/U6-U5 tri-snRNP into the spliceosome. Nat Struct Mol Biol 15, 435–443 (2008). https://doi.org/10.1038/nsmb.1415

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