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Human PRP4 kinase is required for stable tri-snRNP association during spliceosomal B complex formation

Abstract

Reversible protein phosphorylation has an essential role during pre-mRNA splicing. Here we identify two previously unidentified phosphoproteins in the human spliceosomal B complex, namely the pre-mRNA processing factors PRP6 and PRP31, both components of the U4/U6−U5 tri-small nuclear ribonucleoprotein (snRNP). We provide evidence that PRP6 and PRP31 are directly phosphorylated by human PRP4 kinase (PRP4K) concomitant with their incorporation into B complexes. Immunodepletion and complementation studies with HeLa splicing extracts revealed that active human PRP4K is required for the phosphorylation of PRP6 and PRP31 and for the assembly of stable, functional B complexes. Thus, the phosphorylation of PRP6 and PRP31 is likely to have a key role during spliceosome assembly. Our data provide new insights into the molecular mechanism by which PRP4K contributes to splicing. They further indicate that numerous phosphorylation events contribute to spliceosome assembly and, thus, that splicing can potentially be modulated at multiple regulatory checkpoints.

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Figure 1: Human PRP6 and PRP31 are phosphorylated in spliceosomal B complexes.
Figure 2: Phosphorylation of human PRP31 is dependent on formation of the spliceosomal B complex and precedes SF3B155 phosphorylation.
Figure 3: Phosphorylation of human PRP6 and PRP31 is dependent on PRP4 kinase.
Figure 4: Human PRP4 kinase is required for spliceosomal B complex formation.
Figure 5: Tri-snRNP assembly and stability are not affected by PRP4K depletion.

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Acknowledgements

We are grateful to T. Conrad and H. Kohansal for help in preparing HeLa cell nuclear extract. We would also like to thank C. Girard for critical discussions, K. Hartmuth (Max Planck Insitute (MPI) for Biophysical Chemistry) for providing phosphospecific SF3b155 antibody and S. Trowitzsch and G. Weber (MPI for Biophysical Chemistry) for providing purified human U4/U6−U5 tri-snRNPs. This work was supported by grants from the Deutschen Forschungsgemeinschaft, the European Commission (EURASNET-518238), Fonds der Chemischen Industrie and the Ernst Jung Stiftung to R.L. and a Young Investigator Programme grant from EURASNET to H.U.

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M.S., H.-H.H., H.U. and R.L. designed the research; M.S. and H.-H.H. performed the research; R.G. provided anti-PRP4K antibodies; M.S., H.-H.H., C.L.W., H.U. and R.L. analyzed the data; M.S., C.L.W. and R.L. wrote the paper.

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Correspondence to Reinhard Lührmann.

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Schneider, M., Hsiao, HH., Will, C. et al. Human PRP4 kinase is required for stable tri-snRNP association during spliceosomal B complex formation. Nat Struct Mol Biol 17, 216–221 (2010). https://doi.org/10.1038/nsmb.1718

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