Article | Published:

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

Nature Structural & Molecular Biology volume 17, pages 216221 (2010) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Spliceosome structure and function. in The RNA World 3rd edn (eds. Gesteland, R.F., Cech, T.R. & Atkins, J.F.) 369–400 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2006).

  2. 2.

    , , , & Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6*U5 tri-snRNP formation and pre-mRNA splicing. EMBO J. 21, 1148–1157 (2002).

  3. 3.

    , , , & RNAi knockdown of hPRP31 leads to an accumulation of U4/U6 di-snRNPs in Cajal bodies. EMBO J. 23, 3000–3009 (2004).

  4. 4.

    et al. A human homolog of yeast pre-mRNA splicing gene, PRP31, underlies autosomal dominant retinitis pigmentosa on chromosome 19q13.4 (RP11). Mol. Cell 8, 375–381 (2001).

  5. 5.

    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).

  6. 6.

    , & The 65 and 110 kDa SR-related proteins of the U4/U6.U5 tri-snRNP are essential for the assembly of mature spliceosomes. EMBO J. 20, 2553–2563 (2001).

  7. 7.

    et al. SMNrp is an essential pre-mRNA splicing factor required for the formation of the mature spliceosome. EMBO J. 20, 2304–2314 (2001).

  8. 8.

    , , & SPF30 is an essential human splicing factor required for assembly of the U4/U5/U6 tri-small nuclear ribonucleoprotein into the spliceosome. J. Biol. Chem. 276, 31142–31150 (2001).

  9. 9.

    & SR proteins escort the U4/U6.U5 tri-snRNP to the spliceosome. RNA 1, 692–706 (1995).

  10. 10.

    , & Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing. Nucleic Acids Res. 20, 5263–5269 (1992).

  11. 11.

    et al. Thiophosphorylation of U1–70K protein inhibits pre-mRNA splicing. Nature 363, 283–286 (1993).

  12. 12.

    , & Both phosphorylation and dephosphorylation of ASF/SF2 are required for pre-mRNA splicing in vitro. RNA 3, 1456–1467 (1997).

  13. 13.

    & Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing. Genes Dev. 11, 334–344 (1997).

  14. 14.

    & Flipping the switch to an active spliceosome. Cell 96, 599–602 (1999).

  15. 15.

    , & PP1/PP2A phosphatases are required for the second step of Pre-mRNA splicing and target specific snRNP proteins. Mol. Cell 23, 819–829 (2006).

  16. 16.

    & SR proteins and splicing control. Genes Dev. 10, 1569–1579 (1996).

  17. 17.

    Sorting out the complexity of SR protein functions. RNA 6, 1197–1211 (2000).

  18. 18.

    , , , & Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants. Nucleic Acids Res. 18, 4427–4438 (1990).

  19. 19.

    et al. Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis. Genes Dev. 12, 1409–1414 (1998).

  20. 20.

    , , & The [U4/U6.U5] tri-snRNP-specific 27K protein is a novel SR protein that can be phosphorylated by the snRNP-associated protein kinase. RNA 3, 344–355 (1997).

  21. 21.

    , & Multiple roles of the SR protein family in splicing regulation. Prog. Mol. Subcell. Biol. 31, 33–58 (2003).

  22. 22.

    et al. The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. EMBO J. 15, 265–275 (1996).

  23. 23.

    et al. Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I. Nature 381, 80–82 (1996).

  24. 24.

    , , & Purification and characterization of a kinase specific for the serine- and arginine-rich pre-mRNA splicing factors. Proc. Natl. Acad. Sci. USA 91, 10824–10828 (1994).

  25. 25.

    , , & Novel SR-protein-specific kinase, SRPK2, disassembles nuclear speckles. Biochem. Biophys. Res. Commun. 242, 357–364 (1998).

  26. 26.

    , & Genetic analysis of the SR protein ASF/SF2: interchangeability of RS domains and negative control of splicing. Genes Dev. 12, 2222–2233 (1998).

  27. 27.

    , & prp4 from Schizosaccharomyces pombe, a mutant deficient in pre-mRNA splicing isolated using genes containing artificial introns. Mol. Gen. Genet. 226, 305–309 (1991).

  28. 28.

    & Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9, 576–596 (1995).

  29. 29.

    et al. Mammalian PRP4 kinase copurifies and interacts with components of both the U5 snRNP and the N-CoR deacetylase complexes. Mol. Cell. Biol. 22, 5141–5156 (2002).

  30. 30.

    et al. Functional analysis of the fission yeast Prp4 protein kinase involved in pre-mRNA splicing and isolation of a putative mammalian homologue. Nucleic Acids Res. 25, 1028–1035 (1997).

  31. 31.

    , , , & Cloning of human PRP4 reveals interaction with Clk1. J. Biol. Chem. 276, 32247–32256 (2001).

  32. 32.

    et al. Fission yeast Prp4p kinase regulates pre-mRNA splicing by phosphorylating a non-SR-splicing factor. EMBO Rep. 2, 35–41 (2001).

  33. 33.

    , , , & Multiple genetic and biochemical interactions of Brr2, Prp8, Prp31, Prp1 and Prp4 kinase suggest a function in the control of the activation of spliceosomes in Schizosaccharomyces pombe. Curr. Genet. 48, 151–161 (2005).

  34. 34.

    , , & PRP4 is a spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2 localization to the kinetochores. J. Cell Biol. 179, 601–609 (2007).

  35. 35.

    & The biochemical defects of prp4-1 and prp6-1 yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP. Nucleic Acids Res. 21, 1555–1562 (1993).

  36. 36.

    , , & The human homologue of the yeast splicing factor prp6p contains multiple TPR elements and is stably associated with the U5 snRNP via protein-protein interactions. J. Mol. Biol. 298, 567–575 (2000).

  37. 37.

    , & Prp31p promotes the association of the U4/U6 x U5 tri-snRNP with prespliceosomes to form spliceosomes in Saccharomyces cerevisiae. Mol. Cell. Biol. 17, 3580–3588 (1997).

  38. 38.

    , , & The network of protein-protein interactions within the human U4/U6.U5 tri-snRNP. RNA 12, 1418–1430 (2006).

  39. 39.

    et al. Binding of the human Prp31 Nop domain to a composite RNA-protein platform in U4 snRNP. Science 316, 115–120 (2007).

  40. 40.

    et al. Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP. EMBO J. 18, 4535–4548 (1999).

  41. 41.

    , , , & Isolation of an active step I spliceosome and composition of its RNP core. Nature 452, 846–850 (2008).

  42. 42.

    & An RNA switch at the 5′ splice site requires ATP and the DEAD box protein Prp28p. Mol. Cell 3, 55–64 (1999).

  43. 43.

    , & Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).

  44. 44.

    , & Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles. Mol. Cell. Biol. 8, 814–821 (1988).

  45. 45.

    et al. Organization of core spliceosomal components U5 snRNA loop I and U4/U6 Di-snRNP within U4/U6.U5 Tri-snRNP as revealed by electron cryomicroscopy. Mol. Cell 24, 267–278 (2006).

  46. 46.

    et al. Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions. Mol. Cell. Biol. 26, 5528–5543 (2006).

  47. 47.

    , , & RNA structural requirements for the association of the spliceosomal hPRP31 protein with the U4 and U4atac small nuclear ribonucleoproteins. J. Biol. Chem. 281, 28278–28286 (2006).

  48. 48.

    , , & Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).

  49. 49.

    , , , & Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 4, 873–886 (2005).

Download references

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.

Author information

Affiliations

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

    • Marc Schneider
    • , Cindy L Will
    •  & Reinhard Lührmann
  2. Bioanalytical Mass Spectrometry Group, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany.

    • He-Hsuan Hsiao
    •  & Henning Urlaub
  3. Institut de Génétique et Développement de l'Université de Rennes I, Rennes, France.

    • Régis Giet

Authors

  1. Search for Marc Schneider in:

  2. Search for He-Hsuan Hsiao in:

  3. Search for Cindy L Will in:

  4. Search for Régis Giet in:

  5. Search for Henning Urlaub in:

  6. Search for Reinhard Lührmann in:

Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Reinhard Lührmann.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.1718

Further reading