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Signal-dependent regulation of splicing via phosphorylation of Sam68

Abstract

Evolution of human organismal complexity from a relatively small number of genes1,2—only approximately twice that of worm or fly—is explained mainly by mechanisms generating multiple proteins from a single gene, the most prevalent of which is alternative pre-messenger-RNA splicing1,3,4. Appropriate spatial and temporal generation of splice variants demands that alternative splicing be subject to extensive regulation, similar to transcriptional control. Activation by extracellular cues of several cellular signalling pathways can indeed regulate alternative splicing5,6,7,8. Here we address the link between signal transduction and splice regulation. We show that the nuclear RNA-binding protein Sam68 is a new extracellular signal-regulated kinase (ERK) target. It binds exonic splice-regulatory elements of an alternatively spliced exon that is physiologically regulated by the Ras signalling pathway, namely exon v5 of CD44. Forced expression of Sam68 enhanced ERK-mediated inclusion of the v5-exon sequence in mRNA. This enhancement was impaired by mutation of ERK-phosphorylation sites in Sam68, whereas ERK phosphorylation of Sam68 stimulated splicing of the v5 exon in vitro. Finally, Ras-pathway-induced alternative splicing of the endogenous CD44-v5 exon was abolished by suppression of Sam68 expression. Our data define Sam68 as a prototype regulator of alternative splicing whose function depends on protein modification in response to extracellular cues.

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References

  1. 1

    International human genome sequencing consortium Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001)

  2. 2

    Venter, J. C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001)

  3. 3

    Sharp, P. A. Split genes and RNA splicing. Cell 77, 805–815 (1994)

  4. 4

    Maniatis, T. & Tasic, B. Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature 418, 236–243 (2002)

  5. 5

    König, H., Ponta, H. & Herrlich, P. Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator. EMBO J. 17, 2904–2913 (1998)

  6. 6

    van der Houven van Oordt, W. et al. The MKK3/6-p38–signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation. J. Cell Biol. 149, 307–316 (2000)

  7. 7

    Xie, J. & Black, D. L. A CaMK IV responsive RNA element mediates depolarization-induced alternative splicing of ion channels. Nature 410, 936–939 (2001)

  8. 8

    Weg-Remers, S., Ponta, H., Herrlich, P. & König, H. Regulation of alternative pre-mRNA splicing by the ERK MAP-kinase pathway. EMBO J. 20, 4194–4203 (2001)

  9. 9

    Arch, R. et al. Participation in normal immune response of a splice variant of CD44 that encodes a metastasis-inducing domain. Science 257, 682–685 (1992)

  10. 10

    Cooper, D. L. & Dougherty, G. J. To metastasize or not? Selection of CD44 splice sites. Nature Med. 1, 635–637 (1995)

  11. 11

    Sherman, L., Wainright, D., Ponta, H. & Herrlich, P. A splice variant of CD44 expressed in the apical ectodermal ridge presents fibroblast growth factors to limb mesenchyme and is required for limb outgrowth. Genes Dev. 12, 1058–1071 (1998)

  12. 12

    Sherman, L. et al. The CD44 proteins in embryonic development and in cancer. Curr. Top. Microbiol. Immunol. 213, 249–269 (1996)

  13. 13

    Chang, L. & Karin, M. Mammalian MAP kinase signalling cascades. Nature 410, 37–40 (2001)

  14. 14

    Stoss, O. et al. The STAR/GSG family protein rSLM-2 regulates the selection of alternative splice sites. J. Biol. Chem. 276, 8665–8673 (2001)

  15. 15

    Vernet, C. & Artzt, K. STAR, a gene family involved in signal transduction and activation of RNA. Trends Genet. 13, 479–484 (1997)

  16. 16

    Taylor, S. J. & Shalloway, D. An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis. Nature 368, 867–871 (1994)

  17. 17

    Fumagalli, S., Totty, N. F., Hsuan, J. J. & Courtneidge, S. A. A target for Src in mitosis. Nature 368, 871–874 (1994)

  18. 18

    Lin, Q., Taylor, S. J. & Shalloway, D. Specificity and determinants of Sam68 RNA binding. J. Biol. Chem. 272, 27274–27280 (1997)

  19. 19

    Reddy, T. R., Tang, H., Xu, W. & Wong-Staal, F. Sam68, RNA helicase A and Tap cooperate in the post-transcriptional regulation of human immunodeficiency virus and type D retroviral mRNA. Oncogene 19, 3570–3575 (2000)

  20. 20

    Downward, J., Graves, J. D., Warne, P. H., Rayter, S. & Cantrell, D. A. Stimulation of p21ras upon T-cell activation. Nature 346, 719–723 (1990)

  21. 21

    Favata, M. F. et al. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273, 18623–18632 (1998)

  22. 22

    Mermoud, J. E., Cohen, P. & Lamond, A. I. Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing. Nucleic Acids Res. 20, 5263–5269 (1992)

  23. 23

    Summerton, J. Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim. Biophys. Acta 1489, 141–158 (1999)

  24. 24

    Nasevicius, A. & Ekker, S. C. Effective targeted gene ‘knockdown’ in zebrafish. Nature Genet. 26, 216–220 (2000)

  25. 25

    Matter, N. et al. Heterogeneous ribonucleoprotein A1 is part of an exon-specific splice-silencing complex controlled by oncogenic signaling pathways. J. Biol. Chem. 275, 35353–35360 (2000)

  26. 26

    Dignam, J. D., Lebovitz, R. M. & Roeder, R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983)

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Acknowledgements

We thank S. Stamm and O. Stoss for discussions and the gift of antibodies against SLM-2 and Sam68; S. Richard for the murine myc-Sam68 expression construct; S. Weg-Remers for luciferase plasmids; J. Sleeman and G. Dreyfuss for antibodies (2D5 and 4B10, respectively); U. Rahmsdorf and H. Olinger for technical assistance; and J. Valcárcel and I. Mattaj for advice on in vitro splicing and mRNA transport, respectively. This work was supported by the Deutsche Forschungsgemeinschaft.

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Correspondence to Harald König.

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Further reading

Figure 1: Sam68 binds splice-regulatory sequences in CD44 exon v5.
Figure 2: Sam68 regulates the inclusion of CD44 variant exon v5.
Figure 3: Activation and phosphorylation of Sam68 by ERK.
Figure 4: Phosphorylation of Sam68 at ERK-target sites stimulates v5-exon usage in vivo and in vitro.
Figure 5: Sam68 is required for phorbol-ester regulation of v5-exon usage in endogenous CD44.

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