Kinases and phosphatases participate in precursor messenger RNA (pre-mRNA) splicing regulation, but their precise roles and the identities of their cofactors and substrates remain poorly understood. The human Ser/Thr phosphatase PP2Cγ promotes spliceosome assembly. We show that PP2Cγ's distinctive acidic domain is essential for its activity in splicing and interacts with YB-1, a spliceosome-associated factor. Moreover, PP2Cγ is a phosphoprotein in vivo, and its acidic domain is phosphorylated under splicing conditions in vitro. PP2Cγ phosphorylation enhances its interaction with YB-1 and is reversed by the phosphatase in cis. PP2Cγ knockdown leaves constitutive splicing unaffected but inhibits cell proliferation and affects alternative splicing of CD44, a YB-1 target. This effect on splicing regulation is mediated by PP2Cγ's acidic domain, which is essential to promote inclusion of CD44 exons v4 and v5 in vivo. We propose that PP2Cγ modulates alternative splicing of specific pre-mRNAs coregulated by YB-1.
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We thank S. Berget (Baylor College of Medicine) for the CD44 v4-v5 minigene, T. Cooper (Baylor College of Medicine) for YB-1 plasmids, J. Tazi (Institut de Génétique Moléculaire de Montpellier) for plasmid pET14b-hSC35, D. Barford (Chester Beatty Laboratories) for recombinant PP2Cα protein, G. Dreyfuss (University of Pennsylvania School of Medicine) for the hnRNP A1–specific 4B10 antibody, S. Dokudovskaya and C. Muchardt for useful comments on the manuscript, and E. Batsché for helpful advice on the qPCR experiments. This work was supported by US National Institutes of Health grant GM42699 to A.R.K.
The authors declare no competing financial interests.
Alignment of PP2Cγ AD from several species. (PDF 159 kb)
Interaction of PP2Cγ and YB-1 does not require RNA. (PDF 184 kb)
Phosphorylation of the AD of PP2Cγ. (PDF 343 kb)
Stability and dephosphorylation of PP2Cγ. (PDF 435 kb)
Phosphorylation state of inactive phosphatase affects YB-1 interaction. (PDF 191 kb)
Depletion of PP2Cγ affects cell growth. (PDF 164 kb)
Knockdown of PP2Cγ by siRNA treatment. (PDF 1287 kb)
PP2Cγ knockdown does not affect β-globin splicing. (PDF 453 kb)
PP2γ knockdown does not affect E1A splicing. (PDF 341 kb)
PP2Cγ knockdown attenuates the effect of YB-1 on CD44 alternative splicing. (PDF 1515 kb)
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Allemand, E., Hastings, M., Murray, M. et al. Alternative splicing regulation by interaction of phosphatase PP2Cγ with nucleic acid–binding protein YB-1. Nat Struct Mol Biol 14, 630–638 (2007) doi:10.1038/nsmb1257
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