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Jeong-Hoon Joo, William J. Degan, Nicholas W. Dunn, Yong H. Kim, Min Jiang, Stephen P. Sugrue; Role of Pinin in Alternative pre-mRNA Splicing during Corneal Epithelial Development. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4390.
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Pinin (Pnn), a nuclear speckle-associated protein, was previously shown to play an essential role in the differentiation and maintenance of corneal epithelium identity in mice. Epithelial-specific alternative splicing largely mediated by Epithelial splicing regulatory proteins 1 and 2 (Esrp1 and 2) especially on the pre-mRNA of Fibroblast growth factor receptor 2 (Fgfr2) has been shown to be crucial for the epithelial differentiation and maintenance. To further investigate Pnn’s function and its underlying mechanism, we focused on Pnn’s role in alternative pre-mRNA splicing during corneal epithelial development.
Conditional inactivation of Pnn in the developing corneal epithelium was achieved by utilizing Pax6 (lens)-Cre mice. Control and mutant corneas were harvested from E17.5 embryos by dissecting at the corneoscleral junction. RNA samples were then subjected to the alternative splicing-specific RT-PCR assays.
While conditional inactivation of Pnn in developing corneal epithelium did not affect alternative splicing of Pyruvate kinase isozyme type M2 (Pkm2) nor total Fgfr2 transcript level, Pnn depletion resulted in significant disruption of alternative splicing pattern of Fgfr2, leading to the aberrant ratio between IIIb (epithelial-type) and IIIc (mesenchymal-type) isoforms in mutant corneal epithelium. Our quantitative RT-PCR assay revealed three-fold increase in IIIc/IIIb ratio. Pnn-dependent alteration of Fgfr2 pre-mRNA splicing was also observed in Pnn hypomorphic mouse embryonic stem cells. Additionally, pre-mRNA splicing pattern of Cd44, p120-Catenin (CtnnD1), and enabled homolog (Enah), whose splicing was also shown to be dependent on Esrp proteins, was markedly altered in Pnn mutant cornea, suggesting the possibility of Pnn’s role in epithelial-specific alternative splicing.
Our study suggests that Pnn is involved in the regulation of epithelial-specific alternative splicing of Fgfr2, Cd44, CtnnD1, and Enah, and that Pnn may act through modulating alternative pre-mRNA splicing, possibly in coordination with Esrp proteins, during corneal epithelial development.
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