September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells
Author Affiliations & Notes
  • Stephen P Sugrue
    Anatomy & Cell Biology, University of Florida, Gainesville, Florida, United States
  • Debra Akin
    Anatomy & Cell Biology, University of Florida, Gainesville, Florida, United States
  • Jeremy Newman
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida, United States
  • Lauren McIntyre
    Molecular Genetics & Microbiology, University of Florida, Gainesville, Florida, United States
  • Footnotes
    Commercial Relationships   Stephen Sugrue, None; Debra Akin, None; Jeremy Newman, None; Lauren McIntyre, None
  • Footnotes
    Support  NIH Grant R01 EY007883, P30 EY021721
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4369. doi:
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    • Get Citation

      Stephen P Sugrue, Debra Akin, Jeremy Newman, Lauren McIntyre; RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4369.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The specialized corneal epithelium requires differentiation specific for its role at the anterior surface of the eye, thus tight maintenance of the differentiated qualities of the corneal epithelial is essential. Pinin (PNN) is an exon junction component that has dramatic implications on corneal epithelial cell differentiation and may act as a stabilizer of the corneal epithelial cell phenotype. Our studies revealed that PNN is involved in both transcriptional repression complexes and spliceosomal complexes, placing PNN at the fulcrum between chromatin and mRNA splicing. Transcriptome analysis of PNN-knockdown cells revealed clear, reproducible alterations in transcript profiles and splicing patterns of a subset of genes that can significantly impact the epithelial cell phenotype. Here, we further investigate PNN’s role in the regulation of gene expression and alternative splicing (AS) in a corneal epithelial context.

Methods : RNA-seq was used to determine differential gene expression and AS events in authenticated human corneal epithelial cells that carry a doxycycline-inducible PNN-knockdown shRNA vector.

Results : Multiple genes and AS events were identified as differentially expressed between PNN-knockdown and control cells. Genes up-regulated by PNN-knockdown include a large proportion that are associated with enhanced cell migration and ECM remodeling such as MMPs, ADAMs, HAS2, LAMA3, CXCRs and UNC5C. Genes down-regulated in response to PNN depletion include IGFBP5, FGD3, FGFR2, PAX6, RARG and SOX10. AS events in PNN knockdowns compared to controls were also more likely to be detected and up-regulated. In particular, 60% of exon skipping events, detected in only one condition, were detected in PNN-knockdowns and of the shared exon skipping events, 92% of those differentially expressed were more frequent in the PNN-knockdown.

Conclusions : These data suggest that lowering PNN levels in epithelial cells results in dramatic transformation in the amount and composition of splicing variants and that PNN plays a crucial role in the selection of which RNA isoforms differentiating cells produce. Many genes impacted by PNN-knockdown are known to affect epithelial phenotype. This window into the complexity of RNA splicing in the corneal epithelium implies that PNN exerts broad influence over the regulation and maintenance of epithelial cell phenotype.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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