title: RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells.
abstract: OBJECTIVE
  The specialized corneal epithelium requires differentiated properties, 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 (EJC) that has dramatic implications for corneal epithelial cell differentiation and may act as a stabilizer of the corneal epithelial cell phenotype. Our studies revealed that PNN is involved in transcriptional repression complexes and spliceosomal complexes, placing PNN at the fulcrum between chromatin and mRNA splicing. Transcriptome analysis of PNN-knockdown cells revealed clear and reproducible alterations in transcript profiles and splicing patterns of a subset of genes that would significantly impact the epithelial cell phenotype. We further investigated PNN's role in the regulation of gene expression and alternative splicing (AS) in a corneal epithelial context.
  
  
  METHODS
  Human corneal epithelial (HCET) cells that carry the doxycycline-inducible PNN-knockdown shRNA vector were used to perform RNA-seq to determine differential gene expression and differential AS events.
  
  
  RESULTS
  Multiple genes and AS events were identified as differentially expressed between PNN-knockdown and control cells. Genes upregulated by PNN knockdown included a large proportion of genes that are associated with enhanced cell migration and ECM remodeling processes, such as MMPs, ADAMs, HAS2, LAMA3, CXCRs, and UNC5C. Genes downregulated in response to PNN depletion included IGFBP5, FGD3, FGFR2, PAX6, RARG, and SOX10. AS events in PNN-knockdown cells compared to control cells were also more likely to be detected, and upregulated. In particular, 60% of exon-skipping events, detected in only one condition, were detected in PNN-knockdown cells 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 of PNN levels in epithelial cells results in dramatic transformation in the number and composition of splicing variants and that PNN plays a crucial role in the selection of which RNA isoforms differentiating cells produce. Many of the genes affected by PNN knockdown are known to affect the 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 the epithelial cell phenotype.
authors.affiliation: Department of Anatomy and Cell Biology, University of Florida College of Medicine Gainesville, FL.
authors.forename: Debra
authors.initials: D
authors.lastname: Akin
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL.
authors.forename: Jeremy R B
authors.initials: JR
authors.lastname: Newman
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL.
authors.forename: Lauren M
authors.initials: LM
authors.lastname: McIntyre
authors.affiliation: Department of Anatomy and Cell Biology, University of Florida College of Medicine Gainesville, FL.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Sugrue
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D015536:Down-Regulation; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; D009687:Nuclear Proteins; C102646:PNN protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D020782:Matrix Metalloproteinases