Transcriptomic Analysis of PNN- and ESRP1-Regulated Alternative Pre-mRNA Splicing in Human Corneal Epithelial Cells
Copyright policy )Transcriptomic Analysis of PNN- and ESRP1-Regulated Alternative Pre-mRNA Splicing in Human Corneal Epithelial Cells
We investigated the impact of PININ (PNN) and epithelial splicing regulatory protein 1 (ESRP1) on alternative pre-mRNA splicing in the corneal epithelial context.Isoform-specific RT-PCR assays were performed on wild-type and Pnn knockout mouse cornea. Protein interactions were examined by deconvolution microscopy and co-immunoprecipitation. For genome-wide alternative splicing study, immortalized human corneal epithelial cells (HCET) harboring doxycycline-inducible shRNA against PNN or ESRP1 were created. Total RNA was isolated from four biological replicates of control and knockdown HCET cells, and subjected to hGlue3_0 transcriptome array analysis.Pnn depletion in developing mouse corneal epithelium led to disrupted alternative splicing of multiple ESRP-regulated epithelial-type exons. In HCET cells, ESRP1 and PNN displayed close localization in and around nuclear speckles, and their physical association in protein complexes was identified. Whole transcriptome array analysis on ESRP1 or PNN knockdown HCET cells revealed clear alterations in transcript profiles and splicing patterns of specific subsets of genes. Separate RT-PCR validation assays confirmed successfully specific changes in exon usage of several representative splice variants, including PAX6(5a), FOXJ3, ARHGEF11, and SLC37A2. Gene ontologic analyses on ESRP1- or PNN-regulated alternative exons suggested their roles in epithelial phenotypes, such as cell morphology and movement.Our data suggested that ESRP1 and PNN modulate alternative splicing of a specific subset of target genes, but not general splicing events, in HCET cells to maintain or enhance epithelial characteristics.
- University of Florida United States
- Sanford Burnham Prebys Medical Discovery Institute United States
Mice, Knockout, Reverse Transcriptase Polymerase Chain Reaction, Gene Expression Profiling, Immunoblotting, Epithelium, Corneal, Nuclear Proteins, RNA-Binding Proteins, Exons, Alternative Splicing, Mice, Gene Expression Regulation, Microscopy, Fluorescence, Mutation, RNA Precursors, Animals, Humans, RNA, Heterogeneous Nuclear, Cell Adhesion Molecules, Cells, Cultured
Mice, Knockout, Reverse Transcriptase Polymerase Chain Reaction, Gene Expression Profiling, Immunoblotting, Epithelium, Corneal, Nuclear Proteins, RNA-Binding Proteins, Exons, Alternative Splicing, Mice, Gene Expression Regulation, Microscopy, Fluorescence, Mutation, RNA Precursors, Animals, Humans, RNA, Heterogeneous Nuclear, Cell Adhesion Molecules, Cells, Cultured
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