title: Transcriptomic analysis of PNN- and ESRP1-regulated alternative pre-mRNA splicing in human corneal epithelial cells.
abstract: OBJECTIVE
  We investigated the impact of PININ (PNN) and epithelial splicing regulatory protein 1 (ESRP1) on alternative pre-mRNA splicing in the corneal epithelial context.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610, USA. jpjhjh@ufl.edu
authors.forename: Jeong-Hoon
authors.initials: JH
authors.lastname: Joo
authors.forename: Greg P
authors.initials: GP
authors.lastname: Correia
authors.forename: Jian-Liang
authors.initials: JL
authors.lastname: Li
authors.forename: Maria-Cecilia
authors.initials: MC
authors.lastname: Lopez
authors.forename: Henry V
authors.initials: HV
authors.lastname: Baker
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Sugrue
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D005091:Exons; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012332:RNA, Heterogeneous Nuclear / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D015815:Cell Adhesion Molecules; C539364:ESRP1 protein, human; D009687:Nuclear Proteins; C102646:PNN protein, human; D012322:RNA Precursors; D012332:RNA, Heterogeneous Nuclear; D016601:RNA-Binding Proteins