title: Role of Pnn in alternative splicing of a specific subset of lncRNAs of the corneal epithelium.
abstract: OBJECTIVE
  GG-H whole transcriptome array analysis suggested involvement of PININ (PNN) in the alternative splicing of multiple long non-coding RNAs (lncRNAs). To further investigate PNN's role in regulating the alternative splicing of lncRNAs in a corneal epithelial context, we performed detailed analyses for detecting and identifying alternatively spliced lncRNAs.
  
  
  METHODS
  Total RNA was isolated from PNN knockdown human corneal epithelial (HCET) cells or Pnn-deficient mouse corneas, and subjected to real-time-PCR (RT-PCR) assays, and the alternatively spliced lncRNAs were counted. Alternatively spliced lncRNAs were detected with in situ hybridization with variant-specific RNA probes on human cornea sections.
  
  
  RESULTS
  Our analysis uncovered PNN's impact on the transcript levels of several lncRNAs including Linc00085 and HAS2-AS1. Interestingly, a mouse ortholog of HAS2-AS1, Has2as, clearly exhibited a differential splicing pattern among three major splice variants in the Pnn-deficient mouse cornea. The sequence analyses and quantification of splice variants of candidate lncRNAs, including RP11-295B20.2, RP11-18I14.1, and RP11-322M19.1, demonstrated complex configuration of their splicing changes, with a significant impact of PNN on the process. Knockdown of PNN in HCET cells led to specific changes in the inclusion of multiple cassette exons as well as in the use of alternative splice sites in RP11-322M19.1 and RP11-18I14.1, resulting in considerable net changes in the ratio between the splice variants. Finally, in situ hybridization analyses revealed the presence of RP11-295G20.2 in the nuclei of corneal epithelial cells, but not in the stromal cells of the human cornea, while RP11-322M19.1 was present in epithelial and non-epithelial cells.
  
  
  CONCLUSIONS
  The data suggest PNN's role in the alternative splicing of a specific subset of lncRNAs might have a significant impact on the corneal epithelium.
authors.affiliation: Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL.
authors.forename: Jeong Hoon
authors.initials: JH
authors.lastname: Joo
authors.affiliation: Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL.
authors.forename: Danny
authors.initials: D
authors.lastname: Ryu
authors.affiliation: Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL.
authors.forename: Qian
authors.initials: Q
authors.lastname: Peng
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; D000818:Animals; D015815:Cell Adhesion Molecules / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics; D004268:DNA-Binding Proteins; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D005091:Exons; D055785:Gene Knockdown Techniques; D006801:Humans; D007438:Introns; D051379:Mice; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D022821:RNA Splice Sites; D062085:RNA, Long Noncoding / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome
chemical_list: D015815:Cell Adhesion Molecules; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C102646:PNN protein, human; C491175:Pnn protein, mouse; D022821:RNA Splice Sites; D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D034741:RNA, Small Interfering