Purchase this article with an account.
Jeong-Hoon Joo, Stephen Sugrue; Role of PININ in the Regulation of Alternative Splicing of Long Non-coding RNAs. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2581.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
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 the regulation of the alternative splicing of lncRNAs in a corneal epithelial context, we performed detailed analyses for the detection and identification of alternatively spliced lncRNAs.
Total RNA was isolated from PNN knockdown human corneal epithelial (HCET) cells or Pnn-deficient mouse cornea, and subjected to RT-PCR assays. Quantification of alternatively spliced lncRNAs was performed by ChemiDocTM XRS+ and Image Lab™ Software Version 4.0. Detection of alternatively spliced lncRNAs was achieved through in situ hybridization with variant-specific RNA probes on human cornea sections.
The sequence analyses and quantification of splice variants of candidate lncRNAs, such as RP11-322M19.1 and LOC100505761, clearly demonstrated complex configuration of their splicing changes and significant impact of PNN on the process. Knockdown of PNN in HCET cells led to the specific alterations in the inclusion of multiple cassette exons as well as in the usage of alternative splice sites in RP11-322M19.1 and LOC100505761, resulting in the considerable net changes in the ratio between splice variants. Our analysis also uncovered PNN’s impact on the transcript levels of several lncRNAs including Linc00085 and HAS2-AS1 (Hyaluronic acid synthase 2-antisense RNA 1). Interestingly, a mouse ortholog of HAS2-AS1, Has2as, clearly exhibited a differential splicing pattern among three major splice variants in Pnn-deficient mouse cornea. Since hyaluronic acid has been closely implicated in the formation of persistent lens stalk, which has been consistently observed in Pnn-deficient mouse embryos, it will be of great relevance to investigate the mechanism underlying Pnn’s impact on the alternative splicing of Has2as. Finally, in situ hybridization analyses revealed the presence of two splice variants of RP11-295G20.2, one of PNN-regulated lncRNAs, in the nuclei of corneal epithelial cells, but not in the stromal cells of human cornea, suggesting their potential roles in corneal epithelial cells.
The data strongly suggest PNN’s role in the alternative splicing of a specific subset of lncRNAs that might have significant impact on the corneal epithelium. (NIH Grant R01 EY007883, P30 EY021721)
This PDF is available to Subscribers Only