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Stephen Sugrue, Jeong-Hoon Joo; Identification and Validation of PNN-regulated Splicing Events in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3218. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Conditional inactivation of Pnn in developing mouse corneal epithelium resulted in severe disruption in epithelial differentiation. Since PNN is found associated with a large number of splicing proteins and exerts influence on splice site selection in minigenes, we identified PNN-regulated splicing events in a corneal epithelial context and explored the relevance of PNN’s impact on alternative splicing of its target genes.
Isoform-specific RT-PCR assays were performed on control and PNN knockdown human corneal epithelial (HCET) cells. The level of alternatively spliced transcripts was quantified by ChemiDocTM XRS+ and Image Lab™ Software Version 4.0.
PNN knockdown in HCET cells resulted in the increased inclusion of entire intron 11 of FOXJ3, which leads to premature translational termination and most likely nonsense-mediated decay (NMD) of alternatively spliced FOXJ3 transcripts. On the other hand, retained intron 9 of a transcription factor FAM50A upon PNN knockdown is not expected to cause a frame shift nor early termination, thus predicted to add 49 amino acids to FAM50A, highlighting the complexity of splicing-dependent mRNA quality control mechanism and the importance of precise regulation of splicing events. While an alternative cassette exon (24a) of a guanine nucleotide exchange factor, ECT2, is found to be a PNN-silenced exon, inclusion of intron 9 in GLT8D1 is determined to be enhanced by PNN. Most interestingly, our study clearly indicated PNN’s involvement in the regulation of alternative splicing of two essential components of the γ-secretase protein complex, which plays a central role in Alzheimer’s disease and Notch signaling pathway. Knockdown of PNN promotes inclusion of introns 3 and 15 in PSENEN (PEN-2) and NCSTN (nicastrin) transcripts respectively. Since Notch signaling has been shown to play a key role in the corneal epithelial differentiation and maintenance, our findings on PNN’s involvement in the alternative splicing of two major components of γ-secretase protein complex may provide a valuable insight not only to the PNN’s functional mechanism but also to the many aspects of corneal epithelial biology.
Our study identifies an exciting panel of alternatively spliced transcripts to be explored for their biological significance in corneal epithelial development and maintenance. (NIH Grant R01 EY007883, P30 EY021721)
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