Abstract
Purpose:
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.
Methods:
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.
Results:
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.
Conclusions:
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)
Keywords: 482 cornea: epithelium •
480 cornea: basic science •
533 gene/expression