June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Identification and Validation of PNN-regulated Splicing Events in Human Corneal Epithelial Cells
Author Affiliations & Notes
  • Stephen Sugrue
    Anatomy & Cell Biology, University of Florida, Gainesville, FL
  • Jeong-Hoon Joo
    Anatomy & Cell Biology, University of Florida, Gainesville, FL
  • Footnotes
    Commercial Relationships Stephen Sugrue, None; Jeong-Hoon Joo, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3218. doi:https://doi.org/
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    • Get Citation

      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)

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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  
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