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Stephen P Sugrue, Daniel Ryu, Qian Peng, Jeong-Hoon Joo; RNA processing of gamma secretase complex members, Pen2 and Nicastrin, corneal Epithelia. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5546.
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It is now evident that RNA processing has a significant and broad influence over cellular phenotype. The gamma secretase complex, which contains both Pen2 and Nicastrin, functions through the proteolytic cleavage of Notch after ligand binding releasing an intracellular fragment of Notch, which then translocates to the nucleus and activates transcription factors that impact both cell differentiation and morphogenesis. Our previous studies showed that knockdown of PNN promotes inclusion of introns 3 and 15 in presenilin enhancer 2 homolog (Pen-2) and nicastrin (Ncstn) transcripts respectively and hence premature stops in both transcripts.
Here we ask, through RT-PCR and fluorescence in situ hybridization, whether these alternatively spliced mRNA isoforms are expressed in human in vivo corneal epithelia and cultured epithelial explants.
We have observed low levels of intron 3-containing PEN-2 and intron 15-containing Ncstn isoforms in human in vivo epithelia and epithelial explants, suggestive that these isoforms are stabile in vivo. In addition, in situ hybridizations suggest that Ncstn-(i15) message may be exported from the nucleus and accumulates in the cytoplasm in a similar pattern to the WT Ncstn.
Our work demonstrates that normal human corneal epithelial cells express low levels of splicing isoforms of both PEN-2 and Nicastrin.
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