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RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells

PURPOSE: The specialized corneal epithelium requires differentiated properties, specific for its role at the anterior surface of the eye. Thus, tight maintenance of the differentiated qualities of the corneal epithelial is essential. Pinin (PNN) is an exon junction component (EJC) that has dramatic...

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Autores principales: Akin, Debra, Newman, Jeremy R.B., McIntyre, Lauren M., Sugrue, Stephen P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Molecular Vision 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734150/
https://www.ncbi.nlm.nih.gov/pubmed/26900324
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author Akin, Debra
Newman, Jeremy R.B.
McIntyre, Lauren M.
Sugrue, Stephen P.
author_facet Akin, Debra
Newman, Jeremy R.B.
McIntyre, Lauren M.
Sugrue, Stephen P.
author_sort Akin, Debra
collection PubMed
description PURPOSE: The specialized corneal epithelium requires differentiated properties, specific for its role at the anterior surface of the eye. Thus, tight maintenance of the differentiated qualities of the corneal epithelial is essential. Pinin (PNN) is an exon junction component (EJC) that has dramatic implications for corneal epithelial cell differentiation and may act as a stabilizer of the corneal epithelial cell phenotype. Our studies revealed that PNN is involved in transcriptional repression complexes and spliceosomal complexes, placing PNN at the fulcrum between chromatin and mRNA splicing. Transcriptome analysis of PNN-knockdown cells revealed clear and reproducible alterations in transcript profiles and splicing patterns of a subset of genes that would significantly impact the epithelial cell phenotype. We further investigated PNN’s role in the regulation of gene expression and alternative splicing (AS) in a corneal epithelial context. METHODS: Human corneal epithelial (HCET) cells that carry the doxycycline-inducible PNN-knockdown shRNA vector were used to perform RNA-seq to determine differential gene expression and differential AS events. RESULTS: Multiple genes and AS events were identified as differentially expressed between PNN-knockdown and control cells. Genes upregulated by PNN knockdown included a large proportion of genes that are associated with enhanced cell migration and ECM remodeling processes, such as MMPs, ADAMs, HAS2, LAMA3, CXCRs, and UNC5C. Genes downregulated in response to PNN depletion included IGFBP5, FGD3, FGFR2, PAX6, RARG, and SOX10. AS events in PNN-knockdown cells compared to control cells were also more likely to be detected, and upregulated. In particular, 60% of exon-skipping events, detected in only one condition, were detected in PNN-knockdown cells and of the shared exon-skipping events, 92% of those differentially expressed were more frequent in the PNN knockdown. CONCLUSIONS: These data suggest that lowering of PNN levels in epithelial cells results in dramatic transformation in the number and composition of splicing variants and that PNN plays a crucial role in the selection of which RNA isoforms differentiating cells produce. Many of the genes affected by PNN knockdown are known to affect the epithelial phenotype. This window into the complexity of RNA splicing in the corneal epithelium implies that PNN exerts broad influence over the regulation and maintenance of the epithelial cell phenotype.
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spelling pubmed-47341502016-02-19 RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells Akin, Debra Newman, Jeremy R.B. McIntyre, Lauren M. Sugrue, Stephen P. Mol Vis Research Article PURPOSE: The specialized corneal epithelium requires differentiated properties, specific for its role at the anterior surface of the eye. Thus, tight maintenance of the differentiated qualities of the corneal epithelial is essential. Pinin (PNN) is an exon junction component (EJC) that has dramatic implications for corneal epithelial cell differentiation and may act as a stabilizer of the corneal epithelial cell phenotype. Our studies revealed that PNN is involved in transcriptional repression complexes and spliceosomal complexes, placing PNN at the fulcrum between chromatin and mRNA splicing. Transcriptome analysis of PNN-knockdown cells revealed clear and reproducible alterations in transcript profiles and splicing patterns of a subset of genes that would significantly impact the epithelial cell phenotype. We further investigated PNN’s role in the regulation of gene expression and alternative splicing (AS) in a corneal epithelial context. METHODS: Human corneal epithelial (HCET) cells that carry the doxycycline-inducible PNN-knockdown shRNA vector were used to perform RNA-seq to determine differential gene expression and differential AS events. RESULTS: Multiple genes and AS events were identified as differentially expressed between PNN-knockdown and control cells. Genes upregulated by PNN knockdown included a large proportion of genes that are associated with enhanced cell migration and ECM remodeling processes, such as MMPs, ADAMs, HAS2, LAMA3, CXCRs, and UNC5C. Genes downregulated in response to PNN depletion included IGFBP5, FGD3, FGFR2, PAX6, RARG, and SOX10. AS events in PNN-knockdown cells compared to control cells were also more likely to be detected, and upregulated. In particular, 60% of exon-skipping events, detected in only one condition, were detected in PNN-knockdown cells and of the shared exon-skipping events, 92% of those differentially expressed were more frequent in the PNN knockdown. CONCLUSIONS: These data suggest that lowering of PNN levels in epithelial cells results in dramatic transformation in the number and composition of splicing variants and that PNN plays a crucial role in the selection of which RNA isoforms differentiating cells produce. Many of the genes affected by PNN knockdown are known to affect the epithelial phenotype. This window into the complexity of RNA splicing in the corneal epithelium implies that PNN exerts broad influence over the regulation and maintenance of the epithelial cell phenotype. Molecular Vision 2016-01-16 /pmc/articles/PMC4734150/ /pubmed/26900324 Text en Copyright © 2016 Molecular Vision. http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited, used for non-commercial purposes, and is not altered or transformed.
spellingShingle Research Article
Akin, Debra
Newman, Jeremy R.B.
McIntyre, Lauren M.
Sugrue, Stephen P.
RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells
title RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells
title_full RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells
title_fullStr RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells
title_full_unstemmed RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells
title_short RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells
title_sort rna-seq analysis of impact of pnn on gene expression and alternative splicing in corneal epithelial cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734150/
https://www.ncbi.nlm.nih.gov/pubmed/26900324
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