Project description: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.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.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.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.

Project description: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. Our studies have focused on pinin (PNN), an exon junction component (EJC) that has dramatic implications on corneal epithelial cell differentiation and may act as a stabilizer of the corneal epithelial cell phenotype. Our studies revealed that PNN is involved in both transcriptional repression complexes and the 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. Here, we further investigate PNN’s role in the regulation of gene expression and alternative splicing (AS) in a corneal epithelial context. We used human corneal epithelial cells (HCET cells) that carry doxycycline-inducible PNN-knockdown shRNA vector and performed RNA-seq to determine differential gene expression and differential AS events. Multiple genes and AS events were identified as differentially expressed between PNN-knockdown and controls cells. Genes up-regulated by PNN-knockdown included a large proportion of genes that are associated with processes associated with enhanced cell migration and ECM remodeling including: MMPs, ADAMs, HAS2, LAMA3, CXCRs and UNC5C. Genes down-regulated in response to PNN depletion included: IGFBP5. FGD3, FGFR2, PAX6, RARG and SOX10. AS events in PNN compared to controls was also more likely to be detected, and uregulated in PNN-knockdowns. In particular, 60% of exon skipping events detected in only one condition were detected in PNN-knockdowns and of the shared exon skipping events, 92% of those differentially expressed were more frequent in the PNN-knockdown. This suggests that in the absence of PNN the epithelial cells are dramatically transformed in the amount and composition of isoforms and that PNN plays a crucial role in the selection of which isoforms differentiating cells produce. Many of the genes affected by PNN-knockdown are known to affect 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 epithelial cell phenotype. We used HCET cells that carry doxycycline-inducible PNN knockdown shRNA vector and performed RNA-seq to determine differential gene expression and differential alternative splicing events.

Project description: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. Our studies have focused on pinin (PNN), an exon junction component (EJC) that has dramatic implications on corneal epithelial cell differentiation and may act as a stabilizer of the corneal epithelial cell phenotype. Our studies revealed that PNN is involved in both transcriptional repression complexes and the 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. Here, we further investigate PNN’s role in the regulation of gene expression and alternative splicing (AS) in a corneal epithelial context. We used human corneal epithelial cells (HCET cells) that carry doxycycline-inducible PNN-knockdown shRNA vector and performed RNA-seq to determine differential gene expression and differential AS events. Multiple genes and AS events were identified as differentially expressed between PNN-knockdown and controls cells. Genes up-regulated by PNN-knockdown included a large proportion of genes that are associated with processes associated with enhanced cell migration and ECM remodeling including: MMPs, ADAMs, HAS2, LAMA3, CXCRs and UNC5C. Genes down-regulated in response to PNN depletion included: IGFBP5. FGD3, FGFR2, PAX6, RARG and SOX10. AS events in PNN compared to controls was also more likely to be detected, and uregulated in PNN-knockdowns. In particular, 60% of exon skipping events detected in only one condition were detected in PNN-knockdowns and of the shared exon skipping events, 92% of those differentially expressed were more frequent in the PNN-knockdown. This suggests that in the absence of PNN the epithelial cells are dramatically transformed in the amount and composition of isoforms and that PNN plays a crucial role in the selection of which isoforms differentiating cells produce. Many of the genes affected by PNN-knockdown are known to affect 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 epithelial cell phenotype.

Project description:Plasma membrane calcium-ATPases (PMCAs) play a critical role in regulating intracellular calcium concentration. Four genes encode PMCA proteins with alternative splicing of transcripts at three sites (A, B and C) serving to increase isoform diversity. Our previous work shows that all four PMCAs are expressed and have specific locations in human corneal epithelium (hCE). The present work examined which splice variants of PMCAs are expressed in hCE. Total RNA was extracted from hCE scraped from cadaver corneas of five different donors (two females and three males, age range 55-76 years). RT-PCR was performed using PMCA isoform-specific primers designed to amplify transcripts that included either splice site A or splice sites B and C. PMCA cDNAs were sequenced or cloned, and then sequenced. There was uniformity in the PMCA1 and PMCA4 expression profile among the five donors. Specifically, every donor expressed PMCA4 transcripts (4x at site A and 4b at site B/C). Every donor also expressed PMCA1 transcripts at sites B/C, specifically PMCA1b and PMCA1kb. In contrast, PMCA2 and PMCA3 expression varied; PCR DNAs were detected in two of five donors. One donor expressed PMCA2a and a novel PMCA2 variant we termed PMCA2((i)). PMCA3a transcript was demonstrated in a different donor. Finally, for all the donors, bands encoding site A transcripts for PMCA4 were obtained but no PCR transcripts were detected at site A for PMCA1, PMCA2 and PMCA3. This investigation showed that hCE expressed multiple splice variants of PMCA isoforms. Furthermore, this study documented the expression of the PMCA1k variant (PMCA1kb) previously only described in intestine and pancreatic beta cells and describes a novel PMCA2((i)) variant. Finally, this study suggests that the molecular configuration of PMCA1, PMCA2 and PMCA3 in the region of splice site A in hCE must be different than in other tissues since the same primers that produced site A transcripts in several other tissues were ineffective in priming PCR in hCE.

Dataset Information

Role of Pnn in alternative splicing of a specific subset of lncRNAs of the corneal epithelium.

Publications

Role of Pnn in alternative splicing of a specific subset of lncRNAs of the corneal epithelium.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets