Dataset Information

Transcriptomic analysis of PNN- and ESRP1-regulated alternative pre-mRNA splicing in human corneal epithelial cells.

ABSTRACT: PURPOSE:We investigated the impact of PININ (PNN) and epithelial splicing regulatory protein 1 (ESRP1) on alternative pre-mRNA splicing in the corneal epithelial context. METHODS:Isoform-specific RT-PCR assays were performed on wild-type and Pnn knockout mouse cornea. Protein interactions were examined by deconvolution microscopy and co-immunoprecipitation. For genome-wide alternative splicing study, immortalized human corneal epithelial cells (HCET) harboring doxycycline-inducible shRNA against PNN or ESRP1 were created. Total RNA was isolated from four biological replicates of control and knockdown HCET cells, and subjected to hGlue3_0 transcriptome array analysis. RESULTS:Pnn depletion in developing mouse corneal epithelium led to disrupted alternative splicing of multiple ESRP-regulated epithelial-type exons. In HCET cells, ESRP1 and PNN displayed close localization in and around nuclear speckles, and their physical association in protein complexes was identified. Whole transcriptome array analysis on ESRP1 or PNN knockdown HCET cells revealed clear alterations in transcript profiles and splicing patterns of specific subsets of genes. Separate RT-PCR validation assays confirmed successfully specific changes in exon usage of several representative splice variants, including PAX6(5a), FOXJ3, ARHGEF11, and SLC37A2. Gene ontologic analyses on ESRP1- or PNN-regulated alternative exons suggested their roles in epithelial phenotypes, such as cell morphology and movement. CONCLUSIONS:Our data suggested that ESRP1 and PNN modulate alternative splicing of a specific subset of target genes, but not general splicing events, in HCET cells to maintain or enhance epithelial characteristics.

SUBMITTER: Joo JH

PROVIDER: S-EPMC3559072 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Transcriptomic analysis of PNN- and ESRP1-regulated alternative pre-mRNA splicing in human corneal epithelial cells.

Joo Jeong-Hoon JH Correia Greg P GP Li Jian-Liang JL Lopez Maria-Cecilia MC Baker Henry V HV Sugrue Stephen P SP

Investigative ophthalmology & visual science 20130123 1

<h4>Purpose</h4>We investigated the impact of PININ (PNN) and epithelial splicing regulatory protein 1 (ESRP1) on alternative pre-mRNA splicing in the corneal epithelial context.<h4>Methods</h4>Isoform-specific RT-PCR assays were performed on wild-type and Pnn knockout mouse cornea. Protein interactions were examined by deconvolution microscopy and co-immunoprecipitation. For genome-wide alternative splicing study, immortalized human corneal epithelial cells (HCET) harboring doxycycline-inducibl ...[more]

PMID: 23299472

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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.

Dataset Information

Transcriptomic analysis of PNN- and ESRP1-regulated alternative pre-mRNA splicing in human corneal epithelial cells.

Publications

Transcriptomic analysis of PNN- and ESRP1-regulated alternative pre-mRNA splicing in human corneal epithelial cells.

Similar Datasets

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