Abstract
Purpose: :
PININ (PNN) is a nuclear speckle-associated protein that was previously shown to play essential roles in corneal epithelial differentiation. One of PNN’s interaction partners, Epithelial Splicing Regulatory Protein 1 (ESRP1), was shown to modulate alternative splicing in an epithelial-specific manner. To further investigate the impact of PNN and ESRP1 on alternative pre-mRNA splicing in the corneal epithelial context, we performed genome-wide exon array analysis.
Methods: :
Two lines of human corneal epithelial cells (HCET) harboring doxycycline-inducible shRNA against PNN or ESRP1 were created. Cells were cultured for three days with/without doxycycline. Total RNA was isolated from four biological replicates each condition and subjected to Affymetrix hGlue3_0 exon array analysis.
Results: :
Alternative splicing pattern of 254 genes was interrupted upon PNN knockdown with less than 2-fold change in the total expression level compared to non-induced cells. ESRP1 knockdown led to aberrant splicing of 45 genes, while doxycycline alone did not cause any perturbation in alternative splicing. Interestingly, splicing pattern of 11 genes out of 45 affected genes by ESRP1 knockdown was also disrupted by PNN knockdown in a markedly similar fashion. For example, either PNN or ESRP1 knockdown equally promoted alternative 5’ splice site selection on exon 1 of ubiquitin C. As for ESRP1 knockdown, altered exon usage was observed in two epithelial-mesenchymal transition (EMT)-related genes, Rho guanine nucleotide exchange factor 11 (inclusion of exon 38) and solute carrier family 37 member 2 (exclusion of exon 18). Most interestingly, the analysis indicated that PNN knockdown specifically led to 2-fold increase in exon 5a inclusion on PAX6. Increased transcript level of PAX6(5a) upon PNN downregulation was confirmed by separate alternative splicing-specific RT-PCR assays, suggesting PNN’s role as one of factors governing alternative splicing of PAX6.
Conclusions: :
Our data suggest that PNN and ESRP1 modulate alternative splicing of a specific subset of exons, but not general splicing events. PNN and ESRP1 may share functional connection in epithelial-specific alternative splicing. (NIH Grant EY07883)
Keywords: cornea: epithelium • gene/expression • gene microarray