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Stephen P Sugrue, Debra Akin, Leyi Jing; Transcriptome analyses reveal Pnn-knockdown-induced changes in the corneal epithelial alternative splicing of long non-coding RNAs (lncRNAs). Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5648.
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Pnn is involved in both transcriptional repression complexes and the spliceosomal complexes, placing Pnn at the fulcrum point between chromatin and mRNA splicing. PNN knockdown cells exhibit alterations in transcript profiles and splicing patterns of subset of target genes that may impact the epithelial cell phenotype. Previous GG-H transcriptome array revealed Pnn-knockdown-induced changes in the alternative splicing of long non-coding RNAs (lncRNAs).
To investigate PNN’s regulation of the alternative splicing of lncRNAs in a corneal epithelial context, we used human corneal epithelial cells (HCET) with inducible PNN shRNA and performed detailed RNA-seq analyses of alternatively spliced lncRNAs. Total RNA was isolated from knockdowns and controls and libraries were prepared from Ribo(-) RNA. We align data to the genome using the RNA-Seq, and determine feature counts for transcripts, exons, introns, and splice junctions. Using a combination of expression and splice junction data, aberrantly spliced transcripts are identified and quantified.
The RNA-seq data provide deep coverage of Wt and PNN-knockdown transcriptome and statistical power for differential expression analyses. Determination of aberrant splicing events revealed a specific set of candidates. Of particular interest, knockdown of PNN led to the specific alterations in the inclusion of multiple cassette exons as well as in the usage of alternative splice sites resulting in the considerable net changes in the ratio between splice variants (as we reported Joo et al. Mol Vis 2014). These data may indicate a role of Pnn in exon recognition in lncRNAs.
Pnn plays a central role(s) in the establishment and maintenance of the corneal epithelium. This RNA-seq study suggests involvement of PNN in the alternative splicing of multiple long non-coding RNAs (lncRNAs). This is the first RNA-seq study to interrogate the transcriptome in Pnn-depleted cells. The data suggest that while Pnn has broad impact on splicing, there are a specific subsets of lncRNAs affected by PNN knock down. We suggest that some lncRNAs may exert broad influence over the regulation and maintenance of epithelial cell phenotype.
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