Analysis of psoriasis-relevant gene expression and exon usage alterations after silencing of SR-rich splicing regulators

Abstract

In our recent cDNA microarray experiment, three SR-rich splicing factors-SFRS18, PPIG and LUC7L3-were shown to exert altered responsiveness upon T-lymphokine stimulation of psoriatic non-involved and healthy epidermis samples. We have also demonstrated that double silencing LUC7L3 and SFRS18 efficiently decreased production of the psoriasis-associated EDA+ fibronectin isoform. These findings prompted the further investigation of signalling pathways affected by LUC7L3 and SFRS18. To detect gene expression and splicing pattern alterations upon double silencing of LUC7L3 and SFRS18 in an HPV-immortalised keratinocyte cell culture, paired-end RNA sequencing was carried out. Marked changes in exon usage were revealed, in contrast to the modest alterations detected in gene expression, providing a closer delineation of the potential targets of the examined splicing factors. The most prominent gene expression change was detected for IFI6, an interferon-inducible gene highly expressed in psoriasis. Interacting partners of IFI6 and certain psoriasis-associated transcripts also exhibited significantly increased expression upon silencing. In addition to elevated abundance of the EDA+ fibronectin interactor ITGA5, we confirmed decreased EDA domain inclusion, which agrees well with our prior experimental data. Furthermore, differential exon usage was established for the transcription element CREB1, along with HERC6 and CUL1, which are implicated in ubiquitination. Although immortalised keratinocytes express low levels of TINCR, a long non-coding RNA involved in terminal differentiation of keratinocytes, splicing alterations were successfully demonstrated for this RNA as well. We believe that the targeted investigation of mRNA maturation disturbances may help us gain deeper insight into the molecular pathogenesis of psoriasis.

Keywords: RNA-Sequencing; antiviral immunity; inflammation; mRNA maturation; non-coding RNAs.