Alternative splicing of scleroderma‐associated lysyl hydroxylase 2 mRNA is regulated by Fox proteins

Abstract

Scleroderma (SSc) is a complex skin disease with heterogeneous components. No treatment against scleroderma is available and many questions about its biology are unanswered. We focus on extracellular component of SSc and towards understanding the regulation of lysyl hydroxylase 2 (LH2) alternative splicing that plays a significant role in SSc biology.We report that Fox2 RNA binding protein regulates the splicing of SSc‐associated LH2(long) mRNA that includes an extra 63bp exon 13A (E13A). Using an LH2 minigene, we show that overexpression of Fox2 in skin A431 cells increased E13A inclusion in LH2(long) mRNA. As control, overexpression of the Fox2 RNA binding motif deletion mutant did not increase E13A inclusion. Knockdown of Fox2 with siRNAs in Murine Embryonic Fibroblast (MEFs) decreased E13A inclusion. Our data suggests that the Fox2 role in regulating LH2 alternative splicing is neither cell nor species specific. We also knocked down Fox2 in primary cells derived from SSc patients. This decreased the LH2(long) mRNA that is increased in SSc. There are 2 Fox2 motifs in each of the introns flanking E13A. We mutated the Fox2 motifs individually and in combination to determine the relative effect on LH2 splicing. Identification of regulatory factors that play roles in inclusion of E13A in SSc‐associated LH2(long) mRNA offers important insights to therapeutic approaches against SSc.