Vitamin A Metabolite, All-trans-retinoic Acid, Mediates Alternative Splicing of Protein Kinase C δVIII (PKCδVIII) Isoform via Splicing Factor SC35

Vitamin A metabolite, all-trans-retinoic acid (RA), induces cell growth, differentiation, and apoptosis and has an emerging role in gene regulation and alternative splicing events. Protein kinase C delta (PKC delta), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKC delta, PKC delta VIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKC delta VIII via utilization of a downstream 5' splice site of exon 10 on PKC delta pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKC delta VIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKC delta VIII alternative splicing. To identify the cis-elements involved in 5' splice site selection we cloned a minigene, which included PKC delta exon 10 and its flanking introns in the pSPL3 splicing vector. Alternative 5' splice site utilization in the minigene was promoted by RA. Further, co-transfection of SC35 with PKC delta minigene promoted selection of 5 delta splice site II. Mutation of the SC35 binding site in the PKC delta minigene abolished RA-mediated utilization of 5' splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKC delta exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKC delta pre-mRNA alternative splicing. This study demonstrates how a nutrient, vitamin A, via its metabolite RA, regulates alternative splicing and thereby gene expression of the pro-survival protein PKC delta VIII.