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

Abstract

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␦ (PKC␦), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKC␦, PKC␦VIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKC␦VIII via utilization of a downstream 5 splice site of exon 10 on PKC␦ pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKC␦VIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKC␦VIII alternative splicing. To identify the cis-elements involved in 5 splice site selection we cloned a minigene, which included PKC␦ 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␦ minigene promoted selection of 5 splice site II. Mutation of the SC35 binding site in the PKC␦ minigene abolished RA-mediated utilization of 5 splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKC␦ exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKC␦ 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␦VIII.Vitamin A, an important micronutrient and its active metabolite all-trans-retinoic acid (RA) 2 influence a broad range of physiological and pathological processes in the embryonic central nervous system and in the mature brain. Protein kinase C (PKC), a serine/threonine kinase family, consists of 11 isoforms and their splice variants and is involved in the regulation of cellular differentiation, growth, and apoptosis (2). Protein kinase C␦, a member of the novel PKC subfamily, is implicated in both apoptosis (3-10) and cell survival pathways (11-14) Thus, PKC␦ has dual effects and represents a switch that determines cell survival and fate. This can be explained by the expression of alternatively spliced variants of PKC␦ with distinct functions in the apoptotic cascade. The occurrence of PKC␦ isoforms is species-specific. PKC␦I is ubiquitously present in all species while PKC␦II, -␦IV, -␦V, -␦VI, and -␦VII isoforms are present in mouse tissues (15, 16); PKC␦III is present in rats (17) and PKC␦VIII is present in humans (1).An important mechanism of regulating gene expression occurs by alternative splicing which expands the coding capacity of a single gene to produce different proteins with distinct functions (18). It is now established that close to 90% of human genes undergo alternative splicing and encode for at least two isoforms. Divergence observed in gene expression because of alternative splicing may be tissue-specific (19,20), de...

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