title: Vitamin A metabolite, all-trans-retinoic acid, mediates alternative splicing of protein kinase C deltaVIII (PKCdeltaVIII) 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 Cdelta (PKCdelta), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKCdelta, PKCdeltaVIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKCdeltaVIII via utilization of a downstream 5' splice site of exon 10 on PKCdelta pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKCdeltaVIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKCdeltaVIII alternative splicing. To identify the cis-elements involved in 5' splice site selection we cloned a minigene, which included PKCdelta 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 PKCdelta minigene promoted selection of 5' splice site II. Mutation of the SC35 binding site in the PKCdelta minigene abolished RA-mediated utilization of 5' splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKCdelta exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKCdelta 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 PKCdeltaVIII.
authors.affiliation: James A Haley Veterans Hospital, Tampa, Florida 33612, USA.
authors.forename: Hercules
authors.initials: H
authors.lastname: Apostolatos
authors.forename: André
authors.initials: A
authors.lastname: Apostolatos
authors.forename: Timothy
authors.initials: T
authors.lastname: Vickers
authors.forename: James E
authors.initials: JE
authors.lastname: Watson
authors.forename: Shijie
authors.initials: S
authors.lastname: Song
authors.forename: Fernando
authors.initials: F
authors.lastname: Vale
authors.forename: Denise R
authors.initials: DR
authors.lastname: Cooper
authors.forename: Juan
authors.initials: J
authors.lastname: Sanchez-Ramos
authors.forename: Niketa A
authors.initials: NA
authors.lastname: Patel
mesh_terms: D017398:Alternative Splicing; D017209:Apoptosis / Q000235:genetics; D001665:Binding Sites; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D009474:Neurons / Q000166:cytology; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology; D051745:Protein Kinase C-delta / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics* / Q000502:physiology; D000068103:Serine-Arginine Splicing Factors; D014212:Tretinoin / Q000502:physiology*; D014801:Vitamin A / Q000378:metabolism / Q000494:pharmacology
chemical_list: D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D014801:Vitamin A; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; D014212:Tretinoin; C497258:PRKCD protein, human; D051745:Protein Kinase C-delta