The protein kinase C beta II exon confers mRNA instability in the presence of high glucose concentrations.
Previous studies showed that short term exposure of cells to high glucose destabilized protein kinase C (PKC) betaII mRNA, whereas PKCbetaI mRNA levels remained unaltered. Because PKCbeta mRNAs share common sequences other than the PKCbetaII exon encoding a different carboxyl terminus, we examined PKCbetaII mRNA for a cis-acting region that could confer glucose-induced destabilization. A beta-globin/growth hormone reporter con struct containing the PKCbetaII exon was transfected into human aorta and rat vascular smooth muscle cells (A10) to follow glucose-induced destabilization. Glucose (25 mm) exposure destabilized PKCbetaII chimeric mRNA but not control mRNA. Deletion analysis and electrophoretic mobility shift assays followed by UV cross-linking experiments demonstrated that a region introduced by inclusion of the betaII exon was required to confer destabilization. Although a cis-acting element mapped to 38 nucleotides within the betaII exon was necessary to bestow destabilization, it was not sufficient by itself to confer complete mRNA destabilization. Yet, in intact cells antisense oligonucleotides complementary to this region blocked glucose-induced destabilization. These results suggest that this region must function in context with other sequence elements created by exon inclusion involved in affecting mRNA stability. In summary, inclusion of an exon that encodes PKCbetaII mRNA introduces a cis-acting region that confers destabilization to the mRNA in response to glucose.
Patel
Niketa A
NA
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612, USA.
Eichler
Duane C
DC
Chappell
David S
DS
Illingworth
Philip A
PA
Chalfant
Charles E
CE
Yamamoto
Mayumi
M
Dean
Nicholas M
NM
Wyatt
Jacqueline R
JR
Mebert
Konrad
K
Watson
James E
JE
Cooper
Denise R
DR
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D005914:Globins / Q000235:genetics; D005947:Glucose / Q000494:pharmacology*; D006207:Half-Life; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics*; D064546:Protein Kinase C beta; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats
D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D005914:Globins; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D005947:Glucose