Splice variant-specific silencing of angiotensin II type 1a receptor messenger RNA by RNA interference in vascular smooth muscle cells.

Abstract In the rat, two distinct angiotensin II type 1a (rAT 1a ) receptor mRNAs are synthesized from a single rAT 1a receptor gene by alternative splicing. These two transcripts are comprised of exons 1, 2, and 3 (E1,2,3) or exons 1 and 3 (E1,3). Since exon 3 contains the entire coding region, both transcripts encode identical rAT 1a receptors. Real-time PCR revealed that in rat aortic smooth muscle cells (RASMC), E1,2,3 mRNA accounted for 69.5 ± 0.9% of total rAT 1a receptor mRNA. The aim of this study was to use RNA interference (RNAi) to selectively silence the rAT 1a receptor splice variants. Forty-eight hour treatment of RASMC with E1,3-targeting siRNA (10 nM; S1 E1,3 ) resulted in a 91.2 ± 0.5% ( n  = 3, P n  = 4, P 1 receptor specific binding compared with cells treated with a non-silencing control siRNA; under these conditions, no effect was observed on levels of E1,2,3 mRNA. Conversely, treatment with E1,2,3-targeting siRNA (S2 E2 ) had no effect on E1,3 mRNA while reducing E1,2,3 mRNA by 73.9 ± 4.2% ( n  = 3, P 1 receptor binding by 39.4 ± 5.4% ( n  = 4, P 1 receptor expression in RASMC derives from the E1,2,3 splice variant. These data also demonstrate that rAT 1a receptor mRNA can be silenced in a splice-variant specific manner using siRNA in RASMC, thus providing an excellent model system for investigating the role of alternative splicing in the regulation of rAT 1a receptor expression.