Ali Hassan

Ali Hassan, Hong Ji, Yinghua Zhang & Kathryn Sandberg

Georgetown University Medical Center, Washington, DC.

In the rat, two distinct angiotensin type 1a receptor (AT_1aR) mRNAs are synthesized from a single AT_1aR gene by alternative splicing. These two transcripts are comprised of exons 1, 2 & 3 (E1,2,3) or exons 1 & 3 (E1,3). Both E1,2,3 and E1,3 code for identical AT_1aR proteins since exon 3 contains the entire coding region. We found that the E1,3 mRNA variant is translated more efficiently in vitro than the E1,2,3 transcript and that AT_1aR expression is markedly higher in Chinese hamster ovary (CHO) cells stably transfected with the E1,3 splice variant than in cells stably transfected with the E1,2,3 variant. In this study we investigated the role of alternative splicing in AT_1aR expression in rat aortic smooth muscle cells (RASMC) which endogenously express the AT_1aR. Real-time PCR revealed that in these cells the E1,2,3 splice variant was predominant, making up 76 ± 1.7% of total AT_1aR mRNA while the E1,3 splice variant accounted for the remainder. The role of the different splice variants was investigated using an siRNA sequence which was designed to specifically target the junction between exons 1 & 3, a region unique to the E1,3 splice variant. Treatment with this siRNA (S1) for 48 h resulted in a 72% reduction in E1,3 mRNA compared with cells treated with cells treated with a non-silencing (NS) siRNA sequence (pg/μg total RNA: NS, 0.124 ± 0.014, S1 0.035 ± 0.007; P<0.05). In contrast, S1 treatment had no effect on levels of E1,2,3 mRNA (pg/μg total RNA: NS, 0.366 ± 0.022, S1, 0.382 ± 0.067 pg/μg total RNA). Thus the S1 siRNA treatment potently and specifically knocked down the E1,3 splice variant while leaving the E1,2,3 splice variant intact. Following 48 h treatment with S1 siRNA ¹²⁵I[Sar¹Ile⁸]Ang II specific binding was reduced by 52% compared with cells treated with NS siRNA (cpm/well: NS, 788 ± 35; S1, 378 ± 46; P<0.05). This inhibitory effect of S1 treatment on binding is disproportionate given that the E1,2,3 mRNA is predominant in these cells. It demonstrates that in RASMC the majority of AT_1aR protein is translated from a splice variant which makes up less than a quarter of the total mRNA population. These data support our in vitro observations that the E1,3 splice variant is more efficiently translated than the E1,2,3 transcript and suggest that the E1,3 variant accounts for the majority of AT_1aR expression in VSMC even though it makes up only 30% of the total AT_1aR mRNA population. Therefore, regulation of alternative splicing which results in different E1,3 to E1,2,3 ratios would cause marked changes in AT_1aR expression even if total AT_1aR mRNA levels remain the same. These data reveal that AT_1aR expression is translationally controlled by alternative splicing in RASMC.

Presented at the Angiotensin Gordon Research Conference, Ventura, CA, 2004.