Ali Hassan

Ali Hassan, Hong Ji, Yinghua Zhang & Kathryn Sandberg

Department of Medicine, Georgetown University, 4000 Reservoir Rd, NW, Washington, DC 20057.

In the rat, two distinct 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). Since exon 3 contains the entire coding region both transcripts encode identical AT_1aR. Real-time PCR revealed that in RASMC E1,2,3 accounted for 76 ± 2% of total AT_1aR mRNA. The role of the different splice variants in AT_1aR regulation was investigated using an siRNA duplex designed to specifically target the junction between exons 1 & 3, a region unique to the E1,3 transcript. 48 h treatment with this siRNA (S1) resulted in a 72% reduction in E1,3 mRNA compared with cells treated with a non-silencing (NS) siRNA (pg/μg total RNA: NS, 0.124 ± 0.014; S1, 0.035 ± 0.007; P<0.05). 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). Following 48 h treatment with S1 siRNA, ¹²⁵I[Sar¹Ile⁸]Ang II specific binding was reduced by 52% compared to 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 are translated from a splice variant which makes up only 24% of the total AT_1aR mRNA population. Therefore, even small alterations in the ratio of E1,3 and E1,2,3 transcripts could cause marked changes in AT_1aR expression.

Experimental Biology 2004, Washington, DC, Abstract 7230, 2004.