Ali Hassan

Ali Hassan, James W Kim, 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 and 3 (E1,2,3) or exons 1 and 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 have found that the E1,3 mRNA variant is translated more efficiently in vitro than the E1,2,3 transcript and that in 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. The aim of this study was to investigate the role of alternative splicing in >AT_1aR expression in vascular smooth muscle cells (VSMC) using siRNA technology. An siRNA duplex was designed which targets the junction between exons 1 and 3, a region unique to the E1,3 splice variant. To determine the E1,3 siRNA (S1) specificity, CHO cells stably expressing the E1,3 or E1,2,3 variants were transfected with S1 siRNA for 48 h. In the E1,3 expressing cells, ¹²⁵I[Sar¹,Ile⁸]Ang II specific binding was reduced by 53.6 ± 3.9% compared to cells transfected with a non-silencing (i.e. negative control; NC) siRNA sequence (NC, 11421 ± 2005 cpm vs S1, 5306 ± 442 cpm; p<0.05, n=3), whereas the S1 siRNA had no effect on ¹²⁵I[Sar¹,Ile⁸]Ang II binding in the E1,2,3 expressing cells. When rat VSMC were transfected with S1 siRNA, a similar reduction in ¹²⁵I[Sar¹,Ile⁸]Ang II binding was observed. Following a 48 h treatment with S1 siRNA, ¹²⁵I[Sar¹,Ile⁸]Ang II binding was reduced by 49.8 ± 1.1% compared with NC siRNA-treated cells (NC, 7663 ± 328 cpm vs S1, 3921 ± 81 cpm; p<0.001, n=3); however, real-time PCR revealed that the E1,3 transcript accounts for less than 30% of the total AT_1aR 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 since 30% of the total AT_1aR mRNA population accounts for at least 50% of the AT_1aRs expressed on the cell membrane. Furthermore these findings indicate that even if total AT_1aR mRNA levels remain the same, AT_1aR expression could change markedly from alternative splicing that resulted in alterations in E1,3 to E1,2,3 splice variant ratios.

57th Annual Fall Conference of the Council for High Blood Pressure Research, Washington, DC, Abstract 75, 2003.