Abstract 1037: Angiotensin II Type 1 Receptor 1166 Polymorphism A → C Increases mRNA Stability and Steady-State Levels
Angiotensin II type 1 receptor (AT1R) regulation plays an important role in the pathogenesis of hypertension and heart failure. Posttranscriptional regulation of AT1R is largely mediated by its 3′untranslated region (3′UTR). In order to analyze structure-function relationship of the 3′UTR, a number of AT1R 3′UTR constructs were created and tested. Removal of 3′UTR from AT1R message increases mRNA levels by increasing its half-life. Transfer of the 3′UTR of AT1R to 3′end of either bcl-2 or luciferase coding region resulted in a decrease in the steady-state mRNA expression of these fusion constructs and therefore 3′UTR contains all the necessary elements for its inhibitory effect. Functional characterization of 3′UTR deletions and domain swapping constructs showed that 3′UTR contains both stabilizing and destabilizing elements resulting in increased or decreased steady-state mRNA levels. In order to identify functionally active regions, AT1R 3′UTR was subjected to random mutagenesis. After several rounds of screening, two single base alterations were found to have an effect on mRNA levels. Alteration in the base at 1255 resulted in a 3-fold decrease and at 1166 2.5-fold increase in mRNA levels. Thus AT1R 3′UTR 1166 A → C polymorphism results in a partial loss of 3′UTR effect on mRNA destabilization. Interestingly, the carrier status of this single polymorphism located in the 3′UTR of AT1R has been associated with increased incidence of hypertension and higher rate of restenosis. In order to study the RNA-protein interactions, we performed RNA-shift and Northwestern assays using 3′UTR 1081–1181 as a probe and found at least three RNA-interacting proteins at sizes of 17 kDa, 35 kDa, and 45 kDa. It is tempting to speculate that changes in RNA-protein interactions could explain the effects of 1166 A → C polymorphism on mRNA levels.