Binding of human antigen R (HuR) to an AU-rich element (ARE) in the 3′untranslated region (3′UTR) reduces the expression of decay accelerating factor (DAF)

We have investigated the role of the 3′untranslated region (3′UTR) in the expression of decay accelerating factor (DAF), one of the major membrane regulators of Complement activation. We show here that the 3′UTR of DAF contains an adenylate uridine rich element (ARE) AUUUAUUUAUAUUUAUUUA, which belongs to Class II Cluster 4 of the AU-rich element-containing mRNA (ARED) database. Enhanced Green Fluorescent Protein (EGFP) Reporter constructs containing the DAF 3′UTR showed reduced levels of expression when transfected into a variety of cell lines compared to 3′UTR reporter constructs without the ARE sequence. Furthermore, the inhibitor of mRNA transcription Actinomycin D had a much stronger effect on mRNA half-life of the ARE-containing 3′UTR demonstrating that this ARE destabilises the mRNA. Electrophoretic Mobility Shift Assays (EMSA) using biotinylated RNA probes, demonstrated that cytoplasmic Human antigen R (HuR) bound to the DAF ARE. Transfection experiments using HuR specific siRNA increased DAF expression whilst plasmids containing the coding sequence of HuR had the opposite effect, demonstrating that HuR reduced the stability of DAF mRNA and suggesting that it is of importance in regulating the expression of DAF. These data suggest that modulators of HuR could potentially be used to alter DAF expression and therefore increase the susceptibility of malignant cells to immunotherapy.