The luciferase assay used to identify sites within the TFRC mRNA that are required for degradation during iron repletion. (A) The previously minimized TFRC 3′ UTR that supports iron-dependent regulation (Casey et al. 1989) was cloned into the 3′ UTR of the FL gene within the pmirGLO vector. (B) The effect of iron depletion and repletion on the FL enzyme activity of wt FL-TFRC mRNA and the Δ31–108 deletion, which earlier was demonstrated to be nonresponsive to iron (Mullner and Kuhn 1988); the FL activity was normalized to the corresponding RL value. Transfected mouse fibroblasts were treated with either 100 µM DFO or 100 µg/mL FAC for the indicated times prior to the luciferase assays. (C) The change in FL/RL for enzyme activity correlates well with the change in FL/RL for mRNA abundance. FL/RL values are relative to the wt and were measured after 14 h of treatment with 100 µg/mL FAC. Statistical significance was analyzed by two-tailed Student's t-test. (***) P < 0.001, (ns) not significant: P > 0.05. All error bars represent ± SEM of at least three biological replicates.
