Three 5-bp hairpin loops have an additive effect on instability. (A) Summary of mutagenesis study (Tables 2, 3); green, sites of mutations that destabilize mRNA; red, sites of mutations resulting in stabilization; gray, sites of mutations with no significant effect. The endonuclease cleavage site was earlier proposed to be 3′ of G184 (arrow; Binder et al. 1994). Inset shows the UAAC tetraloop substitution in IRE C. (B) Predicted secondary structure of the RNA optimized for instability; green, mutations that are destabilizing. (C) Sequential mutagenesis of the three non-IRE stem–loops results in an additive stabilization of the opt RNA. Values are relative to the wt RNA (dashed line). Luciferase was measured after 14 h treatment with 100 µg/mL FAC. (D). The opt RNA within transfected mouse fibroblasts has decreased stability relative to the wt sequence. Fourteen hours after the transfections, cells were transferred into fresh iron-replete media for 2 h prior to the addition of 5 µM actinomycin D. Total RNA was isolated at the indicated time points and mRNA levels assayed by qRT-PCR. The FL mRNA was normalized to the corresponding RL mRNA, and the FL/RL ratio for the zero hour actinomycin D point was set as 100%. Statistical significance was analyzed by two-tailed Student's t-test. (ns) Not significant: P > 0.05. Unless indicated otherwise, all error bars represent ± SEM of three biological replicates.
