Characterization of the Translation-dependent Step during Iron-regulated Decay of Transferrin Receptor mRNA

Iron regulates the stability of the mRNA encoding the transferrin receptor (TfR). When iron is scarce, iron regulatory proteins (IRPs) stabilize TfR mRNA by binding to the 3'-untranslated region. High levels of iron induce degradation of TfR mRNA; the translation inhibitor cycloheximide prevents this. To distinguish between cotranslational mRNA decay and a trans effect of translation inhibitors, we designed a reporter system exploiting the properties of the selectable marker gene thymidine kinase (TK), The 3'-untranslated region of human transferrin receptor, which contains all elements necessary for iron-dependent regulation of mRNA stability, was fused to the TK cDNA. In stably transfected mouse fibroblasts, the expression of the reporter gene was perfectly regulated by iron. Introduction of stop codons in the TK coding sequence or insertion of stable stem-loop structures in the leader sequence did not affect on the iron-dependent regulation of the reporter mRNA This implies that global translation inhibitors stabilize TfR mRNA in trans. Cycloheximide prevented the destabilization of TfR mRNA only in the presence of active IRPs. Inhibition of IRP inactivation by cycloheximide or by the specific proteasome inhibitor MG132 correlated with the stabilization of TfR mRNA These observations suggest that inhibition of translation by cycloheximide interferes with the rate-limiting step of iron-induced TfR mRNA decay in a transacting mechanism by blocking IRP inactivation.