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Abstract
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 trans-acting mechanism by blocking IRP inactivation.
Highlights
Regulated degradation of mRNA constitutes an important mechanism for differential gene expression
thymidine kinase (TK)-hTfR Reporter mRNA Stability Is Regulated by Iron—To analyze the effect of translation on the iron-dependent decay of RNA molecules containing transferrin receptor (TfR) control elements, we established a selectable reporter system
The coding region of the mouse thymidine kinase cDNA was fused to the entire 3Ј-UTR of human TfR cDNA to give the construct TK-hTfR (Fig. 1A)
Summary
Regulated degradation of mRNA constitutes an important mechanism for differential gene expression (reviewed in Refs. 1 and 2). Mammalian cells induce iron uptake by increasing the number of TfRs on the cell surface (6 –9) This rise in receptor expression is mediated by a dramatic increase in TfR mRNA stability and is dependent on the presence of the 3Ј-untranslated region of the TfR transcript [10]. In iron-deprived cells, specific RNA-binding factors, the iron regulatory proteins (IRPs), bind to these regions and prevent the degradation of TfR mRNA, presumably by masking a rapid turnover determinant [13, 14]. The IRE binding activities of IRP-1 and IRP-2 were shown to be differentially regulated in response to non-iron environmental conditions, such as nitric oxide signaling [37,38,39], oxidation [40, 41], and hypoxia [42,43,44].
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