Iron is an essential element and is required by most cells for survival and growth. In excess, iron is toxic to cells. Consequently, its level in cells is tightly regulated. Mammalian cells maintain stable cytosolic concentrations of free iron both by regulating their uptake of iron via the transferrin receptor (TfR) and by sequestering intracellular iron into ferritin. Ferritin synthesis is induced by iron through a mechanism which shifts latent ferritin mRNA to actively translating polysomes. TfR synthesis is decreased by iron, which promotes destabilization of TfR mRNA. The coordinate regulation of ferritin and TfR synthesis by iron is controlled by iron responsive elements, or IREs. These IREs are located in the 5'- and 3'-untranslated regions of ferritin and TfR mRNAs, respectively. The IREs form a characteristic stem-loop structure and bind cytosolic proteins, the iron responsive proteins or IRE-BPs. Two such proteins, IRE-BP Bl and IRE-BP B2, have been characterized in part. The proposed experiments are aimed at determining the mechanisms through which ferritin and TfR mRNAs are coordinately regulated by the IRE-BPs. The first specific aim is to isolate an IRE-BP cDNA by screening a rat liver lambda cDNA library with oligonucleotides derived from amino acid sequences of IRE-BP, the principal rat IRE-BP. The cDNA sequence will be determined and the deduced amino acid sequence will be compared to amino acid sequences of other proteins to identify conserved regions of the IRE-BP that may be functionally important in RNA-protein recognition. The second specific aim is to study the expression of IRE-BP B1 in rat hepatoma cells. The IRE-BP cDNA will be used to determine if IRE-BP B1 expression responds to changes in intracellular iron and/or heme levels and to determine if transcriptional or post-transcriptional mechanisms are involved in the regulation of IRE-BP B1 synthesis. The third specific aim is test the predicted secondary of the ferritin and TfR IREs by synthesizing IRE RNAs containing altered nucleotide sequences and testing these mutants for IRE-BP B1 binding. These studies will permit the correlation of IRE structure with biological function. The fourth specific aim is to determine the function of a second protein in rat liver, IRE-BP B2. IRE-BP B2 protein from rat liver will be isolated and sequenced, and its function and the functional determinants in its structure will be characterized. The fifth and last specific aim is to determine if there are other factors involved in the regulation of TfR mRNA by iron. A cell-free system will be developed to identify polysomal or cytosolic factors other than IRE-BPS, that may specifically affect the degradation of TfR mRNA.
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