Ferritin mRNA probed, near the iron regulatory region, with protein and chemical (1,10-phenanthroline-Cu) nucleases A possible role for base-paired flanking regions

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Abstract
Iron stimulates ferritin synthesis in whole cells and animals, by increasing the entry of ferritin mRNA into polyribosomes. Dissection of the regulation at the molecular level has identified a 28-nucleotide, conserved, regulatory sequence (IRE=iron regulatory element) in the 5′ non-coding region of ferritin mRNAs, plustrans-acting factor(s), one of which is a 90-kDa protein. The site of iron action is not entirely characterized but may involve heme; sequences in the 3′ non-coding region of ferritin mRNA can modulate regulation. Ferritin mRNA is the first eukaryotic mRNA for which a conserved regulatory sequence and regulator protein have been identified. The same RNA-protein motif is used, through iron-dependent degradation of transferrin receptor mRNA, to decrease synthesis of the receptor and cellular iron uptake. The regulatory structure of the transferrin receptor mRNA is composed, in part, of five copies of the IRE in the 3′ noncoding region. IRE structure, probed by cleavage with RNases T1, V1, 1,10-phenanthroline-Cu or modification with dimethyl sulfate, is a hairpin loop with conformational variations dependent on magnesium; a basepaired region flanking the IRE is also structurally sensitive to magnesium. Similar results were obtained with a synthetic 55-mer containing the IRE and with a full-length in vitro transcript with a G→A substitution in the loop. However, in both cases, the IRE structure was closer to the computer-predicted structure and was less affected by magnesium than in native ferritin mRNA, indicating the importance of the loop sequence and RNA interactions outside the IRE structure. The combined IRE +flanking regions in six different ferritin mRNAs form a structure very close to the cap where interference with translational initiation is likely.