Fe ²⁺ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA repression and activation

Abstract

Iron increases synthesis rates of proteins encoded in iron-responsive element (IRE)-mRNAs; metabolic iron ("free," "labile") is Fe 2þ . The noncoding IRE-RNA structure, approximately 30 nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA riboregulators bind specifically to iron-regulatory proteins (IRP) proteins, inhibiting ribosome binding. Deletion of the IRE-RNA from an mRNA decreases both IRP binding and IRP-independent protein synthesis, indicating effects of other "factors." Current models of IRE-mRNA regulation, emphasizing iron-dependent degradation/modification of IRP, lack answers about how iron increases IRE-RNA/IRP protein dissociation or how IRE-RNA, after IRP dissociation, influences protein synthesis rates. However, we observed Fe 2þ (anaerobic) or Mn 2þ selectively increase the IRE-RNA/IRP K D . Here we show: (i) Fe 2þ binds to the IRE-RNA, altering its conformation (by 2-aminopurine fluorescence and ethidium bromide displacement); (ii) metal ions increase translation of IRE-mRNA in vitro; (iii) eukaryotic initiation factor (eIF)4F binds specifically with high affinity to IRE-RNA; (iv) Fe 2þ increased eIF4F/IRE-RNA binding, which outcompetes IRP binding; (v) exogenous eIF4F rescued metal-dependent IRE-RNA translation in eIF4F-depeleted extracts. The regulation by metabolic iron binding to IRE-RNA to decrease inhibitor protein (IRP) binding and increase activator protein (eIF4F) binding identifies IRE-RNA as a riboregulator.ferrous ion regulation | metabolic riboregulator I ron increases rates of ferritin protein synthesis in animals by facilitating messenger RNA/ribosome binding; metabolic iron (i.e., "labile" or "free" iron in cells) is considered to be ferrous (1). The iron response requires a noncoding riboregulator called the "iron-responsive element" (IRE), which is approximately 30 nt, folded into a distorted, bulged helix loop (2-5). This riboregulatory structure is also found in mRNAs for proteins of iron traffic (6-9), cell cycling (10), and the nervous system (11). IRP proteins bind with different stabilities to IRE-RNAs of the IRE-RNA family (12, 13), creating a graded or hierarchal set of mRNA responses to iron in vivo. Deletion of the 30 nt IRE-RNA not only removes IRP regulation but also decreases the rate of IRP-independent protein synthesis (14). A number of current models of IRE-RNA/IRP regulation feature iron-dependent degradation/modification of the IRP proteins as the main control point (8,9,15,16). Such models do not answer two important questions: (i) How does iron increase release of IRP protein for [4Fe-4S]-modification and/or degradation? Overlap of the IRE-RNA and the Fe-S binding sites on IRP1 prevents Fe-S insertion in the IRP1/IRE-RNA complex (5). (ii) How does the IRE-RNA control rates of IRP-independent protein synthesis (14)? In an earlier study, we showed that Fe 2þ ions (anaerobic) selectively increased the dissociation constant for the IRE-RNA/IRP1 complex in solution (12). Here we r...

Regulators of Iron Homeostasis: New Players in Metabolism, Cell Death, and Disease

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