Binding of a Cytosolic Protein to the Iron-Responsive Element of Human Ferritin Messenger RNA

Rouault, TA; Hentze, Matthias W.; Caughman, S. Wright; Harford, J B; Klausner, R D

doi:10.1126/science.3413484

Cited by 354 publications

(201 citation statements)

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“…Similar results were observed when wild type IRE (pGl3-1236) was compared with a nonfunctional IRE point mutant with a deletion of a single "C" in the loop (Ref. 12 and data not shown). To confirm that knockdown of IRPs affected translation of luciferase rather than luciferase mRNA levels, we also measured luciferase mRNA in transient transfections.…”

Section: Effects Of Irp Knockdowns On Endogenous Proteins Of Ironsupporting

confidence: 69%

Excess Capacity of the Iron Regulatory Protein System

Wang

D’Agostino

et al. 2007

Journal of Biological Chemistry

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Iron regulatory proteins (IRP1 and IRP2) are master regulators of cellular iron metabolism. IRPs bind to iron-responsive elements (IREs) present in the untranslated regions of mRNAs encoding proteins of iron storage, uptake, transport, and export. Because simultaneous knockout of IRP1 and IRP2 is embryonically lethal, it has not been possible to use dual knockouts to explore the consequences of loss of both IRP1 and IRP2 in mammalian cells. In this report, we describe the use of small interfering RNA to assess the relative contributions of IRP1 and IRP2 in epithelial cells. Iron is an essential element required for the function of numerous critical enzymes and is essential for cell survival. However, because of the ability of iron to catalyze the formation of reactive oxygen species, excess iron is harmful. Consequently, levels of intracellular iron must be tightly controlled (1). Iron-responsive elements (IREs) 2 play a central role in the regulation of iron homeostasis. IREs are mRNA hairpin elements present in the untranslated region of mRNAs encoding proteins of iron storage (ferritin) (2-4), iron uptake (TfR1) (5, 6), iron transport (DMT1) (7,8), and iron export (ferroportin) (9 -11).The role of IRE elements in ferritin and TfR1 has been particularly well studied. In ferritin H and L, an IRE is present in the 5Ј-UTR of the mRNA, whereas in TfR1, five IRE elements are present in the 3Ј-UTR. In both cases, the IRE can be bound by IRE-binding proteins. When IRE-binding proteins bind to the IRE in the 5Ј-UTR of ferritin, they inhibit translation (3, 4). When IRE-binding proteins bind to the 3Ј-UTR of TfR1, they stabilize the mRNA (6).Two IRE-binding proteins have been identified, IRP1 (12, 13) and IRP2 (14). They are highly homologous but respond to iron challenge via different mechanisms. IRP1 is a bifunctional protein; its conformational and functional changes depend on cellular iron status. When iron levels are high, IRP1 forms a 4Fe-4S cluster and functions as a cytosolic aconitase. Under these conditions, IRP1 cannot bind the IRE. Conversely, when iron levels are low, the Fe-S cluster disassembles, and IRP1 binds to IREs. Recently, it has been suggested that iron-dependent regulation of IRP1 abundance represents an additional mechanism of control of IRP1 (15). IRP2 differs from IRP1 by the presence of an additional 73-amino acids in the N terminus. When iron levels are high, IRP2 undergoes iron-dependent degradation (16,17). The consequence of this coordinate regulation of IRP proteins is that in iron-replete conditions, there is less IRP1 and IRP2 available to bind to IREs, leading to an increase in ferritin and a decrease in TfR1. Conversely, under conditions of iron limitation, increases in the activity and level of IRP1 and IRP2 lead to decreased ferritin and increased TfR1. Because increased ferritin increases iron storage, whereas decreased TfR1 decreases iron transport, these IRP-dependent events act in concert to maintain iron homeostasis. IRPs are also regulated by other physiological stimuli, inc...

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Section: Effects Of Irp Knockdowns On Endogenous Proteins Of Ironsupporting

confidence: 69%

Excess Capacity of the Iron Regulatory Protein System

Wang

D’Agostino

et al. 2007

Journal of Biological Chemistry

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“…The spontaneous or diamide-induced formation of disulfide bonds between cysteines 437 and 503 or 437 and 506, in apo-IRF, as well as its alkylation by N-ethylmaleiniide, inhibit (Kuhn and Hentze, 1992;Leibold and Guo, 1992;Klausner et al, 1993). A soluble 98 kDa protein, iron regulatory factor (IRF), also called iron-responsive element-binding protein (IRE-BP), binds to specific RNA hairpin structures known as iron-responsive elements (IREs) (Leibold et al, 1988;Rouault et al, 1988;Koeller et al, 1989;Muillner et al, 1989). The IRE consists of a stable stem -10 nucleotides long, which is interrupted by an unpaired C positioned five nucleotides 5' of the loop.…”

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confidence: 99%

Mutational analysis of the [4Fe-4S]-cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase.

1994

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“…All H-chain and L-chain ferritin mRNAs [3, 7, 81 and erythroid A1S mRNA [9, 101 have in their 5'-untranslated regions a single copy of a 28-nucleotide stem loop, designated as an iron-responsive element (IRE) [ll]. Very similar structures are found in the 3'-untranslated regions of mammalian transferrin-receptor mRNAs [ 11 -141. A cytosolic protein variously called iron-regulatory factor (IRF) [ 121, iron-responsive-element-binding protein [ 151, or ferritin-repressor protein [16], can bind to IRE in mature mRNAs [12,[17][18][19]. In the high-affinity apo-form, IRF Correspondence to R. R. Crichton, Unit6 de Biochimie, UniFax: +32 1047 2796.…”

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Control of cellular iron homeostasis by iron‐responsive elements in vivo

Ward

Kühn²,

Kaldy³

et al. 1994

European Journal of Biochemistry

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It has recently been proposed that cellular iron homeostasis in mammalian cells is regulated at the post-transcriptional level by the reciprocal control of transferrin receptor and fenitin mRNA expression via an iron-regulatory factor. This iron-regulatory factor has been shown to be a cytoplasmic aconitase which can bind to iron-responsive elements in the corresponding mRNAs with greater or lesser affinity as a function of the iron status of the cell. In the present study, we show that in vivo the affinity of iron-regulatory factor for iron-responsive elements in liver reflects the long-term iron status of the tissue in animal models for iron overloading and iron deficiency, when combined with altered transferrin saturation and serum iron levels. In contrast hepatic iron overload achieved without altering such haematopoeitic indices, had a less pronounced effect. In both spleen and heart, the affinities of iron-regulatory factor changed in parallel with both altered iron status and haematological markers. In brain and duodenum, there were no consistent changes in ironregulatory-factor activity with iron loading or depletion. Iron-regulatory-factor activity in kidney responded in an as yet unexplained manner.Iron is an essential element for the growth, differentiation and well-being of most living organisms [l]. It is therefore of paramount importance to understand at the molecular, cellular and whole-organism level how iron homeostasis is regulated. Cellular iron homeostasis can be defined as the mechanisms whereby the intracellular concentration of the metal ion is maintained at a level adequate for the cell's requirements, but not sufficiently high to cause toxic effects. This is achieved by regulating the uptake of the metal ion in concert with its intracellular utilisation, storage and eventual externalization. It has been suggested that the expression of several key proteins of iron metabolism is regulated by intracellular iron levels [2-61. The post-transcriptional regulation of the iron-storage protein ferritin, the erythroid 5-aminolaevulinate synthase (AlS) and the transfenin receptor operate at the level of mRNA. All H-chain and L-chain ferritin mRNAs [3, 7, binds tightly to the IRE of transferrin-receptor mRNA, thereby ensuring its protection against nuclease digestion [20] and translation of the transferrin receptor, thus enhancing the uptake of iron by the cell. The same iron-depleted form of IRF binds strongly to the femtin and A1S mRNAs and thus blocks the biosynthesis of ferritin erythroid AlS. When cells are replete in iron the IRF binds poorly to femtin and erythroid AIS mRNAs ensuring their expression, and dissociates from the transfenin-receptor mRNA ensuring that it will be degraded by cellular nucleases. Thus, the model implies a coordinate regulation at the level of translation of mRNAs for ferritin and erythroid AlS, on the one hand, and transferrin receptor on the other.The IRF has been clearly shown to be a cytosolic aconitase which, in its active 4Fe-4S form, has a turnover number s...

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Binding of a Cytosolic Protein to the Iron-Responsive Element of Human Ferritin Messenger RNA

Cited by 354 publications

References 17 publications

Excess Capacity of the Iron Regulatory Protein System

Excess Capacity of the Iron Regulatory Protein System

Mutational analysis of the [4Fe-4S]-cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase.

Control of cellular iron homeostasis by iron‐responsive elements in vivo

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