Review
Iron-regulatory proteins, iron-responsive elements and ferritin mRNA translation

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Abstract

Iron plays a central role in the metabolism of all cells. This is evident by its major contribution to many diverse functions, such as DNA replication, bacterial pathogenicity, photosynthesis, oxidative stress control and cell proliferation. In mammalian systems, control of intracellular iron homeostasis is largely due to posttranscriptional regulation of binding by iron-regulatory RNA-binding proteins (IRPs) to iron-responsive elements (IREs) within ferritin and transferrin receptor (TfR) mRNAs. The TfR transports iron into cells and the iron is subsequently stored within ferritin. IRP binding is under tight control so that it responds to changes in intracellular iron requirements in a coordinate manner by differentially regulating ferritin mRNA translational efficiency and TfR mRNA stability. Several different stimuli, as well as intracellular iron levels and oxidative stress, are capable of regulating these RNA–protein interactions. In this mini-review, we shall concentrate on the mechanisms underlying modulation of the interaction of IRPs and the ferritin IRE and its role in regulating ferritin gene expression.

Introduction

Iron is an essential nutrient and a potential toxin. The cloning of the iron-regulatory proteins (IRPs) and understanding of their interaction with iron-responsive elements (IREs) has revealed a fascinating and exquisite system for controlling iron homeostasis in mammalian cells [1]. In vertebrate species, uptake of transferrin bound iron into cells occurs via transferrin-receptor (TfR) mediated endocytosis [2], [3]. An uncharacterised low molecular weight iron chelate mediates distribution of iron within the cell and iron released from transferrin is incorporated into newly formed iron containing proteins and/or the iron storage protein, ferritin [4], [5], [6] (Fig. 1). Iron is found stored in cells in the ferric state as ferric oxyhydroxide (Fe(III)OOH) bound to ferritin [6]. Ferritin is a mixture of 24 heavy (H-, 21 kDa) and light (L-, 19 kDa) chain subunits and these subunits are coded for by their corresponding mRNAs [6]. Maintenance of iron homeostasis is obviously complex and a finely tuned system for the regulation and sensing of cellular iron requirements is, therefore, paramount to all cells.

Central to this theme in iron homoestasis is the existence of two mammalian IRPs, IRP1 and IRP2, whose function is modulated by iron. IRPs behave as cytoplasmic trans-acting mRNA-binding proteins. At least one type of IRP has been found in every mammalian cell type studied, and more recently in the invertebrates, Drosophila melanogaster and Caenorhabditis elegans[7]. Posttranslational modification of these RBPs alters their binding affinity to their specific cognate mRNA hairpin structures, the iron responsive elements (IREs), and controls expression of target genes posttranscriptionally. IREs present in the 5′-untranslated region (UTR) of ferritin high (H) and low (L) chain [6], erythroid 5-aminolaevulinate synthase (eALAS) [8], mammalian mitochondrial aconitase (mt-acon) [9] and D.melanogaster succinate dehydrogenase subunit b [10] mediate the translational efficiency of these mRNAs (Fig. 1). 5 individual IRE motifs present in the 3′-UTR of the transferrin receptor (TfR) [11] mediate mRNA stability (Fig. 2). The coordinate but divergent posttranscriptional regulation of IRP/IRE RNA–protein interactions in these genes governing iron uptake, storage and usage in cellular iron metabolism maintains intracellular iron at steady state levels (see Fig. 1, Fig. 2).

Much work has focussed on understanding how the IRP/IRE RNA–protein interaction is modulated as it forms the foundation of the cellular iron sensory and regulatory network. Recent evidence suggests that IRP binding is regulated differently in different cell types and that there are a number of effectors that modulate IRP/IRE RNA–protein interactions. Indeed, if IRPs are central regulators of iron homeostasis and iron is required for multiple diverse cellular functions, then responses to multiple effectors, other than iron, should be apparent. Important modulators of IRP binding and/or function include nitric oxide (NO) [12], oxidative stress by H2O2 [13] and hypoxia [14], erythropoietin [15], hepatic α-antitrypsin [16], phosphorylation by protein kinase Cs (PKCs) [17], thyroid hormone (T3) [18] and heme [19]. We have also found that thyrotropin releasing hormone (TRH) and phosphatases also modulate IRP1 and IRP2 binding in pituitary cells. As there is a major review in this issue (by Ponka and Lok) on the regulation of TfR gene expression, including the role of IRPs in controlling TfR mRNA stability, we will concentrate on the role of IRPs in the translational regulation of ferritin gene expression in this mini-review.

Section snippets

IREs, IRPs and translational control

The IRE stem-loop structure (Fig. 3A) acts as both a repressor and an enhancer of translation. The position of the IRE relative to the 5′UTR mRNA cap structure (<60 nt) has been shown to be a crucial factor regulating translation [20]. Binding of an IRP to the IRE is thought to sterically hinder the ability of the 43 S initiation complex (consisting of eIF4E, eIF4G, eIF3 and the 40 S ribosomal subunit) from forming (blocking recruitment of the 40 S ribosomal subunit to the mRNA) or from binding

Iron induced modulation

IRP1 was the first IRP to be described and, until recently, was most intensively investigated. Its central importance in intracellular iron homeostasis was demonstrated when IRP1 overexpression rendered the cell unable to regulate its iron levels [35]. IRP1 is a bifunctional protein, acting either as a cytoplasmic aconitase (haloprotein) in iron replete cells or as a RNA-binding protein (apoprotein) in iron depleted cells. This functional switch is dependent upon the assembly of a iron-sulphur

Iron induced modulation

The discovery of a second vertebrate IRP, IRP2, led to the postulate that two IRPs are essential for high fidelity control of iron homeostasis in cells that are exposed to multiple internal and external stimuli [47], [49], [50], [51]. As a consequence, there has been a resurgence of interest in understanding the mechanisms controlling the regulation of IRP2 in mammalian cells in the last few years. Indeed, recent studies have demonstrated the important role it plays in regulating iron

Invertebrate IRP1-isoforms

Interestingly, IRP1-isoforms, but no IRP2-isoforms, have recently been identified in D.melanogaster and C.elegans[7]. One IRP was found in C.elegans and two IRPs, IRP1A and IRP1B (86% identical to each other), in D.melanogaster. IRP1A has been localised to position 94C1-8 and IRP1B to position 86B3-6 on the right arm of chromosome 3 and both show 67% homology to IRP1. Human IRP1 has been localised to human chromosome 9 [55]. The invertebrate IRPs do not contain an IRP2-like amino acid

Non-IRP/IRE regulation of ferritin translation — the acute box

There has long been a clinical association between serum iron levels, serum ferritin levels, intracellular ferritin levels and cytokine induced inflammation [56]. Indeed, elevated serum ferritin levels have been used as a diagnostic for inflammation and disease. IL-1 is the major inflammatory cytokine that lowers blood serum iron levels [57], and an increase in ferritin synthesis immediately precedes a lowering in serum iron levels [58]. The elevated ferritin levels lead to a lowering of serum

Concluding remarks

Vertebrates and invertebrates have devised elaborate and exquisitely controlled systems to regulate iron homeostasis. The IRPs, which are conserved across species, play a central role as bifunctional regulators of ferritin mRNA translational efficiency and TfR mRNA stability. Acting in concert, these IRP/IRE interactions are a superb example of how cells have crafted a master control system based on a single high affinity RNA–protein interaction that has great complexity, due to the expanding

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