Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation

Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60–100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90–156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that shorten the effective distance between the IRE and cap and allow full translational repression by IRP1. In this way some cap-distal IREs may function like cap-proximal ones.     

Coordinating responses to iron and oxygen stress with DNA and mRNA promoters: The ferritin story

Combinations of DNA antioxidant response element and mRNA iron responsive element regulate ferritin expression in animals in response to oxidant and iron stress, or normal developmental signals. Ferritins are protein nanocages, found in animals, plants, bacteria, and archaea, that convert iron and oxygen to ferric oxy biominerals in the protein central cavity; the mineral traps potentially toxic reactants and concentrates iron for the future synthesis of other iron/heme proteins. Regulatory signals and the nanocage gene products are the same throughout biology, but the genetic mechanisms, DNA versus DNA + mRNA, vary. The number of genes, temporal regulation, tissue distribution in multi-cellular organisms, and gene product size (maxi-ferritins have 24 subunits and mini-ferritins, or Dps proteins, have 12 subunits and are restricted to bacteria and archaea) suggest an overwhelming diversity and variability. However, common themes of regulation and function are described which indicate not only that the three-dimensional protein structure and the functions of the ferritins are conserved, but also that broad features of genetic regulation are conserved relative to organismal and/or community needs. The analysis illustrates the centrality of the ferritins to life with iron and oxygen and models how Nature harnesses potentially dangerous chemistry for biology.     

Accommodating variety in iron-responsive elements: Crystal structure of transferrin receptor 1 B IRE bound to iron regulatory protein 1

Iron responsive elements (IREs) are short stem-loop structures found in several mRNAs encoding proteins involved in cellular iron metabolism. Iron regulatory proteins (IRPs) control iron homeostasis through differential binding to the IREs, accommodating any sequence or structural variations that the IREs may present. Here we report the structure of IRP1 in complex with transferrin receptor 1 B (TfR B) IRE, and compare it to the complex with ferritin H (Ftn H) IRE. The two IREs are bound to IRP1 through nearly identical protein-RNA contacts, although their stem conformations are significantly different. These results support the view that binding of different IREs with IRP1 depends both on protein and RNA conformational plasticity, adapting to RNA variation while retaining conserved protein-RNA contacts.     

Iron and exercise induced alterations in antioxidant status. Protection by dietary milk proteins

Lipid peroxidation stress induced by iron supplementation can contribute to the induction of gut lesions. Intensive sports lead to ischemia reperfusion, which increases free radical production. Athletes frequently use heavy iron supplementation, whose effects are unknown. On the other hand, milk proteins have in vitro antioxidant properties, which could counteract these potential side effects. The main aims of the study were: (1) to demonstrate the effects of combined exercise training (ET) and iron overload on antioxidant status; (2) to assess the protective properties of casein in vivo; (3) to study the mechanisms involved in an in vitro model.

Antioxidant status was assessed by measuring the activity of antioxidant enzymes (superoxide dismutase (SOD); glutathione peroxidase (GSH-Px)), and on the onset of aberrant crypts (AC) in colon, which can be induced by lipid peroxidation. At day 30, all ET animals showed an increase in the activity of antioxidant enzymes, in iron concentration in colon mucosa and liver and in the number of AC compared to untrained rats. It was found that Casein's milk protein supplementation significantly reduced these parameters. Additional information on protective effect of casein was provided by measuring the extent of TBARS formation during iron/ascorbate-induced oxidation of liposomes. Free casein and casein bound to iron were found to significantly reduce iron-induced lipid peroxidation. The results of the overall study suggest that Iron supplementation during intensive sport training would decrease anti-oxidant status. Dietary milk protein supplementation could at least partly prevent occurrence of deleterious effects to tissue induced by iron overload.     

Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins

One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.     

Identification and characterization of the iron regulatory element in the ferritin gene of a plant (soybean)

Iron increases ferritin synthesis, targeting plant DNA and animal mRNA. The ferritin promoter in plants has not been identified, in contrast to the ferritin promoter and mRNA iron-responsive element (IRE) in animals. The soybean leaf, a natural tissue for ferritin expression, and DNA, with promoter deletions and luciferase or glucuronidase reporters, delivered with particle bombardment, were used to show that an 86-base pair fragment (iron regulatory element (FRE)) controlled iron-mediated derepression of the ferritin gene. Mutagenesis with linkers of random sequence detected two subdomains separated by 21 base pairs. FRE has no detectable homology to the animal IRE or to known promoters in DNA and bound a trans-acting factor in leaf cell extracts. FRE/factor binding was abrogated by increased tissue iron, in analogy to mRNA (IRE)/iron regulatory protein in animals. Maximum ferritin derepression was obtained with 50 microm iron citrate (1:10) or 500 microm iron citrate (1:1) but Fe-EDTA was ineffective, although the leaf iron concentration was increased; manganese, zinc, and copper had no effect. The basis for different responses in ferritin expression to different iron complexes, as well as the significance of using DNA but not mRNA as an iron regulatory target in plants, remain unknown.     

Interaction of anthracyclines with iron responsive element mRNAs

Double-stranded sections of mRNA are often inviting sites of interaction for a wide variety of proteins and small molecules. Interactions at these sites can serve to regulate, or disrupt, the homeostasis of the encoded protein products. Such ligand target sites exist as hairpin-loop structures in the mRNAs of several of the proteins involved in iron homeostasis, including ferritin heavy and light chains, and are known as iron responsive elements (IREs). These IREs serve as the main control mechanism for iron metabolism in the cell via their interaction with the iron regulatory proteins (IRPs). Disruption of the IRE/IRP interaction could greatly affect iron metabolism. Here, we report that anthracyclines, a class of clinically useful chemotherapeutic drugs that includes doxorubicin and daunorubicin, specifically interact with the IREs of ferritin heavy and light chains. We characterized this interaction through UV melting, fluorescence quenching and drug-RNA footprinting. Results from footprinting experiments with wild-type and mutant IREs indicate that anthracyclines preferentially bind within the UG wobble pairs flanking an asymmetrically bulged C-residue, a conserved base that is essential for IRE-IRP interaction. Additionally, drug-RNA affinities (apparent K(d)s) in the high nanomolar range were calculated from fluorescence quenching experiments, while UV melting studies revealed shifts in melting temperature (DeltaT(m)) as large as 10 degrees C. This anthracycline-IRE interaction may contribute to the aberration of intracellular iron homeostasis that results from anthracycline exposure.     

Regulation of the 75-kDa subunit of mitochondrial complex I by iron

Iron homeostasis is tightly regulated, as cells work to conserve this essential but potentially toxic metal. The translation of many iron proteins is controlled by the binding of two cytoplasmic proteins, iron regulatory protein 1 and 2 (IRP1 and IRP2) to stem loop structures, known as iron-responsive elements (IREs), found in the untranslated regions of their mRNAs. In short, when iron is depleted, IRP1 or IRP2 bind IREs; this decreases the synthesis of proteins involved in iron storage and mitochondrial metabolism (e.g. ferritin and mitochondrial aconitase) and increases the synthesis of those involved in iron uptake (e.g. transferrin receptor). It is likely that more iron-containing proteins have IREs and that other IRPs may exist. One obvious place to search is in Complex I of the mitochondrial respiratory chain, which contains at least 6 iron-sulfur (Fe-S) subunits. Interestingly, in idiopathic Parkinson's disease, iron homeostasis is altered, and Complex I activity is diminished. These findings led us to investigate whether iron status affects the Fe-S subunits of Complex I. We found that the protein levels of the 75-kDa subunit of Complex I were modulated by levels of iron in the cell, whereas mRNA levels were minimally changed. Isolation of a clone of the 75-kDa Fe-S subunit with a more complete 5'-untranslated region sequence revealed a novel IRE-like stem loop sequence. RNA-protein gel shift assays demonstrated that a specific cytoplasmic protein bound the novel IRE and that the binding of the protein was affected by iron status. Western blot analysis and supershift assays showed that this cytosolic protein is neither IRP1 nor IRP2. In addition, ferritin IRE was able to compete for binding with this putative IRP. These results suggest that the 75-kDa Fe-S subunit of mitochondrial Complex I may be regulated by a novel IRE-IRP system.     

Overexpression of H ferritin and up-regulation of iron regulatory protein genes during differentiation of 3T3-L1 pre-adipocytes

The role of iron-dependent oxidative metabolism in protecting the oxidable substrates contained in mature adipocytes is still unclear. Because differentiation increases ferritin formation in several cell types, thereby leading to an accumulation of H-rich isoferritins, we investigated whether differentiation affects iron metabolism in 3T3-L1 pre-adipocytes. To this aim, we evaluated the expression of the genes coding for the H and L ferritin subunits and for cytoplasmic iron regulatory protein (IRP) during the differentiation of 3T3-L1 cells in adipocytes induced by the addition of isobutylmethylxanthine, insulin, and dexamethasone. Differentiation enhanced ferritin formation and caused overexpression of the H subunit, thus altering the H/L subunit ratio. Northern blot analysis showed increased levels of H subunit mRNA. A gel retardation assay of cytoplasmic extract from differentiated cells, using an iron-responsive element as a probe, revealed enhanced an RNA binding capacity of IRP1, which correlated with the increase of IRP1 mRNA. The observed correlation between differentiation and iron metabolism in adipocytes suggests that an accumulation of H-rich isoferritin may limit the toxicity of iron in adipose tissue, thus exerting an antioxidant function.     

Differential regulation of iron regulatory element-binding protein(s) in cell extracts of activated lymphocytes versus monocytes-macrophages.

The intracellular iron level exerts a negative feedback on transferrin receptor (TfR) expression in cells requiring iron for their proliferation, in contrast to the positive feedback observed in monocytes-macrophages. It has been suggested recently that modulation of TfR and ferritin synthesis by iron is mediated through a cytoplasmic protein(s) (iron regulatory element-binding protein(s) (IRE-BP)), which interacts with ferritin and TfR mRNA at the level of hairpin structures (IRE), thus leading to inhibition of transferrin mRNA degradation and repression of ferritin mRNA translation. In the present study we have evaluated in parallel the level of TfR expression, ferritin, and IRE-BP in cultures of: (i) circulating human lymphocytes stimulated to proliferate by phytohemagglutinin (PHA) and (ii) circulating human monocytes maturing in vitro to macrophages. The cells were grown in either standard or iron-supplemented culture. TfR and ferritin expression was evaluated at both the protein and mRNA level. IRE-BP activity was measured by gel retardation assay in the absence or presence of beta-mercaptoethanol (spontaneous or total IRE-BP activity, respectively). Spontaneous IRE-BP activity, already present at low level in quiescent T lymphocytes, shows a gradual and marked increase in PHA-stimulated T cells from day 1 of culture onward. This increase is directly and strictly correlated with the initiation and gradual rise of TfR expression, which is in turn associated with a decrease of ferritin content. Both the rise of TfR and spontaneous IRE-BP activity are completely inhibited in iron-supplemented T cell cultures. In contrast, the total IRE-BP level is similar in both quiescent and PHA-stimulated lymphocytes, grown in cultures supplemented or not with iron salts. Monocytes maturing in vitro to macrophages show a sharp increase of spontaneous and, to a lesser extent, total IRE-BP; the addition of iron moderately stimulates the spontaneous IRE-BP activity but not the total one. Here again, the rise of spontaneous IRE-BP from very low to high activity is strictly related to the parallel increase of TfR expression and, suprisingly, also with a very pronounced rise of ferritin expression observed at both the mRNA and protein level. It is noteworthy the effect of beta-mercaptoethanol is cell specific, i.e. the ratio of total versus spontaneous IRE-BP activity is different in activated lymphocytes and maturing monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)