Conservation in the Iron Responsive Element Family

Iron responsive elements (IREs) are mRNA stem-loop targets for translational control by the two iron regulatory proteins IRP1 and IRP2. They are found in the untranslated regions (UTRs) of genes that code for proteins involved in iron metabolism. There are ten “classic” IRE types that define the conserved secondary and tertiary structure elements necessary for proper IRP binding, and there are 83 published “IRE-like” sequences, most of which depart from the established IRE model. Here are structurally-guided discussions regarding the essential features of an IRE and what is important for IRE family membership.

Iron-sulfur cluster deficiency can be sensed by IRP2 and regulates iron homeostasis and sensitivity to ferroptosis independent of IRP1 and FBXL5

Intracellular iron levels are strictly regulated to support homeostasis and avoid iron-mediated ROS production. Loss of iron-sulfur cluster (ISC) synthesis can increase iron loading and promote cell death by ferroptosis. Iron-responsive element-binding proteins IRP1 and IRP2 posttranscriptionally regulate iron homeostasis. IRP1 binding to target mRNAs is competitively regulated by ISC occupancy. However, IRP2 is principally thought to be regulated at the protein level via E3 ubiquitin ligase FBXL5–mediated degradation. Here, we show that ISC synthesis suppression can activate IRP2 and promote ferroptosis sensitivity via a previously unidentified mechanism. At tissue-level O2 concentrations, ISC deficiency enhances IRP2 binding to target mRNAs independent of IRP1, FBXL5, and changes in IRP2 protein level. Deletion of both IRP1 and IRP2 abolishes the iron-starvation response, preventing its activation by ISC synthesis inhibition. These findings will inform strategies to manipulate ferroptosis sensitivity and help illuminate the mechanism underlying ISC biosynthesis disorders, such as Friedreich’s ataxia.

Profilin2 Is Controlled By the Iron Regulatory Proteins and Modulates Iron Homeostasis

Objective: The IRPs/IRE regulatory network plays a central role in the control of cellular iron homeostasis. Using a high throughput approach, we have previously identified novel IRP1 and IRP2 interacting mRNAs. Among the identified mRNAs, we studied more in depth Profilin2 (Pfn2), a protein involved in endocytosis and neurotransmitters release. The aim of this work is to characterize Pfn2 as a novel IRPs target mRNA and study its role in iron homeostasis. Materials and Methods: Mouse and human Pfn2 mRNAs were tested by non-radioactive competitive electrophoretic mobility shift assays (EMSA) for the binding to IRP1 and IRP2. To test the responsiveness of Pfn2 to IRP activity, Pfn2 mRNA levels were analyzed in mice with intestinal IRP1 and IRP2 deficiency. The labile iron pool (LIP) was measured in HeLa and Hepa1-6 cell lines with transient or stable overexpression of Pfn2. Tissues derived from Pfn2 knock-out mice were analyzed for iron content, measured by atomic absorption or colorimetric assay, and for mRNA and protein levels of iron-related genes. Results: Combination of EMSA experiments and bioinformatic analyses allowed the identification of a novel and conserved 3’UTR iron responsive element in Pfn2 mRNA with an atypical hexanucleotide apical loop (AAGUGG). Pfn2 mRNA levels were significantly reduced (~20-25%) in duodenal samples from mice with IRP1 and IRP2 intestinal specific ablation, suggesting that IRPs exert a positive effect on Pfn2 mRNA expression in vivo. Overexpression of Pfn2 cDNA in HeLa and Hepa1-6 cells reduces LIP levels compared to control cells. Finally, analysis of Pfn2 KO mice showed iron accumulation in discrete areas of the brain (olfactory bulb, hippocampus and midbrain) together with an hepatic iron deficiency with ferritin reduction. Conclusion: Our results indicate that Pfn2 is controlled by the IRP regulatory system in vivo and that Pfn2 modulates iron homeostasis in cell lines and mice. Funding: Work supported by grant SAF2012-40106 from Spanish Secretary of Research, Development and Innovation (MINECO) and grant CIVP16A1857 “Ayudas a proyectos de Investigación en Ciéncias de la Vida - Fundación Ramón Areces” to M.S. M.S. held a research contract under the Ramón y Cajal program from the Spanish Ministry of Science and Innovation (RYC-2008-02352). Disclosures No relevant conflicts of interest to declare.

Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins

Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.

A Closer Look at Cellular Iron Metabolism in IRP2 Deficient Erythroblasts.

Developing red blood cells are the major consumers of body iron which is indispensable for the enormous production of heme for hemoglobin synthesis. The uptake of iron occurs via binding of iron-loaded transferrin to its cognate receptor (TfR). Thereafter the iron is shuttled to the mitochondria where it is incorporated into protoporphyrin IX to form heme. Excess iron is enclosed within the iron storage protein ferritin. Coordinated control between iron uptake and storage is mainly achieved by the post-transcriptional regulation of TfR1 and ferritin synthesis by the iron regulatory proteins IRP1 and IRP2. Recently, two groups independently created mice lacking either IRP1 or IRP2 and showed that only IRP2 deficient mice developed microcytic hypochromic anemia. Both groups observed a reduction in TfR1 protein expression levels in the developing red blood cells of IRP2 knockout animals and suggested that the decrease in receptor levels is responsible for the development of anemia. For a more detailed analysis of how the loss of IRP2 expression influences iron metabolism and hemoglobinization during terminal erythroid differentiation, we isolated CFU-E-like erythroid cells from mouse fetal liver of wild type, IRP1 and IRP2 knock out animals. In vitro cultivation of these primary erythroid cells and their synchronous induction for differentiation allowed us to study their cellular iron metabolism at different time points. We analyzed the extent of hemoglobinization and cell size as well as the expression of ferritin and TfR1 during various stages of erythroid differentiation in IRP1, IRP2 and wild type cells. In agreement with the published phenotype of microcytic hypochromic anemia, only erythroblasts lacking IRP2 exhibited a reduction in hemoglobinization and showed a significant increase in ferritin protein levels before and after induction of differentiation. In contrast, TfR1 protein expression levels on the cell surface were significantly decreased in IRP2 deficient cells until 24h of differentiation, but converged with those of wild type cells at 48h of differentiation at the time point at which hemoglobinization is fully in progress. Moreover, measurement of 59Fe uptake and its cellular distribution showed that there is significantly more 59Fe located in cytosolic ferritin of IRP2 knock out cells at all time points compared to their wild type counterpart. In summary, these results suggest that not only the reduced expression of TfR1, but also the up-regulation of ferritin, play important roles in the development of anemic phenotype in IRP2 knock out mice. This work was supported by the Canadian Institutes of Health Research and the Canadian Blood Services.

Low Cytosolic Non-Heme Iron Levels in Erythroid Cells Prevent IRP2-Mediated Ferritin Upregulation during Differentiation.

Erythroid cells are the major consumers of iron in the human body. Differentiating erythroid cells shuttle the metal with very high efficiency towards mitochondria for the formation of heme. To satisfy their high iron needs, developing red blood cells (RBC) have to sustain high expression of transferrin receptor 1 (TfR) despite increasing cellular iron concentration. Moreover, synthesis of ferritin must not be activated by incoming iron, since this would represent a counterproductive storage during the phase of high iron demand. Recently we have demonstrated that during terminal differentiation primary erythroid cells satisfy their exceptionally high requirements for iron by switching to a mode where the post-transcriptional, iron-dependent regulatory system, formed by iron responsive proteins (IRP1 and IRP2) and iron responsive elements (IREs), seems to sense a low-iron state. This occurs despite a massive net increase of iron import into the cell (Schranzhofer et al., Blood107:4159, 2006). To examine the hypothesis that erythroid cells have low non-heme iron levels in their cytosol, we experimentally increased the cytosolic iron pool by either inhibiting heme biosynthesis or overloading cells with iron. Both block of heme synthesis by either succinylacetone or isonicotinic acid hydrazide (INH) or administration of ferric ammonium citrate, resulted in a clear increase in ferritin levels. This increase was directly proportional to the increase in the cellular concentration of non-heme-iron. Moreover, the effect of INH, the inhibitor of 5-aminolevulinic acid (ALA) synthase, could be reversed by the addition of ALA. Strikingly, increases in ferritin expression upon perturbation of cellular iron homeostasis strongly correlated with the loss of IRE-binding activity of IRP2 but not IRP1, as determined by mobility shift assays. This suggests that IRP2 is the major regulator of ferritin expression in erythroid cells. To further elaborate on this observation, we cultured primary erythroblasts derived from IRP1−/− and IRP2−/− mice (kindly provided by Drs. M. Hentze and B. Galy). In agreement with the published phenotype of microcytic hypochromic anemia, only erythroblasts lacking IRP2 exhibited a reduction in hemoglobinization. Moreover, only IRP2−/− cells showed a significant increase in ferritin expression, whereas developing RBC lacking IRP1 had levels of ferritin protein equal to wild type cells. We conclude that in erythroid cells efficient shuttling of incoming iron towards mitochondria and its prompt use for heme formation is important to keep the cytosol in an iron-deprived state and consequently ferritin protein levels low. This translational repression seems to be mainly achieved by IRP2. Together with the observation that surface expression of TfR was reduced in IRP2−/− erythroblasts during self renewal but not during terminal differentiation, our results suggest that not only down-regulation of TfR, but also up-regulation of ferritin may be a major factor for the anemic phenotype observed in IRP2−/− mice.

Modulation of IRP Binding Activity by Oxygen in Primary Erythroid Cells.

Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are cytoplasmic RNA-binding proteins that target specific stem-loop RNA structures known as iron responsive elements (IRE). Binding of IRPs to IREs inhibits translation of ferritin mRNA and stabilizes transferrin receptor (TfR) mRNA. Various factors have been reported to regulate binding activity of IRPs, such as iron, phosphorylation, nitric oxide and hypoxia. While there is a consistent agreement on the negative effect of iron on the interaction between IRPs and IREs, reports regarding the influence of hypoxia on the IRE-binding activity of IRPs vary in a species and cell specific manner. It was the aim of this work to study the effect of hypoxic (3% oxygen) and normoxic (20% oxygen) conditions on IRP binding activity in primary erythroid cells. The cells were induced for differentiation and incubated under physiological, low (Desferrioxamine) and high (ferric ammonium citrate) iron conditions. Binding activity of IRPs and protein levels of ferritin and TfR as well as cell proliferation and differentiation parameters were determined to analyze the regulation of iron metabolism during terminal differentiation. The data show, that in developing red blood cells binding activities of IRP1 and IRP2 are reduced at 3% oxygen. This reduction correlates with increased ferritin protein levels and decreased TfR protein levels. Moreover, incubation under hypoxia strongly decreased cell expansion and reduces hemoglobinization. These results suggest that terminal erythroid differentiation in the bone marrow might occur under normoxic rather than hypoxic conditions.