Untranslated Region Of Ferritin mRNA Research Articles

Regulation of transferrin receptor-1 mRNA by the interplay between IRE-binding proteins and miR-7/miR-141 in the 3'-IRE stem-loops.

Intracellular iron is tightly regulated by coordinated expression of iron transport and storage genes, such as transferrin receptor-1 (TfR1) and ferritin. They are primarily regulated by iron through iron-induced dissociation of iron-regulatory proteins (IRPs) from iron-responsive elements (IREs) in the 3′-UTR (untranslated region) of TfR1 or 5′-UTR of ferritin mRNA, resulting in destabilization of TfR1 mRNA and release of ferritin translation block. Thus high iron decreases iron transport via TfR1 mRNA degradation and increases iron storage via ferritin translational up-regulation. However, the molecular mechanism of TfR1 mRNA destabilization in response to iron remains elusive. Here, we demonstrate that miR-7-5p and miR-141-3p target 3′-TfR1 IREs and down-regulate TfR1 mRNA and protein expression. Conversely, miR-7-5p and miR-141-3p antagomiRs partially but significantly blocked iron- or IRP knockdown-induced down-regulation of TfR1 mRNA, suggesting the interplay between these microRNAs and IRPs along with involvement of another uncharacterized mechanism in TfR1 mRNA degradation. Luciferase reporter assays using 3′-UTR TfR1 IRE mutants suggested that the IREs C and E are targets of miR-7-5p and miR-141-3p, respectively. Furthermore, miR-7 expression was inversely correlated with TfR1 mRNA in human pancreatic adenocarcinoma patient samples. These results suggest a role of microRNAs in the TfR1 regulation in the IRP–IRE system.

Divergent modulation of iron regulatory proteins and ferritin biosynthesis by hypoxia/reoxygenation in neurones and glial cells

Ferritin, the main iron storage protein, exerts a cytoprotective effect against the iron-catalyzed production of reactive oxygen species, but its role in brain injury caused by hypoxia/reoxygenation is unclear. Ferritin expression is regulated mainly at post-transcriptional level by iron regulatory proteins (IRP1 and IRP2) that bind specific RNA sequences (IREs) in the 5'untranslated region of ferritin mRNA. Here, we show that hypoxia decreases IRP1 binding activity in glial cells and enhances it in cortical neurons. These effects were reversed by reoxygenation in both cell types. In glial cells there was an early increase of ferritin synthesis during hypoxia and reoxygenation. Conversely, in cortical neurons, ferritin synthesis increased during the late phase of reoxygenation. Steady-state analysis of ferritin mRNA levels suggested that ferritin synthesis is regulated mainly post-transcriptionally by IRPs in glioma cells, both transcriptionally and post-transcriptionally in type-1 astrocytes, and mainly at transcriptional level in an IRP-independent way in neurons. The different regulation of ferritin expression may account for the different vulnerability of neurons and glial cells to the injury elicited by oxygen and glucose deprivation (OGD)/reoxygenation. The greater vulnerability of cortical neurons to hypoxia-reoxygenation was strongly attenuated by the exogenous administration of ferritin during OGD/reoxygenation, suggesting the possible cytoprotective role exerted by this iron-segregating protein.

Molecular cloning, expression and isolation of ferritins from two tick species— Ornithodoros moubata and Ixodes ricinus

Genes encoding ferritins were isolated and cloned from cDNA libraries of hard tick Ixodes ricinus and soft tick Ornithodoros moubata. Both tick ferritins are composed of 172 amino-acid residues and their calculated mass is 19,667.2 Da and 19,974.5 Da for I. ricinus and O. moubata, respectively . The sequences of both proteins are closely related to each other as well as to the ferritin from another tick species Dermacentor variabilis (>84% similarity) . The proteins contain the conserved motifs for ferroxidase center typical for heavy chains of vertebrate ferritins. The stem-loop structure of a putative iron responsive element was found in the 5′ untranslated region of ferritin mRNA of both ticks. Antibodies against fusion ferritin from O. moubata were raised in a rabbit and used to monitor the purification of a small amount of ferritins from both tick species. The authenticity of ferritin purified from O. moubata was confirmed by mass-fingerprinting analysis. In the native state, the tick ferritins are apparently larger (~500 kDa) than horse spleen ferritin (440 kDa). On SDS-PAGE tick ferritins migrate as a single band of about 21 kDa. These results suggest that tick ferritins are homo-oligomers of 24 identical subunits of heavy-chain type. The Northern blot analysis revealed that O. moubata ferritin mRNA level is likely not up-regulated after ingestion of a blood meal.

Nitrogen monoxide-mediated control of ferritin synthesis: implications for macrophage iron homeostasis.

Intracellular iron homeostasis is regulated posttranscriptionally by iron regulatory proteins 1 and 2 (IRP1 and IRP2). In the absence of iron in the labile pool, IRPs bind to specific nucleotide sequences called iron responsive elements (IREs), which are located in the 5' untranslated region of ferritin mRNA and the 3' untranslated region of transferrin receptor mRNA. IRP binding to the IREs suppresses ferritin translation and stabilizes transferrin receptor mRNA, whereas the opposite scenario develops in iron-replete cells. Binding of IRPs to the IREs is also affected by nitrogen monoxide (NO), but there are conflicting reports regarding the effect of NO on ferritin synthesis. In this study, we demonstrated that a short exposure of RAW 264.7 cells (a macrophage cell line) to the NO+ donor, sodium nitroprusside (SNP), resulted in a dramatic increase in ferritin synthesis. The SNP-mediated increase of ferritin synthesis could be blocked by MG132, an inhibitor of proteasome-dependent protein degradation, which also prevented the degradation of IRP2 caused by SNP treatment. Moreover, treatment of RAW 264.7 cells with IFN-gamma and lipopolysaccharide caused IRP2 degradation and stimulated ferritin synthesis, changes that could be prevented by specific inhibitors of inducible nitric oxide synthase. Furthermore, the SNP-mediated increase in ferritin synthesis was associated with a significant enhancement of iron incorporation into ferritin. These observations indicate that NO+-mediated modulation of IRP2 plays an important role in controlling ferritin synthesis and iron metabolism in murine macrophages.

Cellular iron metabolism

Iron is essential for oxidation-reduction catalysis and bioenergetics, but unless appropriately shielded, iron plays a key role in the formation of toxic oxygen radicals that can attack all biological molecules. Hence, specialized molecules for the acquisition, transport (transferrin), and storage (ferritin) of iron in a soluble nontoxic form have evolved. Delivery of iron to most cells, probably including those of the kidney, occurs following the binding of transferrin to transferrin receptors on the cell membrane. The transferrin-receptor complexes are then internalized by endocytosis, and iron is released from transferrin by a process involving endosomal acidification. Cellular iron storage and uptake are coordinately regulated post-transcriptionally by cytoplasmic factors, iron-regulatory proteins 1 and 2 (IRP-1 and IRP-2). Under conditions of limited iron supply, IRP binding to iron-responsive elements (present in 5' untranslated region of ferritin mRNA and 3' untranslated region of transferrin receptor mRNA) blocks ferritin mRNA translation and stabilizes transferrin receptor mRNA. The opposite scenario develops when iron in the transit pool is plentiful. Moreover, IRP activities/levels can be affected by various forms of "oxidative stress" and nitric oxide. The kidney also requires iron for metabolic processes, and it is likely that iron deficiency or excess can cause disturbed function of kidney cells. Transferrin receptors are not evenly distributed throughout the kidney, and there is a cortical-to-medullary gradient in heme biosynthesis, with greatest activity in the cortex and least in the medulla. This suggests that there are unique iron/heme metabolism features in some kidney cells, but the specific aspects of iron and heme metabolism in the kidney are yet to be explained.

Regulation of Ferritin Synthesis and Iron Regulatory Protein 1 by Oxygen in Mouse Peritoneal Macrophages

Ferritin is an intracellular iron storage protein whose synthesis is regulated post-transcriptionally by a mechanism that involves binding of cytoplasmic iron regulatory protein (IRP) to iron-responsive element (IRE) in the 5′ untranslated region of ferritin mRNA. In this study, we have shown that in mouse peritoneal macrophages, the synthesis of ferritin was enhanced and the IRE binding activity of IRP-1 was diminished when the oxygen tension was decreased. Iron is known to induce ferritin synthesis and even in the presence of a low concentration of iron, synthesis of ferritin was enhanced and the activity of IRP-1 was decreased under hypoxia. The enhanced synthesis of ferritin under hypoxia was abolished by the addition of O−2-generating agents but not H2O2. The decreased activity of IRP-1 under hypoxia was reversed by adding O−2-generating agents. These data suggest that O−2generated in the cell is involved in alterations of ferritin synthesis and the activity of IRP-1 by oxygen.

Hereditary Hyperferritinemia-Cataract Syndrome Caused by a 29-Base Pair Deletion in the Iron Responsive Element of Ferritin L-Subunit Gene

AbstractIron availability regulates ferritin synthesis posttranscriptionally by the interaction between iron-regulatory proteins (IRPs) and an iron responsive element (IRE), a stem-loop sequence located on the 5′ untranslated region of ferritin mRNA. IRPs recognize IREs as a sequence/structure motif, blocking ferritin translation. Recently, we and others independently described families with a combination of hyperferritinemia (serum L-ferritin ≥ 1,000 μg/L, without iron overload) and congenital bilateral cataract, transmitted as an autosomal-dominant trait. The molecular basis were two distinct point mutations in the highly conserved CAGUG(X) hexaloop of L-ferritin IRE on chromosome 19. A new three-generation family with a similar phenotype and a unique genotype is here reported. DNA amplification by polymerase chain reaction and sequence analysis showed a 29-base pair deletion in the L-ferritin IRE, involving the whole 5′ sequence essential to the base pairing of the IRE stem. This deletion is predicted to cause the disruption of IRE stem-loop secondary structure and the nearly complete abolition of the negative control of ferritin synthesis by IRE/IRP binding. Hereditary Hyperferritinemia-Cataract Syndrome (HHCS) appears as a new genetic disorder with a unique phenotype associated with at least four different mutations in the L-ferritin IRE. Hematologists should take into account HHCS in the differential diagnosis of unexplained hyperferritinemia.

Resistance to the Antitumor Agent Gallium Nitrate in Human Leukemic Cells Is Associated with Decreased Gallium/Iron Uptake, Increased Activity of Iron Regulatory Protein-1, and Decreased Ferritin Production

The mechanism of drug resistance to gallium nitrate is not known. Since gallium can be incorporated into ferritin, an iron storage protein that protects cells from iron toxicity, we investigated whether ferritin expression was altered in gallium-resistant (R) CCRF-CEM cells. We found that the ferritin content of R cells was decreased, while heavy chain ferritin mRNA levels and iron regulatory protein-1 (IRP-1) RNA binding activity were increased. IRP-1 protein levels were similar in gallium-sensitive (S) and R cells, indicating that R cells contain a greater proportion of IRP-1 in a high affinity mRNA binding state. 59Fe uptake and transferrin receptor expression were decreased in R cells. In both S and R cells, gallium inhibited cellular 59Fe uptake, increased ferritin mRNA and protein, and decreased IRP-1 binding activity. Gallium uptake by R cells was markedly diminished; however, the sensitivity of R cells to gallium could be restored by increasing their uptake of gallium with excess transferrin. Our results suggest that R cells have developed resistance to gallium by down-regulating their uptake of gallium. In parallel, iron uptake by R cells is also decreased, leading to changes in iron homeostasis. Furthermore, since gallium has divergent effects on iron uptake and ferritin synthesis, its action may also include a direct effect on ferritin mRNA induction and IRP-1 activity.

Regulation of Ferritin Synthesis in Macrophages by Oxygen and Sulfhydryl-Reactive Agent

Ferritin synthesis is known to be regulated translationally by specific mRNA-protein interactions between an iron-responsive element (IRE) localized in the 5′ untranslated region of ferritin mRNA and IRE-binding protein (IRE-BP). Binding of IRE-BP to IRE depresses its translation. In the present study, we demonstrated that ferritin synthesis in macrophages is strongly induced under hypoxic conditions by diethylmaleate, a sulfhydryl-reactive agent. The induction by diethylmaleate decreased as the oxygen tension rose. O − 2 is involved in this oxygen effect, because the induction was prevented when O − 2-generating agents were present. Actinomycin D did not inhibit the ferritin synthesis induced by diethylmaleate under hypoxi. These results suggest that O − 2 is ivolved in post-transcriptional regulation of ferritin sythesis.

The iron regulatory region of ferritin mRNA is also a positive control element for iron-independent translation.

The iron regulatory element (IRE) in the 5'-untranslated region of ferritin mRNA interacts with a specific regulator protein (P-90, IRE-BP, or FRP) to block translation. High cellular iron changes the IRE/P-90 interaction to relax the translational block and allow polyribosome formation. We now show that the IRE and base-paired flanking regions also enhance translation in the absence of P-90, explaining the high translational efficiency of deregulated ferritin mRNA observed previously. The effect of the IRE on translational efficiency was examined by comparing four sets of mRNAs: (1) +/- IRE in animal (frog) ferritin, regulated translationally by iron in vivo; (2) +/- animal IRE fused with plant (soybean) ferritin, regulated transcriptionally by iron in vivo; (3) repositioned IRE in animal ferritin; (4) mutated IRE in animal ferritin with G16A substitution, which decreases P-90 binding (negative control). The IRE region increased translational efficiency of both the animal ferritin and the heterologous IRE/soybean ferritin fusion mRNAs; the effect was observed in cell-free translation systems from either plants (wheat germ) or animals (rabbit reticulocyte). Repositioning the IRE further from the 5' cap eliminated positive control of translation. The single base mutation had no effect, indicating that positive and negative translational control involves different sections of the IRE region. Thus, the IRE region in ferritin mRNA encodes both positive translational control and, when combined with the regulator protein P-90, negative translational control.

Regulation of ferritin and transferrin receptor mRNAs.

Iron regulates the synthesis of two proteins critical for iron metabolism, ferritin and the transferrin receptor, through novel mRNA/protein interactions. The mRNA regulatory sequence (iron-responsive element (IRE)) occurs in the 5'-untranslated region of all ferritin mRNAs and is repeated as five variations in the 3'-untranslated region of transferrin receptor mRNA. When iron is in excess, ferritin synthesis and iron storage increase. At the same time, transferrin receptor synthesis and iron uptake decrease. Location of the common IRE regulatory sequence in different noncoding regions of the two mRNAs may explain how iron can have opposite metabolic effects; when the IRE is in the 5'-untranslated region of ferritin mRNA, translation is enhanced by excess iron whereas the presence of the IREs in the 3'-untranslated region of the transferrin receptor mRNA leads to iron-dependent degradation. How and where iron actually acts is not yet known. A soluble 90-kDa regulatory protein which has been recently purified to homogeneity from liver and red cells specifically blocks translation of ferritin mRNA and binds IRE sequences but does not appear to be an iron-binding protein. The protein is the first specific eukaryotic mRNA regulator identified and confirms predictions 20 years old. Concerted regulation by iron of ferritin and transferrin receptor mRNAs may also define a more general strategy for using common mRNA sequences to coordinate the synthesis of metabolically related proteins.

Requirements for the Translational Repression of Ferritin Transcripts in Wheat Germ Extracts by a 90-kDa Protein from Rabbit Liver

A specific repressor of ferritin mRNA translation originally detected in rabbit reticulocyte lysates has now been purified to homogeneity from rabbit liver, as described in a companion paper (Walden, W. E., Patino, M. M., and Gaffield, L. (1989) J. Biol. Chem. 264, 13765-13769). This repressor is a 90-kDa protein that binds to a sequence in the 5'-untranslated region of ferritin mRNA. In this communication we describe the molecular features of a ferritin light chain transcript that are required for the repression of its translation by this protein. Addition of small amounts of the 90-kDa ferritin repressor protein (FRP) completely inhibited translation of ferritin transcripts in a wheat germ system. This repression did not require mRNA sequences contained in the 3'-untranslated region or in the majority of the ferritin coding region. In contrast, the first 130 nucleotides of the 5'-untranslated region, which contains the 28-nucleotide "iron responsive element" (IRE), was required for the repressive effect. Moreover, repression of full length transcripts was relieved by addition of a molar excess of a 92-nucleotide transcript of the 5'-untranslated region which also contained the IRE. These results suggest that no sequence information other than a portion of the 5'-untranslated region containing the IRE sequence is required for action of the 90-kDa FRP. In addition, a quantitative comparison of the repression of transcript with that of poly(A+) RNAs indicates that no post-transcriptional modifications of the latter (other than cap addition) are involved in the action of the 90-kDa FRP.

Multiple red cell ferritin mRNAs, which code for an abundant protein in the embryonic cell type, analyzed by cDNA sequence and by primer extension of the 5'-untranslated regions.

Ferritin maintains iron in a bioavailable, nontoxic form for vertebrates and invertebrates, higher plants, fungi, and bacteria; the protein is formed from two classes of subunits (H and L) in ratios which vary in different cell types. Ferritin may be an abundant, differentiation-specific protein or a "housekeeping" protein. The red cells of embryos are specialized for iron storage and have abundant ferritin; iron regulates the synthesis of ferritin in such cells translationally by recruitment of stored, ferritin mRNA and by translational competition. To characterize mRNA regulated in such a manner, we prepared cDNA from reticulocytes of bullfrog tadpoles, a readily available source of embryonic red cells; moreover, no protein sequence information was available for nonmammalian ferritin. An almost full-length (817 base pairs) cDNA (pJD5F12) was isolated and sequenced, the 5' end was analyzed by primer extension, and the cloned DNA was used as a hybridization probe. We have shown that ferritin mRNA is stored in the cytoplasm and that the 5' end of the mRNA is heterogeneous. The 5'-untranslated region of ferritin mRNA consisted of 143 nucleotides in the major (65%) species and 146 or 152 in the minor species (approximately 17% each). (Heterogeneity is characteristic of some other abundant mRNAs, e.g. globin, which is also translationally regulated.) Since excess iron had no detectable effect on the heterogeneity of the 5' end of ferritin mRNA, the feature is more likely associated with mRNA abundance and/or cell specialization than translational control. In the bullfrog, as in humans and rats, ferritin is encoded by multiple genomic sequences (four to eight) which specify proteins of considerable homology. For example, 75 of the 81 amino acids present in all mammalian ferritins sequenced are also present in the frog; the overall homology between frogs and humans or rats is 59-66%. Ferritin H and L subunits in humans are distinct (overall homology 56%) and appear to have diverged from a common precursor relatively recently. In contrast, ferritin H and L subunits have high homology in tadpole red cells, determined by hybrid select translation, which suggests that bullfrog red cell ferritin may be close to the primordial sequence.