Inactivation of both RNA binding and aconitase activities of iron regulatory protein-1 by quinone-induced oxidative stress

Iron regulatory protein-1 (IRP-1) controls the expression of several mRNAs by binding to iron-responsive elements (IREs) in their untranslated regions. In iron-replete cells, a 4Fe-4S cluster converts IRP-1 to cytoplasmic aconitase. IRE binding activity is restored by cluster loss in response to iron starvation, NO, or extracellular H2O2. Here, we study the effects of intracellular quinone-induced oxidative stress on IRP-1. Treatment of murine B6 fibroblasts with menadione sodium bisulfite (MSB), a redox cycling drug, causes a modest activation of IRP-1 to bind to IREs within 15-30 min. However, IRE binding drops to basal levels within 60 min. Surprisingly, a remarkable loss of both IRE binding and aconitase activities of IRP-1 follows treatment with MSB for 1-2 h. These effects do not result from alterations in IRP-1 half-life, can be antagonized by the antioxidant N-acetylcysteine, and regulate IRE-containing mRNAs; the capacity of iron-starved MSB-treated cells to increase transferrin receptor mRNA levels is inhibited, and MSB increases the translation of a human growth hormone indicator mRNA bearing an IRE in its 5'-untranslated region. Nonetheless, MSB inhibits ferritin synthesis. Thus, menadione-induced oxidative stress leads to post-translational inactivation of both genetic and enzymatic functions of IRP-1 by a mechanism that lies beyond the "classical" Fe-S cluster switch and exerts multiple effects on cellular iron metabolism.     

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.     

Recycling of RNA binding iron regulatory protein 1 into an aconitase after nitric oxide removal depends on mitochondrial ATP

Iron regulatory proteins (IRPs) control iron metabolism by specifically interacting with iron-responsive elements (IREs) on mRNAs. Nitric oxide (NO) converts IRP-1 from a [4Fe-4S] aconitase to a trans-regulatory protein through Fe-S cluster disassembly. Here, we have focused on the fate of IRE binding IRP1 from murine macrophages when NO flux stops. We show that virtually all IRP-1 molecules from NO-producing cells dissociated from IRE and recovered aconitase activity after re-assembling a [4Fe-4S] cluster in vitro. The reverse change in IRP-1 activities also occurred in intact cells no longer exposed to NO and did not require de novo protein synthesis. Likewise, inhibition of mitochondrial aconitase via NO-induced Fe-S cluster disassembly was also reversed independently of protein translation after NO removal. Our results provide the first evidence of Fe-S cluster repair of NO-modified aconitases in mammalian cells. Moreover, we show that reverse change in IRP-1 activities and repair of mitochondrial aconitase activity depended on energized mitochondria. Finally, we demonstrate that IRP-1 activation by NO was accompanied by both a drastic decrease in ferritin levels and an increase in transferrin receptor mRNA levels. However, although ferritin expression was recovered upon IRP-1-IRE dissociation, expression of transferrin receptor mRNA continued to rise for several hours after stopping NO flux.     

Ligand-induced structural alterations in human iron regulatory protein-1 revealed by protein footprinting.

Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.     

Multiple determinants within iron-responsive elements dictate iron regulatory protein binding and regulatory hierarchy

Iron regulatory proteins (IRPs) are iron-regulated RNA binding proteins that, along with iron-responsive elements (IREs), control the translation of a diverse set of mRNA with 5′ IRE. Dysregulation of IRP action causes disease with etiology that may reflect differential control of IRE-containing mRNA. IREs are defined by a conserved stem–loop structure including a midstem bulge at C8 and a terminal CAGUGH sequence that forms an AGU pseudo-triloop and N19 bulge. C8 and the pseudo-triloop nucleotides make the majority of the 22 identified bonds with IRP1. We show that IRP1 binds 5′ IREs in a hierarchy extending over a ninefold range of affinities that encompasses changes in IRE binding affinity observed with human L-ferritin IRE mutants. The limits of this IRE binding hierarchy are predicted to arise due to small differences in binding energy (e.g., equivalent to one H-bond). We demonstrate that multiple regions of the IRE stem not predicted to contact IRP1 help establish the binding hierarchy with the sequence and structure of the C8 region displaying a major role. In contrast, base-pairing and stacking in the upper stem region proximal to the terminal loop had a minor role. Unexpectedly, an N20 bulge compensated for the lack of an N19 bulge, suggesting the existence of novel IREs. Taken together, we suggest that a regulatory binding hierarchy is established through the impact of the IRE stem on the strength, not the number, of bonds between C8 or pseudo-triloop nucleotides and IRP1 or through their impact on an induced fit mechanism of binding.     

Excess capacity of the iron regulatory protein system

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. Stable cell lines were created in which either IRP1, IRP2, or both were knocked down. Knockdown of IRP1 decreased IRE binding activity but did not affect ferritin H and transferrin receptor 1 (TfR1) expression, whereas knockdown of IRP2 marginally affected IRE binding activity but caused an increase in ferritin H and a decrease in TfR1. Knockdown of both IRPs resulted in a greater reduction of IRE binding activity and more severe perturbation of ferritin H and TfR1 expression compared with single IRP knockdown. Even though the knockdown of IRP-1, IRP-2, or both was efficient, resulting in nondetectable protein and under 5% of wild type levels of mRNA, all stable knockdowns retained an ability to modulate ferritin H and TfR1 appropriately in response to iron challenge. However, further knockdown of IRPs accomplished by transient transfection of small interfering RNA in stable knockdown cells completely abolished the response of ferritin H and TfR1 to iron challenge, demonstrating an extensive excess capacity of the IRP system.     

Multiple,conserved iron-responsive elements in the 3'-untranslated region of transferrin receptor mRNA enhance binding of iron regulatory protein 2

Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3'-untranslated region (TfR-3'-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3'-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen)(2) binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3'-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.     

Structure of the 5'' untranslated regulatory region of ferritin mRNA studied in solution.

Ferritin mRNAs are the first eukaryotic mRNAs for which a conserved, translational regulatory sequence has been identified. The sequence of twenty-eight nucleotides, called the IRE (iron regulatory element), is found in the 5'-noncoding region and is required for enhanced translation of ferritin mRNA by excess cellular iron; regulation occurs at initiation. The prediction of secondary structure in the IRE is a hairpin loop. We now report an analysis of the IRE structure in solution studied in natural ferritin mRNAs [H and H'(M) subunits] by primer extension, after modification or cleavage by dimethyl sulfate, RNAases T1 and V1, and the chemical nuclease 1, 10-phenanthroline-copper (OPCu) which cleaves single-stranded and bulged regions of RNA. Overall, the structure in solution of the ferritin mRNA regulatory region is a hairpin loop, with magnesium-sensitive features, in which half the stem is provided by the IRE and half by flanking regions; only secondary structure is conserved in the flanking regions. Predicted bulges or internal loops along the stem were clearly detected by OPCu but were missed by the more bulky probe RNAase T1, indicating the efficacy of OPCu in probing subtle features of RNA structure. Magnesium-dependent deviations from the predicted structure were observed in the stem between the hairpin loop and the bulge at C6. The location of the IRE in relation to the initiator AUG or the cap is variable in different ferritin mRNAs. However, the number of nucleotides in the base-paired flanking regions of known ferritin mRNAs is proportional to the distance of the IRE from the cap and places the secondary/tertiary structure 8-10 nucleotides from the cap where interference with initiation is likely.     

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.     

The importance of a single G in the hairpin loop of the iron responsive element (IRE) in ferritin mRNA for structure: an NMR spectroscopy study.

Noncoding sequences regulate the function of mRNA and DNA. In animal mRNAs, iron responsive elements (IREs) regulate the synthesis of proteins for iron storage, uptake and red cell heme formation. Folding of the IRE was indicated previously by reactivity with chemical and enzymatic probes. 1H- and 31P-NMR spectra now confirm the IRE folding; an atypical 31P-spectrum, differential accessibility of imino protons to solvents, multiple long-range NOEs and heat stable subdomains were observed. Biphasic hyperchromic transitions occurred (52 and 73 degrees C). A G-C base pair occurs in the hairpin loop (HL) (based on dimethylsulfate, RNAse T1 previously used, and changes in NMR imino proton resonances typical of G-C base pairs after G/A substitution). Mutation of the hairpin loop also decreased temperature stability and changed the 31P-NMR spectrum; regulation and protein (IRP) binding were previously shown to change. Alteration of IRE structure shown by NMR spectroscopy, occurred at temperatures used in studies of IRE function, explaining loss of IRP binding. The effect of the HL mutation on the IRE emphasizes the importance of HL structure in other mRNAs, viral RNAs (e.g. HIV-TAR), and ribozymes.     

Modulation of iron regulatory protein-1 by various metals.

Iron regulatory protein-1 (IRP-1) is known as a cytosolic aconitase and a central regulator of iron (Fe) homeostasis. IRP-1 regulates the expression of Fe metabolism-related proteins by interacting with the Fe-responsive element (IRE) in the untranslated regions of mRNAs of these proteins. However, it is less known whether IRP-1 modulates various non-Fe metals. In the present study, we showed that treatment of homogenously purified IRP-1 with non-Fe metals decreased the affinity to IRE in RNA band shift assays and increased aconitase activity. Non-Fe metals also inhibited (55)Fe incorporation into the fourth labile position of the Fe-S cluster of IRP-1. In PLC hepatoma cells, metal loading inactivated binding activity and activated enzyme activity. It also suppressed transferrin receptor mRNA expression in the cells. These results suggest that various non-Fe metals modulate IRP-1 by conversion of the 3Fe-4S apo-form to a [1 non-Fe metal + 3Fe]-4Fe holo-form.     

Evolution of the iron-responsive element

An RNA hairpin structure referred to as the iron-responsive element (IRE) and iron regulatory proteins (IRPs) are key players in the control of iron metabolism in animal cells. They regulate translation initiation or mRNA stability, and the IRE is found in a variety of mRNAs, such as those encoding ferritin, transferrin receptor (Tfr), erythroid aminolevulinic acid synthase (eALAS), mitochondrial aconitase (mACO), ferroportin, and divalent metal transporter 1 (DMT1). We have studied the evolution of the IRE by considering all mRNAs previously known to be associated with this structure and by computationally examining its occurrence in a large variety of eukaryotic organisms. More than 100 novel sequences together with approximately 50 IREs that were previously reported resulted in a comprehensive view of the phylogenetic distribution of this element. A comparison of the different mRNAs shows that the IREs of eALAS and mACO are found in chordates, those of ferroportin and Tfr1 are found in vertebrates, and the IRE of DMT1 is confined to mammals. In contrast, the IRE of ferritin occurs in a majority of metazoa including lower metazoa such as sponges and Nematostella (sea anemone). These findings suggest that the ferritin IRE represents the ancestral version of this type of translational control and that during the evolution of higher animals the IRE structure was adopted by other genes. On the basis of primary sequence comparison between different organisms, we suggest that some of these IREs developed by "convergent evolution" through stepwise changes in sequence, rather than by recombination events.     

Selective Translational Control of the Alzheimer Amyloid Precursor Protein Transcript by Iron Regulatory Protein-1

Iron influx increases the translation of the Alzheimer amyloid precursor protein (APP) via an iron-responsive element (IRE) RNA stem loop in its 5′-untranslated region. Equal modulated interaction of the iron regulatory proteins (IRP1 and IRP2) with canonical IREs controls iron-dependent translation of the ferritin subunits. However, our immunoprecipitation RT-PCR and RNA binding experiments demonstrated that IRP1, but not IRP2, selectively bound the APP IRE in human neural cells. This selective IRP1 interaction pattern was evident in human brain and blood tissue from normal and Alzheimer disease patients. We computer-predicted an optimal novel RNA stem loop structure for the human, rhesus monkey, and mouse APP IREs with reference to the canonical ferritin IREs but also the IREs encoded by erythroid heme biosynthetic aminolevulinate synthase and Hif-2α mRNAs, which preferentially bind IRP1. Selective 2′-hydroxyl acylation analyzed by primer extension analysis was consistent with a 13-base single-stranded terminal loop and a conserved GC-rich stem. Biotinylated RNA probes deleted of the conserved CAGA motif in the terminal loop did not bind to IRP1 relative to wild type probes and could no longer base pair to form a predicted AGA triloop. An AGU pseudo-triloop is key for IRP1 binding to the canonical ferritin IREs. RNA probes encoding the APP IRE stem loop exhibited the same high affinity binding to rhIRP1 as occurs for the H-ferritin IRE (35 pm). Intracellular iron chelation increased binding of IRP1 to the APP IRE, decreasing intracellular APP expression in SH-SY5Y cells. Functionally, shRNA knockdown of IRP1 caused increased expression of neural APP consistent with IRP1-APP IRE-driven translation.