Differences in the RNA binding sites of iron regulatory proteins and potential target diversity (original) (raw)
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Requirements for iron-regulated degradation of the RNA binding protein, iron regulatory protein 2
The EMBO Journal, 1995
Iron regulatory proteins (IRPs) regulate the expression of genes involved in iron metabolism whose transcripts contain RNA stem-loop motifs known as ironresponsive elements (IREs). When iron concentrations are low, IRPs bind to IREs in the 5' untranslated region (UTR) of transcripts where they repress translation, or the 3' UTR of transcripts where they inhibit degradation. The RNA binding activities of the homologous proteins IRP1 and IRP2 are both regulated posttranslationally. The binding activity of IRP2 is regulated by the degradation of the protein when cells are iron-replete. Here, we demonstrate that a 73 amino acid sequence that corresponds to a unique exon in IRP2 contains a sequence required for rapid degradation in iron-replete cells. The deletion of this sequence eliminates the rapid turnover of IRP2, whereas the transfer of this sequence to the corresponding position in the homologous protein IRP1 confers the capacity for iron-dependent degradation upon IRP1. Site-directed mutagenesis has demonstrated that specific cysteines within the IRP2 exon are required for iron-dependent degradation. The degradation of IRP2 appears to be mediated by the proteasome in iron-replete cells. When degradation is prevented, the RNA binding activity of IRP2 is not regulated by iron concentration. Thus, degradation is required for the regulation of the RNA binding activity of IRP2.
Molecular and cellular biology, 1989
The 5' untranslated region of the ferritin heavy-chain mRNA contains a stem-loop structure called an iron-responsive element (IRE), that is solely responsible for the iron-mediated control of ferritin translation. A 90-kilodalton protein, called the IRE binding protein (IRE-BP), binds to the IRE and acts as a translational repressor. IREs also explain the iron-dependent control of the degradation of the mRNA encoding the transferrin receptor. Scatchard analysis reveals that the IRE-BP exists in two states, each of which is able to specifically interact with the IRE. The higher-affinity state has a Kd of 10 to 30 pM, and the lower affinity state has a Kd of 2 to 5 nM. The reversible oxidation or reduction of a sulfhydryl is critical to this switching, and the reduced form is of the higher affinity while the oxidized form is of lower affinity. The in vivo rate of ferritin synthesis is correlated with the abundance of the high-affinity form of the IRE-BP. In lysates of cells treate...
Proceedings of the National Academy of Sciences, 1990
Iron-responsive elements (IREs) are stemloop structures found in the mRNAs encoding ferritin and the transferrin receptor. These elements participate in the ironinduced regulation of the translation offerritin and the stability of the transferrin receptor mRNA. Regulation in both instances is mediated by binding of a cytosolic protein to the IREs. High-affinity binding is seen when cells are starved of iron and results in repression of ferritin translation and inhibition of transferrin receptor mRNA degradation. The IRE-binding protein (IRE-BP) has been identified as an-90-kDa protein that has been purified by both affinity and conventional chromatography. In this report we use RNA affinity chromatography and two-dimensional gel electrophoresis to isolate the IRE-BP for protein sequencing. A degenerate oligonucleotide probe derived from a single peptide sequence was used to isolate a cDNA clone that encodes a protein containing 13 other sequenced peptides obtained from the IRE-BP. Consistent with previous characterization of the IRE-BP, the cDNA encodes a protein of 87 kDa with a slightly acidic pl, and the corresponding mRNA of-3.6 kilobases is found in a variety of cell types.
Nucleic Acids Research, 1993
To assess the influence of RNA sequence/structure on the interaction RNAs with the iron-responsive element binding protein (IRE-BP), twenty eight altered RNAs were tested as competitors for an RNA corresponding to the ferritin H chain IRE. All changes in the loop of the predicted IRE hairpin and in the unpaired cytosine residue characteristically found in IRE stems significantly decreased the apparent affinity of the RNA for the IRE-BP. Similarly, alteration in the spacing and/or orientation of the loop and the unpaired cytosine of the stem by either increasing or decreasing the number of base pairs separating them significantly reduced efficacy as a competitor. It is inferred that the IRE-BP forms multiple contacts with its cognate RNA, and that these contacts, acting in concert, provide the basis for the high affinity of this interaction.
Identification of RNA-binding surfaces in iron regulatory protein-1
The EMBO Journal, 1999
Post-transcriptional regulation of mRNA translation and stability in iron metabolism involves the interaction between the transacting cytoplasmic iron regulatory proteins (IRP-1 and IRP-2) and cis-acting iron-responsive elements (IREs) in mRNA 5Ј-or 3Јuntranslated regions. IRP-1 can adopt two conformations: one with a [4Fe-4S]-cluster, unable to bind IREs, which functions as a cytoplasmic aconitase; one lacking this cluster, which accumulates in iron-deprived cells and binds mRNA firmly. We investigated which surfaces of IRP-1 interact with IREs. Surface areas were predicted on the basis of the crystallized porcine mitochondrial aconitase structure. We selected nine sequences absent or different in mitochondrial and Escherichia coli aconitases, both being devoid of RNAbinding properties. Mutations in two regions of domain 4 of IRP-1 lowered the affinity for a wild-type IRE up to 7-fold in vitro, whereas the aconitase activity, a control for structural integrity, was not affected. Scatchard plot analysis with mutant IREs indicated that domain 4 is involved in the binding specificity. This conclusion was confirmed with hybrid proteins in which IRP-1 surface loops were grafted into IRP-2. The results indicate that arginines 728 and 732 contact the IRE bulge, whereas region 685-689 is necessary for recognition of the IRE loop.
Blood, 2011
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.
Proceedings of the National Academy of Sciences, 1992
Several mech s of posttranscriptional gene regulation are involved in regulation of the expression of essential proteins of iron metabolism. Coordinate regulation of ferritin and transferrin receptor expression is produced by binding of a cytosolic protein, the iron-responsive element binding protein (IRE-BP) to specific stem-oop structures present in target RNAs. The affinity of this protein for its cognate RNA is regulated by the cell in response to changes in iron availability. The IRE-BP demonstrates a striking level of amino acid sequence identity to the iron-sulfur (Fe-S) protein mitochondrial aconitase. Moreover, the recombinant IRE-BP has aconitase function. The lability of the Fe-S cluster in mitochondrial aconitase has led us to propose that the mechnism by which iron levels are sensed by the IRE-BP involves changes in an Fe-S cluster in the IRE-BP. In this study, we demonstrate that procedures aimed at altering the IRE-BP Fe-S cluster in vitro reciprocally alter the RNA binding and aconitase activity of the IRE-BP. The changes in the RNA big of the protein produced in vitro appear to match the previously described alterations of the protein in response to iron availability in the cell. Furthermore, iron manipulation ofcells correlates with the activation or inactivation of the IRE-BP aoitas activity. The results are consistent with a model for the ponslatonal regulation of the IRE-BP in which the Fe-S cluster is altered in response to the availability of intracellular iron and this, in turn, regulates the RNA-binding activity.
Biochemistry, 2000
Iron-responsive elements (IREs), a natural group of mRNA-specific sequences, bind iron regulatory proteins (IRPs) differentially and fold into hairpins [with a hexaloop (HL) CAGUGX] with helical distortions: an internal loop/bulge (IL/B) (UGC/C) or C-bulge. C-bulge iso-IREs bind IRP2 more poorly, as oligomers (n ) 28-30), and have a weaker signal response in vivo. Two trans-loop GC base pairs occur in the ferritin IRE (IL/B and HL) but only one in C-bulge iso-IREs (HL); metal ions and protons perturb the IL/B ) Biochemistry 37, 1505-1512. IRE function (translation) and physical properties (T m and accessibility to nucleases) are now compared for IL/B and C-bulge IREs and for HL mutants. Conversion of the IL/B into a C-bulge by a single deletion in the IL/B or by substituting the HL CG base pair with UA both derepressed ferritin synthesis 4-fold in rabbit reticulocyte lysates (IRP1 + IRP2), confirming differences in IRP2 binding observed for the oligomers. Since the engineered C-bulge IRE was more helical near the IL/B [Cu(phen) 2 resistant] and more stable (T m increased) and the HL mutant was less helical near the IL/B (ribonuclease T1 sensitive) and less stable (T m decreased), both CG trans-loop base pairs contribute to maximum IRP2 binding and translational regulation. The 1 H NMR spectrum of the Mg-IRE complex revealed, in contrast to the localized IL/B effects of Co(III) hexaammine observed previously, perturbation of the IL/B plus HL and interloop helix. The lower stability and greater helix distortion in the ferritin IL/B-IRE compared to the C-bulge iso-IREs create a combinatorial set of RNA/protein interactions that control protein synthesis rates with a range of signal sensitivities. † Partial support for this work came from NIH R01-DK-20251 and the CHORI Foundation. IRP, iron regulatory protein; IL/B, internal loop/bulge; HL, iron-responsive element hairpin hexaloop (Hairpin Loop); RRL, rabbit reticulocyte lysate; TfR, transferrin receptor; WGE, wheat germ extract; WT, wild type.