Abstract
The iron responsive element binding protein (IRE-BP) regulates iron storage and uptake in response to iron. This control results from the interaction of the IRE-BP with the iron responsive element (IRE), a conserved sequence/structure element located near the 5' end of all ferritin mRNAs and in the 3' UTR of transferrin receptor mRNAs. Proteolysis was used to probe for functional elements of the IRE-BP. Partial chymotrypsin digestion generates a simple digestion pattern yielding fragments of 68, 56, 41, and 30 kDa. The 68 and 30 kDa fragments are derived from a single cleavage at Trp623. Further cleavages of the 68 kDa polypeptide yield the 56 and 41 kDa peptides. A combination of UV-crosslinking and chymotrypsin digestion was used to localize an RNA binding element within the C-terminus of the 68 kDa fragment, between amino acid residues 480 and 623. This region includes cysteine residues 503 and 506 which have been shown to be required for iron-sulfur cluster assembly and for iron regulation of the IRE-BP. Proteolytic fragments of the IRE-BP that contain this RNA binding region can be crosslinked to the IRE but do not bind with high affinity, suggesting that elements within the IRE-BP, in addition to those located between residues 480 and 623, are required for high affinity binding to the IRE.
Full text
PDF






Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aziz N., Munro H. N. Iron regulates ferritin mRNA translation through a segment of its 5' untranslated region. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8478–8482. doi: 10.1073/pnas.84.23.8478. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bandziulis R. J., Swanson M. S., Dreyfuss G. RNA-binding proteins as developmental regulators. Genes Dev. 1989 Apr;3(4):431–437. doi: 10.1101/gad.3.4.431. [DOI] [PubMed] [Google Scholar]
- Barton H. A., Eisenstein R. S., Bomford A., Munro H. N. Determinants of the interaction between the iron-responsive element-binding protein and its binding site in rat L-ferritin mRNA. J Biol Chem. 1990 Apr 25;265(12):7000–7008. [PubMed] [Google Scholar]
- Basilion J. P., Rouault T. A., Massinople C. M., Klausner R. D., Burgess W. H. The iron-responsive element-binding protein: localization of the RNA-binding site to the aconitase active-site cleft. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):574–578. doi: 10.1073/pnas.91.2.574. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bettany A. J., Eisenstein R. S., Munro H. N. Mutagenesis of the iron-regulatory element further defines a role for RNA secondary structure in the regulation of ferritin and transferrin receptor expression. J Biol Chem. 1992 Aug 15;267(23):16531–16537. [PubMed] [Google Scholar]
- Brown P. H., Daniels-McQueen S., Walden W. E., Patino M. M., Gaffield L., Bielser D., Thach R. E. Requirements for the translational repression of ferritin transcripts in wheat germ extracts by a 90-kDa protein from rabbit liver. J Biol Chem. 1989 Aug 15;264(23):13383–13386. [PubMed] [Google Scholar]
- Carey J., Cameron V., de Haseth P. L., Uhlenbeck O. C. Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site. Biochemistry. 1983 May 24;22(11):2601–2610. doi: 10.1021/bi00280a002. [DOI] [PubMed] [Google Scholar]
- Casey J. L., Hentze M. W., Koeller D. M., Caughman S. W., Rouault T. A., Klausner R. D., Harford J. B. Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation. Science. 1988 May 13;240(4854):924–928. doi: 10.1126/science.2452485. [DOI] [PubMed] [Google Scholar]
- Casey J. L., Koeller D. M., Ramin V. C., Klausner R. D., Harford J. B. Iron regulation of transferrin receptor mRNA levels requires iron-responsive elements and a rapid turnover determinant in the 3' untranslated region of the mRNA. EMBO J. 1989 Dec 1;8(12):3693–3699. doi: 10.1002/j.1460-2075.1989.tb08544.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Constable A., Quick S., Gray N. K., Hentze M. W. Modulation of the RNA-binding activity of a regulatory protein by iron in vitro: switching between enzymatic and genetic function? Proc Natl Acad Sci U S A. 1992 May 15;89(10):4554–4558. doi: 10.1073/pnas.89.10.4554. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cox L. A., Adrian G. S. Posttranscriptional regulation of chimeric human transferrin genes by iron. Biochemistry. 1993 May 11;32(18):4738–4745. doi: 10.1021/bi00069a007. [DOI] [PubMed] [Google Scholar]
- Cox T. C., Bawden M. J., Martin A., May B. K. Human erythroid 5-aminolevulinate synthase: promoter analysis and identification of an iron-responsive element in the mRNA. EMBO J. 1991 Jul;10(7):1891–1902. doi: 10.1002/j.1460-2075.1991.tb07715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Crichton R. R. Proteins of iron storage and transport. Adv Protein Chem. 1990;40:281–363. doi: 10.1016/s0065-3233(08)60288-0. [DOI] [PubMed] [Google Scholar]
- Dandekar T., Stripecke R., Gray N. K., Goossen B., Constable A., Johansson H. E., Hentze M. W. Identification of a novel iron-responsive element in murine and human erythroid delta-aminolevulinic acid synthase mRNA. EMBO J. 1991 Jul;10(7):1903–1909. doi: 10.1002/j.1460-2075.1991.tb07716.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Emery-Goodman A., Hirling H., Scarpellino L., Henderson B., Kühn L. C. Iron regulatory factor expressed from recombinant baculovirus: conversion between the RNA-binding apoprotein and Fe-S cluster containing aconitase. Nucleic Acids Res. 1993 Mar 25;21(6):1457–1461. doi: 10.1093/nar/21.6.1457. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gait M. J., Karn J. RNA recognition by the human immunodeficiency virus Tat and Rev proteins. Trends Biochem Sci. 1993 Jul;18(7):255–259. doi: 10.1016/0968-0004(93)90176-n. [DOI] [PubMed] [Google Scholar]
- Gray N. K., Quick S., Goossen B., Constable A., Hirling H., Kühn L. C., Hentze M. W. Recombinant iron-regulatory factor functions as an iron-responsive-element-binding protein, a translational repressor and an aconitase. A functional assay for translational repression and direct demonstration of the iron switch. Eur J Biochem. 1993 Dec 1;218(2):657–667. doi: 10.1111/j.1432-1033.1993.tb18420.x. [DOI] [PubMed] [Google Scholar]
- Haile D. J., Hentze M. W., Rouault T. A., Harford J. B., Klausner R. D. Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements. Mol Cell Biol. 1989 Nov;9(11):5055–5061. doi: 10.1128/mcb.9.11.5055. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haile D. J., Rouault T. A., Harford J. B., Kennedy M. C., Blondin G. A., Beinert H., Klausner R. D. Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11735–11739. doi: 10.1073/pnas.89.24.11735. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haile D. J., Rouault T. A., Tang C. K., Chin J., Harford J. B., Klausner R. D. Reciprocal control of RNA-binding and aconitase activity in the regulation of the iron-responsive element binding protein: role of the iron-sulfur cluster. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7536–7540. doi: 10.1073/pnas.89.16.7536. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harrell C. M., McKenzie A. R., Patino M. M., Walden W. E., Theil E. C. Ferritin mRNA: interactions of iron regulatory element with translational regulator protein P-90 and the effect on base-paired flanking regions. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4166–4170. doi: 10.1073/pnas.88.10.4166. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hentze M. W., Argos P. Homology between IRE-BP, a regulatory RNA-binding protein, aconitase, and isopropylmalate isomerase. Nucleic Acids Res. 1991 Apr 25;19(8):1739–1740. doi: 10.1093/nar/19.8.1739. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hentze M. W., Caughman S. W., Rouault T. A., Barriocanal J. G., Dancis A., Harford J. B., Klausner R. D. Identification of the iron-responsive element for the translational regulation of human ferritin mRNA. Science. 1987 Dec 11;238(4833):1570–1573. doi: 10.1126/science.3685996. [DOI] [PubMed] [Google Scholar]
- Hirling H., Emery-Goodman A., Thompson N., Neupert B., Seiser C., Kühn L. C. Expression of active iron regulatory factor from a full-length human cDNA by in vitro transcription/translation. Nucleic Acids Res. 1992 Jan 11;20(1):33–39. doi: 10.1093/nar/20.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirling H., Henderson B. R., Kühn L. C. Mutational analysis of the [4Fe-4S]-cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase. EMBO J. 1994 Jan 15;13(2):453–461. doi: 10.1002/j.1460-2075.1994.tb06280.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keene J. D., Query C. C. Nuclear RNA-binding proteins. Prog Nucleic Acid Res Mol Biol. 1991;41:179–202. doi: 10.1016/s0079-6603(08)60009-4. [DOI] [PubMed] [Google Scholar]
- Kenan D. J., Query C. C., Keene J. D. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci. 1991 Jun;16(6):214–220. doi: 10.1016/0968-0004(91)90088-d. [DOI] [PubMed] [Google Scholar]
- Kennedy M. C., Mende-Mueller L., Blondin G. A., Beinert H. Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11730–11734. doi: 10.1073/pnas.89.24.11730. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klausner R. D., Rouault T. A., Harford J. B. Regulating the fate of mRNA: the control of cellular iron metabolism. Cell. 1993 Jan 15;72(1):19–28. doi: 10.1016/0092-8674(93)90046-s. [DOI] [PubMed] [Google Scholar]
- Leibold E. A., Laudano A., Yu Y. Structural requirements of iron-responsive elements for binding of the protein involved in both transferrin receptor and ferritin mRNA post-transcriptional regulation. Nucleic Acids Res. 1990 Apr 11;18(7):1819–1824. doi: 10.1093/nar/18.7.1819. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leibold E. A., Munro H. N. Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated region of ferritin heavy- and light-subunit mRNAs. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2171–2175. doi: 10.1073/pnas.85.7.2171. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mattaj I. W. A binding consensus: RNA-protein interactions in splicing, snRNPs, and sex. Cell. 1989 Apr 7;57(1):1–3. doi: 10.1016/0092-8674(89)90164-5. [DOI] [PubMed] [Google Scholar]
- Melefors O., Goossen B., Johansson H. E., Stripecke R., Gray N. K., Hentze M. W. Translational control of 5-aminolevulinate synthase mRNA by iron-responsive elements in erythroid cells. J Biol Chem. 1993 Mar 15;268(8):5974–5978. [PubMed] [Google Scholar]
- Mengaud J. M., Horwitz M. A. The major iron-containing protein of Legionella pneumophila is an aconitase homologous with the human iron-responsive element-binding protein. J Bacteriol. 1993 Sep;175(17):5666–5676. doi: 10.1128/jb.175.17.5666-5676.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neupert B., Thompson N. A., Meyer C., Kühn L. C. A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta. Nucleic Acids Res. 1990 Jan 11;18(1):51–55. doi: 10.1093/nar/18.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Owen D., Kühn L. C. Noncoding 3' sequences of the transferrin receptor gene are required for mRNA regulation by iron. EMBO J. 1987 May;6(5):1287–1293. doi: 10.1002/j.1460-2075.1987.tb02366.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pashev I. G., Dimitrov S. I., Angelov D. Crosslinking proteins to nucleic acids by ultraviolet laser irradiation. Trends Biochem Sci. 1991 Sep;16(9):323–326. doi: 10.1016/0968-0004(91)90133-g. [DOI] [PubMed] [Google Scholar]
- Patino M. M., Walden W. E. Cloning of a functional cDNA for the rabbit ferritin mRNA repressor protein. Demonstration of a tissue-specific pattern of expression. J Biol Chem. 1992 Sep 15;267(26):19011–19016. [PubMed] [Google Scholar]
- Philpott C. C., Haile D., Rouault T. A., Klausner R. D. Modification of a free Fe-S cluster cysteine residue in the active iron-responsive element-binding protein prevents RNA binding. J Biol Chem. 1993 Aug 25;268(24):17655–17658. [PubMed] [Google Scholar]
- Prodromou C., Artymiuk P. J., Guest J. R. The aconitase of Escherichia coli. Nucleotide sequence of the aconitase gene and amino acid sequence similarity with mitochondrial aconitases, the iron-responsive-element-binding protein and isopropylmalate isomerases. Eur J Biochem. 1992 Mar 1;204(2):599–609. doi: 10.1111/j.1432-1033.1992.tb16673.x. [DOI] [PubMed] [Google Scholar]
- Robbins A. H., Stout C. D. The structure of aconitase. Proteins. 1989;5(4):289–312. doi: 10.1002/prot.340050406. [DOI] [PubMed] [Google Scholar]
- Rouault T. A., Hentze M. W., Haile D. J., Harford J. B., Klausner R. D. The iron-responsive element binding protein: a method for the affinity purification of a regulatory RNA-binding protein. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5768–5772. doi: 10.1073/pnas.86.15.5768. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rouault T. A., Stout C. D., Kaptain S., Harford J. B., Klausner R. D. Structural relationship between an iron-regulated RNA-binding protein (IRE-BP) and aconitase: functional implications. Cell. 1991 Mar 8;64(5):881–883. doi: 10.1016/0092-8674(91)90312-m. [DOI] [PubMed] [Google Scholar]
- Rouault T. A., Tang C. K., Kaptain S., Burgess W. H., Haile D. J., Samaniego F., McBride O. W., Harford J. B., Klausner R. D. Cloning of the cDNA encoding an RNA regulatory protein--the human iron-responsive element-binding protein. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7958–7962. doi: 10.1073/pnas.87.20.7958. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scherly D., Boelens W., Dathan N. A., van Venrooij W. J., Mattaj I. W. Major determinants of the specificity of interaction between small nuclear ribonucleoproteins U1A and U2B'' and their cognate RNAs. Nature. 1990 Jun 7;345(6275):502–506. doi: 10.1038/345502a0. [DOI] [PubMed] [Google Scholar]
- Shull G. E., Theil E. C. Translational control of ferritin synthesis by iron in embryonic reticulocytes of the bullfrog. J Biol Chem. 1982 Dec 10;257(23):14187–14191. [PubMed] [Google Scholar]
- Swenson G. R., Patino M. M., Beck M. M., Gaffield L., Walden W. E. Characteristics of the interaction of the ferritin repressor protein with the iron-responsive element. Biol Met. 1991;4(1):48–55. doi: 10.1007/BF01135557. [DOI] [PubMed] [Google Scholar]
- Walden W. E., Daniels-McQueen S., Brown P. H., Gaffield L., Russell D. A., Bielser D., Bailey L. C., Thach R. E. Translational repression in eukaryotes: partial purification and characterization of a repressor of ferritin mRNA translation. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9503–9507. doi: 10.1073/pnas.85.24.9503. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walden W. E., Patino M. M., Gaffield L. Purification of a specific repressor of ferritin mRNA translation from rabbit liver. J Biol Chem. 1989 Aug 15;264(23):13765–13769. [PubMed] [Google Scholar]
- Wang Y. H., Sczekan S. R., Theil E. C. Structure of the 5' untranslated regulatory region of ferritin mRNA studied in solution. Nucleic Acids Res. 1990 Aug 11;18(15):4463–4468. doi: 10.1093/nar/18.15.4463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weeks K. M., Ampe C., Schultz S. C., Steitz T. A., Crothers D. M. Fragments of the HIV-1 Tat protein specifically bind TAR RNA. Science. 1990 Sep 14;249(4974):1281–1285. doi: 10.1126/science.2205002. [DOI] [PubMed] [Google Scholar]
- Yu Y., Radisky E., Leibold E. A. The iron-responsive element binding protein. Purification, cloning, and regulation in rat liver. J Biol Chem. 1992 Sep 15;267(26):19005–19010. [PubMed] [Google Scholar]
- Zheng L., Andrews P. C., Hermodson M. A., Dixon J. E., Zalkin H. Cloning and structural characterization of porcine heart aconitase. J Biol Chem. 1990 Feb 15;265(5):2814–2821. [PubMed] [Google Scholar]
- Zheng L., Kennedy M. C., Blondin G. A., Beinert H., Zalkin H. Binding of cytosolic aconitase to the iron responsive element of porcine mitochondrial aconitase mRNA. Arch Biochem Biophys. 1992 Dec;299(2):356–360. doi: 10.1016/0003-9861(92)90287-7. [DOI] [PubMed] [Google Scholar]
- Zähringer J., Baliga B. S., Munro H. N. Novel mechanism for translational control in regulation of ferritin synthesis by iron. Proc Natl Acad Sci U S A. 1976 Mar;73(3):857–861. doi: 10.1073/pnas.73.3.857. [DOI] [PMC free article] [PubMed] [Google Scholar]