Proc. Natl. Acad. Sci. USAVol. 87, pp. 7958-7%2, October 1990Biochemistry

Cloning of the cDNA encoding an RNA regulatory protein-thehuman iron-responsive element-binding protein

(posttranscriptional gene regulation/iron)

TRACEY A. ROUAULT*, CAREEN K. TANG*, STAMATINA KAPTAIN*, WILSON H. BURGESSt, DAVID J. HAILE*,FELIPE SAMANIEGO*, 0. WESLEY MCBRIDEt, JOE B. HARFORD*, AND RICHARD D. KLAUSNER**Cell Biology and Metabolism Branch, National Institute of Child Health, Bethesda, MD 20892; tLaboratory of Molecular Biology, Jerome H. HollandLaboratory, American Red Cross, Rockville, MD 20855; and tLaboratory of Biochemistry, Division of Cancer Biology and Diagnosis, NationalCancer Institute, Bethesda, MD 20892

Communicated by G. D. Aurbach, July 23, 1990 (receivedfor review May 20, 1990)

ABSTRACT Iron-responsive elements (IREs) are stem-loop structures found in the mRNAs encoding ferritin and thetransferrin receptor. These elements participate in the iron-induced regulation of the translation offerritin and the stabilityof the transferrin receptor mRNA. Regulation in both instancesis mediated by binding of a cytosolic protein to the IREs.High-affinity binding is seen when cells are starved of iron andresults in repression of ferritin translation and inhibition oftransferrin receptor mRNA degradation. The IRE-bindingprotein (IRE-BP) has been identified as an -90-kDa proteinthat has been purified by both affinity and conventionalchromatography. In this report we use RNA affinity chroma-tography and two-dimensional gel electrophoresis to isolate theIRE-BP for protein sequencing. A degenerate oligonucleotideprobe derived from a single peptide sequence was used toisolate a cDNA clone that encodes a protein containing 13 othersequenced peptides obtained from the IRE-BP. Consistent withprevious characterization of the IRE-BP, the cDNA encodes aprotein of 87 kDa with a slightly acidic pl, and the correspond-ing mRNA of -3.6 kilobases is found in a variety of cell types.The encoded protein contains a nucleotide-binding consensussequence and regions of cysteine and histidine clusters. ThismRNA is encoded by a single gene on human chromosome 9,a finding consistent with previous localization by functionalmapping. The protein contains no previously defined consensusmotifs for either RNA or DNA binding. The simultaneouscloning of a different, but highly homologous, cDNA suggeststhat the IRE-BP is a member of a distinct gene family.

Studies on the regulation of cellular iron metabolism in highereukaryotes have revealed an intriguing system of posttran-scriptional gene control (1, 2). The mRNAs encoding two ofthe major proteins that participate in iron homeostasis containregulatory structures that have been termed iron-responsiveelements (IREs). These stem-loop structures, first identified inthe 5'-untranslated regions (UTRs) of ferritin mRNAs, weresubsequently identified in the 3' UTR of the transfemn re-ceptor (TfR) mRNA (3-5). In ferritin, a single IRE is respon-sible for iron-dependent control of translation, whereas mul-tiple IREs in the TfR 3' regulatory region mediate iron-dependent control of mRNA half-life (6, 7). IREs function asthe binding site for a cytosolic protein as demonstrated inRNAmobility shift and UV crosslinking experiments (8, 9). Itappears that both ferritin and TfR IREs bind to the sameprotein in vitro (10). Experiments conducted both in vivo andin vitro demonstrate that binding of the protein is responsiblefor repressing ferritin mRNA translation (9, 11). In TfRmRNA, the IREs are part of a more complex RNA instabilityelement. Binding of the protein to the TfR 3' UTR inhibits

degradation of this otherwise rapidly degraded mRNA (6, 7).IREs and a specific cytosolic binding protein have beendetected across a wide number of species ofhigher eukaryotes(12, 13). Several protocols have been reported for use inisolating proteins of similar activity and apparent molecularweight, and these isolated proteins have been named theferritin repressor protein (11), the iron-regulatory factor (14),and the IRE-binding protein (IRE-BP) (9). We favor the lattername, as it encompasses a role in both ferritin and TfRregulation and more directly reflects the known function oftheprotein. Based upon the difference in mobility of human androdent IRE-IRE-BP complexes, we have localized the geneencoding human IRE-BP to chromosome 9 (15).

After initial characterization of IRE-BP, the amount ofbinding activity in a cell lysate was found to increase underconditions of iron deprivation and to decrease in response toan iron load (9, 16). The magnitude of the change in thebinding activity in cell lysates after manipulations of intra-cellular iron was reflected in the quantitative change offerritin translation in response to iron perturbations. A timecourse showing progressive changes in the rates of ferritinbiosynthesis demonstrated concomitant changes in the ex-tent of binding activity (17). Thus, the level of IRE-BPactivity provided a stable record of the iron status of the cell.The lower binding activity seen in iron-replete cells wasdemonstrated to be entirely recovered in vitro by reductionwith thiol agents (17). This observation led us to propose a"sulfhydryl switch" mechanism, whereby the level of cellu-lar iron switched the protein from a lower-affinity (oxidized)form to a high-affinity (reduced) form(s). Heme has beenproposed to mediate the inhibition of the interaction ofIRE-BP with its target RNA (18, 19). However, heme cannonspecifically inhibit numerous nucleic acid-protein inter-actions, and the significance of inhibition of binding by hemein this setting is unclear (20, 21).We have used an IRE RNA affinity-resin purification from

a human liver cytosolic extract and two-dimensional gelelectrophoresis to isolate the IRE-BP for protein sequencing.After sequencing of peptides, a 3.4-kilobase (kb) cDNA clonewas isolated by hybridization with a degenerate oligonucle-otide corresponding to one partial peptide sequence. An openreading frame (ORF) of 790 amino acids was identified thatcontained 14 of the sequenced peptides. A second, distinctcDNA clone selected with the same degenerate oligonucle-otide was found to encode a closely related protein, althoughnone of the sequences predicted by DNA sequencing of this

Abbreviations: IRE, iron-responsive element; IRE-BP, IRE-bindingprotein; UTR, untranslated region; TfR, transferrin receptor; RFLP,restriction fragment length polymorphism; ORF, open readingframe.The sequences reported in this paper have been deposited in theGenBank data base (accession nos. M37836 for IRE-BP and M37835for clone 10.1).

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Proc. Natl. Acad. Sci. USA 87 (1990) 7959

second clone perfectly matched any of the peptides derivedfrom protein isolated by the IRE-affinity resin.

MATERIALS AND METHODSIRE-BP Purification. Purification by means of a single

round of IRE-affinity chromatography was accomplishedusing described methods (22). Material bound to the affinitycolumn was eluted in 9.5 M urea/0.1% SDS/0.5 mM dithio-threitol/0.2% Nonidet P-40/4% Ampholytes, pH 3-10(Serva), incubated for 2 hr at 420C, and subjected to isoelec-tric focusing in a tube gel (23). Proteins were subjected toelectrophoresis in the second dimension according to themethods of Laemmli (24). Proteins were transferred electro-phoretically to nitrocellulose (Schleicher & Schuell). Aftertransfer, the membrane was immersed for 60 sec in 0.1%Ponceau S dye (Sigma)/1% aqueous acetic acid. The specificregion corresponding to the IRE-BP was identified. Thisregion was cut from the nitrocellulose and incubated for 30min at 370C in 1.2 ml of 0.'5% polyvinylpyrrolidone-40 dis-solved in 100 mM acetic acid to prevent subsequent adsorp-tion of protease to the nitrocellulose during digestion. Excesspolyvinylpyrrolidone-40 was removed by extensive washingwith water (25, 26). The protein (100-200 pmol) was digestedwith either Asp-N or Lys-C sequencing grade proteases(Boehringer Mannheim) at an estimated enzyme-to-proteinratio of 1:10. Asp-N digestion was done in 50 mM NaHPO4,pH 8, at 370C for 18 hr, and Lys-C digestion was done in 25mM Tris/1 mM EDTA/5% CH3CN, pH 8.5, at 370C for 18 hr.Resulting peptides were separated on an Applied Biosystemsmodel 130A microbore HPLC system with a 2.1 x 210-mmApplied Biosystems RP-300 column equilibrated in 98%solvent A (0.1% trifluoroacetic acid in water)/2% solvent B[70% (vol/vol) acetonitrile and 0.09% trifluoroacetic acid inwater] using a linear gradient from 2% solvent B to 80%solvent B over 55 min. Amino acid sequences were deter-mined by using an Applied Biosystems model 477A se-quencer and modified Edman chemistry. Cleaved amino acidderivatives were identified with an on-line Applied Biosys-tems model 120A phenylthiohydantoin analyzer. Peaks con-taining double peptide sequences were reduced with 10 mMdithiothreitol at 37C for 60 min and rechromatographed asdescribed above.

Library Screening. A 64-fold degenerate 20-mer derivedfrom a peptide containing the amino acid sequenceNMCPEYG was synthesized on an Applied Biosystems 381ADNA synthesizer and subsequently labeled with [yy-32P]ATP(ICN) and polynucleotide kinase (Boehringer Mannheim)(27). A cDNA A Zap II library (Stratagene) derived fromhuman YT cells (28) was probed with a probe of 500,000cpm/ml. Duplicate filters (DuPont/NEN) from plates con-taining a total of 400,000 phage plaques were hybridized afterprehybridization in 6x SSC (lx SSC is 0.15 M sodiumchloride/0.015 M sodium citrate, pH 7), 2x Denhardt'ssolution (lx Denhardt's solution is 0.02% polyvinylpyrroli-done/0.02% Ficoll/0.02% bovine serum albumin)/1% SDS/salmon sperm DNA at 100 ,ug/ml