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Originally published In Press as doi:10.1074/jbc.M207918200 on August 27, 2002

J. Biol. Chem., Vol. 277, Issue 45, 42579-42587, November 8, 2002
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Multiple, Conserved Iron-responsive Elements in the 3'-Untranslated Region of Transferrin Receptor mRNA Enhance Binding of Iron Regulatory Protein 2*

Ronit Erlitzki, Joanne C. Long, and Elizabeth C. TheilDagger

From the Children's Hospital Oakland Research Institute, The Research Institute of the Children's Hospital and Medical Center Oakland, 5700 Martin Luther King, Jr. Way, Oakland, California 94609-1673

Received for publication, August 4, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The best characterized mechanisms of mRNA regulation are changes in translation where ribosome binding is the regulatory target, or mRNA stability, which is mediated by various cis-acting elements located in the coding sequences or in the untranslated region (UTR)1 of the messages (1). The transferrin receptor (TfR) mRNA provides an example of an mRNA that contains both specific stability elements, the iron-responsive elements (IREs) (2-5) and AU-rich instability elements, that are common to a large family of short-lived mRNAs involved in growth regulation and cell proliferation, such as granulocyte-macrophage colony-stimulating factor (GMC-SF) or interleukins. This combination characterizes other mRNAs, such as c-myc mRNA, although in this case the specific elements are localized in the coding region (1, 6, 7). AU-rich elements mediate decay by promoting the deadenylation of the mRNA, a general mechanism for decreasing the stability of many eukaryotic mRNAs (1, 8, 9). In the TfR mRNA, by contrast, the degradation pathway is uncoupled from deadenylation (10, 11) and involves an endonucleolytic cleavage, possibly related to the TfR-IRE interaction with iron regulatory proteins (IRP1 and IRP2) that are known to stabilize TfR mRNA from nuclease-mediated degradation (4, 12).

The stability element of TfR mRNA is located in the 3'-UTR and composed of ~700 nucleotides (13) with five AUUUA elements, several U stretches, and five conserved palindromic sequences that fold to form the IRE hairpin loops (a, b, c, d, and e) (14). The apical loop of all IREs have a conserved base pair, C14 and G18 (15-18), required for both IRP1 and IRP2 binding. When environmental iron is low, IRP binding to TfR-IREs confers iron-dependent stabilization on the TfR mRNA, up-regulating receptor synthesis and iron entry into cells (19-21). In addition to iron, signals such as peroxide, anoxia, and NO alter the IRP·IRE interactions (22).

IREs are present in a number of mRNAs (23), integrating iron and oxygen metabolism by regulating the tricarboxylic acid cycle (m-aconitase mRNA), iron storage and efflux (ferritin and ferroportin mRNAs), heme synthesis (erythroid-aminolevulinate synthase mRNA) and non-transferrin iron uptake by DMT1 (24) through a novel combinatorial mechanism (2). Homologous, non-IRE mRNAs exist for all known IRE mRNAs (25-29) except ferroportin (30, 31). Selectivity of IRP1 and IRP2 binding depends on bulge or bulge/loop structure in the IRE helix. The large helix distortion created by the internal loop/bulge, unique to ferritin IRE, contributes to greater IRP2 binding relative to weak interaction of the protein with C-bulge IREs (32, 33). High affinity to IRP1 is mainly determined by the apical loop (22, 34).

In the TfR mRNA stability element, high conservation of all five IREs (14, 23, 35) suggests that all of them are essential for function. Many studies of IRP interaction with different IREs, including the TfR-IREs, have used a single IRE (14,16,32-34,36-38) or a 3xIRE construct (IREb, IREc, and IREd) with truncated linking sequences and the less selective binding protein, IRP1 (11, 18, 39).

To understand the conservation of five C-bulge IREs in the TfR mRNA stability element, the interactions of the full length element (5xIRE) with IRP1 and with the more selective IRP2 were examined in vitro and compared with fragments containing only 3 or 2 IREs (Fig. 1). The results showed that in the context of the full length TfR stability element, all 5 IREs appear to be available for IRP binding and that IRP2 binding to multiple-IRE RNA, was comparable with IRP1 binding, i.e. enhanced relative to a single TfR-IRE binding by IRP2. As the IRE copy number decreased from 5, to 3 or 2, the IRP-mediated nuclease resistance of the RNA decreased. Enhanced IRP2 binding to TfR-IREs in the context of 5xIRE was correlated with distortion in the IRE helices, detected by Cu-(phen)2 binding/cleavage, which coincides with similar structure in the ferritin IRE that is related to high IRP2 binding, as previously observed (33, 40). The change in TfR-IRE structures within the context of multiple-IRE RNA, and the variations we observed in cell and developmental expression of IRP1 and IRP2, with IRP2 mRNA predominating, would enable TfR synthesis and iron uptake for growth in IRP1-deficient cell types. Thus, cell-specific variations in IRP and IRP1/IRP2 expression will have a smaller impact on TfR mRNA (multiple IRE) or ferritin mRNA (distinctive IRE) relative to other IRE containing mRNAs, increasing cell specificity and the range of IRE-mRNA response for NO, iron, and oxidative stress signals.


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  		Fig. 1.   Scheme of human TfR-3'-UTR RNA constructs used in the study. TfR-3'-UTR RNA constructs were cloned and in vitro transcribed as described under "Experimental Procedures." Each RNA had different TfR-IRE (gray oval) copy number (5xIRE, 3xIRE, or 2xIRE) linked by the native linking sequences (linker d/e, black box), the only linker deleted in shorter constructs.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of TfR-3'-UTR Stability Element Fragment-- A 662-bp cDNA sequence coding for a part of TfR-3'-UTR responsible for iron regulation of TfR mRNA (13, 41) was amplified by PCR with Advantage® cDNA PCR kit (Clontech) and the primers: sense, 5'-AGCTTTCTGTCCTTTTGGCA-3' and antisense, 5'-CAAGCTTTGAAGATGTCATTGC-3', from Human HeLa cells Marathon-Ready cDNA (Clontech). This sequence (5xIRE), which included all five TfR-IREs (IREs a-e) and the native linking sequences (Fig. 1), served as a template for PCR amplification of fragments with only 2 or 3 IREs: 3xIRE, a 518-bp fragment including TfR-IREb, IREc, and IREd and the linking sequences a/b, b/c, and c/d, which was amplified with the primers: sense, 5'-AATAGAATATAATTATCGGAAGCAGTG-3'; antisense, 5'-CCCTTAGTGTAACATATGGAGATCA-3'. 2xIRE, a 477-bp fragment including TfR-IREb and TfR-IREc and the linking sequences a/b, b/c, and c/d was amplified with the primers: sense, 5'-AATAGAATATAATTATCGGAAGCAGTG-3'; antisense, 5'-CACTGTGGTAGGTAAAAACTACCTTG-3'. PCR products were subcloned into pCR®II-TOPO vector (Invitrogen) and confirmed by sequencing. Cloning and expression requirements added 102 nt (69 nt at the 5' end and 36 nt at the 3' end) of vector sequences to the 5xIRE, 3xIRE, and 2xIRE transcripts. TfR-IREb (chemically synthesized by Dharmacon Research, Inc.) was selected to represent a single TfR-IRE RNA form (1xIRE) because of its relatively high affinity to IRPs (14) and because IREb is one of three TfR-IREs in TRS-1, a minimal TfR-3'-UTR sequence that displays iron-dependent regulation of TfR expression (42).

In Vitro Transcription, Annealing, and Labeling of TfR mRNA Stability Element Fragments-- Confirmed TfR-3'-UTR clones were linearized with SpeI (Roche Molecular Biochemicals) and served as templates for in vitro transcription of uniformly 32P-labeled T7 transcripts with MEGAscriptTM kit (Ambion) in the presence of 7.5 mM each ATP, CTP, and GTP, 3.25 mM UTP, 20 µCi [alpha -32P]UTP (Amersham Biosciences) and 0.75-1.5 µg of DNA template, for 2-3 h at 37 °C. After enzymatic removal of DNA template by an additional 10-min incubation of in vitro transcription reaction with 2 units of DNase (Ambion), unincorporated NTPs were removed by gel filtration (ProbeQuantTM G-50 Micro columns; Amersham Biosciences). Samples were quantified by UV spectroscopy. Synthesis and integrity of full length transcripts were confirmed by denaturing gel electrophoresis of the uniformly labeled RNA (4% acrylamide/8 M urea) calibrated with RNA CenturyTM Marker Plus template set (Ambion). RNA transcripts were melted in water (5 min at 85 °C) and annealed in the presence of Na+ or Mg2+ while slowly cooling to room temperature. Annealing at increased ionic strength (50 mM Na+ or 5 mM Mg2+) eliminated a slower electrophoretically migrating RNA species observed with all three TfR-RNA constructs (2xIRE, 3xIRE, and 5xIRE). A faster migrating, compactly folded conformation, which was unaffected by higher ionic concentration or by the IRP binding solution (see below) was routinely obtained with 50 mM NaCl, aliquoted, frozen in liquid nitrogen, and stored at -80 °C. The annealed RNA was used to analyze IRP binding, nuclease resistance, and Cu-(phen)2 cleavage. When single TfR-IREb RNA and DNA oligomers were used, they were labeled at the 5' end with [gamma -32P]ATP (3000 or 6000 Ci/mmol, respectively; Amersham Biosciences) and bacteriophage T4 polynucleotide kinase (Promega) and purified by gel filtration (ProbeQuantTM G-50 Micro columns; Amersham Biosciences). Secondary structure modeling of the RNA was performed with RNAstructure version 3.71 (D.H. Mathews, M. Zuker, and D.H. Turner).

Purification of Iron Regulatory Proteins (IRP1 and IRP2)-- IRP clones were kindly provided by W. E. Walden (rabbit IRP1) and E. A. Leibold (rat IRP2) to use with the human TfR-3'-UTR RNA sequences. Phylogenetic sequence conservation of the IRPs is >90%, although IRP1 and IRP2 are only ~60% identical (14, 19, 23, 43). The His-tagged IRPs were purified from the cytosol of Saccharomyces cerevisiae as previously described (33) with His-Bind Resin and Buffer kit (Novagen). Purified proteins were stored as small aliquots at -80 °C and used within 4-6 months.

Electrophoretic Mobility-shift Analysis (EMSA) of TfR mRNA Stability Element Complexes with IRPs-- 0.01-0.1 pmol of uniformly 32P-labeled 5xIRE transcript, pre-annealed in 50 mM NaCl, was incubated with various amounts of recombinant IRP (0.01-8 pmol) for 30 min at 4 °C. The binding reaction mixture comprised 24 mM Hepes pH 7.2, 60 mM KCl, 5% glycerol, 2% beta -mercaptoethanol, and 0.004 units/µl RNasin (Promega) in a final volume of 25 µl. Protein-RNA complexes were resolved from unbound RNA by non-denaturing gel electrophoresis as follows. Glycerol-based loading buffer was added to the sample and loaded onto 4% or 6% acrylamide gel (acrylamide: Bis = 19:1). Gels were run in 1× Tris borate-EDTA buffer for 3-7 h at 4 °C and 5-8 V/cm. Images of dried gels were visualized with a PhosphorImager (Amersham Biosciences). Comparison to single TfR-IRE binding used a single, chemically synthesized TfR-IREb (1xIRE) at the same IRP and IRE concentration (2 nM IRE) and binding conditions.

Filter-binding Assay for IRP Protection of TfR mRNA Stability Element Fragments-- Binding to nitrocellulose filters (36) was used for quantitative analysis of IRP interactions with TfR-3'-UTR RNA. To compare RNA with different IRE copy numbers, equal IRE concentrations (2 nM) but different concentrations of total RNA (5xIRE, 0.4 nM; 3xIRE, 0.67 nM; and 2xIRE, 1.0 nM) were used with IRP concentrations of 0.4-120 nM. Binding conditions used for the EMSA assay as described above were scaled up to a final volume of 50 µl for the nuclease resistance/filter binding analysis. Retention of RNA by nitrocellulose membranes (type BA79, 0.1 µm; Schleicher and Schull) in a slot-blot apparatus, with or without RNase T1 digestion (Roche Molecular Biochemicals or Ambion; 10 min with 3-196 units of nuclease) was analyzed after washing filters with 5 volumes of 50 mM KCl and vacuum drying (15 min). Under conditions of 100-200 units of RNase T1/ng RNA, defined by us as the "limit digest," >95% of the unbound RNA was digested to fragments shorter then 35 nt (detected by denaturing gel electrophoresis). RNase T1 was selected to assess IRP-dependent nuclease protection of the RNAs because 1) G residues, targeted by the enzyme, are scattered throughout the TfR-3'-UTR IREs and linking sequences; 2) at the salt concentration used (<100 mM NaCl), RNase T1 cleavage of RNA is relatively nonspecific (both single- or double-stranded G residues) (44); and 3) RNase T1 was active under the standard IRP binding conditions. Retained radioactivity was quantified with a PhosphorImager (Amersham Biosciences) and ImageQuant 5.0 software (Amersham Biosciences). Retention of free RNA (background values) was 1-2% of total applied radioactivity after RNase T1 digestion and 4-6% before digestion. Maximum filter-binding results were normalized to 100% binding for each IRP (60-80% of total applied radioactivity with all RNA constructs).

To analyze IRP protection of the linkers, filter binding was performed as described above with unlabeled in vitro transcribed 5xIRE RNA and saturating amounts of IRPs, as confirmed by filter-binding assay and total shift of the RNA in a non-denaturing gel. IRP·RNA complexes were fully digested with 196 units of RNase T1 (limit digest). Mixtures were filtered through a nitrocellulose filter as described above, and retained RNA was UV cross-linked to the membranes (StrataLinker; Stratagene). Membranes were hybridized with ExpressHyb® hybridization solution according to the manufacturer's recommendations (Clontech) to 10 pmol of 32P-end labeled antisense DNA oligomers (Invitrogen), specifically targeted to linker sequences between IREa and IREb, IRE b and IREc, and IRE c and IRCd (Fig. 1): bc1, nt 3513-3531; bc2, nt 3602-3621; bc3, nt 3682-3701; bc4, nt 3822-3841; bc5, nt 3864-3883; cd1, 3915-3932; cd2, 3933-3950 (numbering according to Ref. 41). No significant sequence similarity was found between the different probes. No hybridization signal was detected with an unrelated DNA oligomer probe.

Cu-(phen)2 Probing of IRE Structure in the Full Length (5xIRE) Transferrin Receptor mRNA Stability Element-- In vitro-transcribed TfR-3'-UTR RNA (5xIRE) in a final concentration of 50 nM was reacted with 18 µM Cu-(phen)2 as previously described (32). The RNA cleavage products were used as templates for 32P-labeled cDNA synthesis with the primer 5'-CACTGTGGTAGGTAAAAACTACCTTG-3', which bound 38 nt downstream of TfR-IREc, and the primer 5'-CAAGCTTTGAAGATGTCATTGC-3', which bound 32 nt downstream of TfR-IREe. The cDNAs were resolved on 8% acrylamide/8 M urea gels, calibrated by dideoxynucleotide sequencing of TfR-3'-UTR with reverse transcriptase, as previously described (45). Radioactivity was located and quantified with ImageQuant 5.0 software (Amersham Biosciences). Significant cleavage was determined as a signal which was threefold different than background values, i.e. cleavage in the absence of Cu-(phen)2 (45).

Northern Blot Analysis of IRP-mRNA Expression in Human Tissues-- Expression of IRP1, IRP2, and TfR was examined using poly(A)+ from a variety of human tissues and cell lines in the multiple tissue expression array filter (MTETM array; Clontech). Probes were prepared by PCR filter amplification of HeLa-cell cDNA (Clontech) with specific primers for IRP1 (sense, 5'-CAAGCAGGCACCACAGACTA-3'; antisense, 5'-ACCCTCAGTGCTCACTTGCT-3') and IRP2 (sense, 5'-GAATGCACCAAATCCTGGAG3-'; antisense, 5'-GAGAAACTGGCACAAGC-3'). Probes were designed based on alignment results of the nucleotide sequences of IRP1 (accession number AF261088) and IRP2 (accession number M58511) to yield PCR products from non-homologous regions: IRP1, 599 bp (2411-3009) and IRP2, 515 bp (420-934); this fragment of IRP2 included the coding region for the unique IRP2 73-amino acid insert (19, 43). Following purification (PCR purification kit; Qiagen), PCR products were confirmed by electrophoresis in agarose gels calibrated with molecular weight markers and stained with ethidium bromide. 200 ng of the DNA were labeled with [32P]dCTP (3000 Ci/mmol; Amersham Biosciences) and Klenow Random Priming labeling kit (Bio-Rad). Unincorporated NTPs were removed by gel filtration (ProbeQuantTM G-50 Micro columns; Amersham Biosciences). Hybridization (ExpressHyb® hybridization solution) and washing and stripping of the multiple tissue expression membrane were performed according to the manufacturer's recommendations (Clontech). No hybridization signals were detected with the yeast RNA or Escherichia coli RNA or DNA controls, confirming the specificity of the probes. Bound radioactivity was analyzed with a PhosphorImager (Amersham Biosciences) and quantified with ImageQuant 5.0. Radioactivity on each filter was normalized to constant specific activity (cpm/A260) to allow comparisons among the different cDNA probes in different tissues.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The IRE structure (~ 30 nt) is the site of IRP binding (46). Single IREs from a variety of mRNAs showed differential IRP1 and IRP2 binding, associating differences in IRE helix structure with different ranges of signal response (12, 47, 48). For a multiple-IRE regulatory structure such as the TfR-3'-UTR mRNA, the simplest model is independent binding of IRPs by each IRE (11, 14, 49). This model does not provide a satisfying explanation to the high evolutionary conservation of the IRE sequences (>90%; (2, 23) or the linkers (>80%),2 nor to the wide range of response of both TfR and ferritin mRNAs to signals such as iron (4), suggesting that TfR-IRE structure/function in the context of the multi-IRE TfR mRNA may vary from that of a single TfR-IRE.

The purpose of the present study was to evaluate the contributions of five TfR-IRE elements, in the context of the natural linking sequences, to the interaction with IRP1 and IRP2. A set of three RNAs with different IRE copy numbers was synthesized by in vitro transcription of DNA cloned from HeLa-cell cDNA as illustrated in Fig. 1. The native, full length TfR mRNA 3'-UTR regulatory element (5xIRE), with all five IREs and the wild-type linking sequences, was employed for IRP-binding experiments and for chemical cleavage by Cu-(phen)2. Truncated RNA constructs of the TfR mRNA 3'-UTR, with only 3 or 2 IREs (3xIRE and 2xIRE) were used to study the effect of IRE copy number on IRP-dependent nuclease resistance of TfR-3'-UTR.

Multiple Intermediates of IRP Binding to the Native TfR-3'-UTR RNA Sequence (5xIRE)-- With a multiple IRP binding sites RNA, binding of a protein to one IRE could affect the binding to other IRE sequences in the same RNA molecule, providing a possible explanation to the high conservation of five IREs in the TfR-3'-UTR RNA. To determine whether IRP binding to RNA with multiple binding sites is "all or none," the effect of multiple-IRE RNA on protein binding below saturation was analyzed by EMSA. An "all or none" interaction would result in a free RNA at sub-saturating IRP concentrations and a single shifted band representing fully bound RNA, when IRP concentrations are high enough to saturate all binding sites. Mobility shift analysis of complexes of 0.1 pmol of uniformly 32P-labeled TfR-3'-UTR regulatory element with increasing amounts of IRP1 (Fig. 2A) and IRP2 (Fig. 2B) revealed a number of distinct RNA/protein complexes, compared with the unbound 5xIRE RNA, and to a single shifted band observed at saturating IRP concentrations (2.5-8 pmol of IRP). Interestingly, with IRP1 (Fig. 2A), the number of complexes corresponded to the number of IRE sequences in the RNA, varying between 2 and 4 with complete conversion to a single, fifth complex, at saturating IRP1 amounts, suggesting that all five IREs were functional binding sites in the context of the native TfR-3'-UTR RNA regulatory element. However, in the case of IRP2, only 3 intermediates of IRP2 complexes with 5xIRE RNA could be detected as well as aggregates, especially at high IRP·RNA ratios, which may have prevented detection of the same number of intermediates observed with IRP1. It is also possible that IRP binding could induce a conformational change that affected the migration of IRP·RNA complexes. Nonetheless, formation of intermediates at sub-saturating concentration of both IRPs indicated that IREs could bind IRP independently when the protein was limiting and that protein could bind to fewer than five IREs at the same time.


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  		Fig. 2.   Intermediates of IRP complexes with the native TfR-3'-UTR RNA (5xIRE). EMSA of TfR-3'-UTR RNA (5xIRE) complexes with IRP1 (A) and IRP2 (B). 0.1 pmol of uniformly 32P-labeled 5xIRE transcript were incubated with increasing amounts of IRP (0.01-8 pmol of IRP) under standard conditions, and complexes were resolved in 4% polyacrylamide gels (see "Experimental Procedures"). The experiments were performed 4 times with IRP1 and 2 times with IRP2. Shown are representative gels with intermediates of IRP·5xIRE complexes at sub-saturating protein concentrations to demonstrate that IRP·RNA interaction is not "all or none." At saturating protein concentrations (IRP·IRE molar ratios: 5-16), a single shifted band was observed.

Multiple IREs in the TfR-3'-UTR RNA Stability Element Enhance IRP2 Binding Relative to a Single TfR-IRE-- To further compare the interaction of IRP1 and IRP2 with multiple-IRE TfR-RNA and their interaction with single TfR-IRE, we employed EMSA at saturating amounts of IRP1 and IRP2. The results showed that total binding of multiple TfR-IREs, in the context of TfR-3'-UTR RNA native sequence (5xIRE), was equivalent for both IRP1 and IRP2, thus eliminating previously observed differences between IRP1 and IRP2 binding by single TfR-IRE or other C-bulge IREs (DMT1, erythroid-aminolevulinate synthase, and m-aconitase) (33, 40). For example, as shown in Fig. 3A, IRP2 activity that shifted 100% of the 5xIRE could shift only ~30% of a single TfR-IREb relative to maximally IRP1-bound TfR-IREb. Clearly, the multiple TfR-IRE RNA sequence enhanced IRP2·IRE interaction compared with single TfR-IRE binding by this protein.


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  		Fig. 3.   Differential IRP1 and IRP2 binding to single TfR-IRE is eliminated in the context of the multiple-IRE TfR-3'-UTR RNA (5xIRE). A, IRP interaction with the multiple-IRE TfR-3'-UTR RNA (5xIRE) was compared with the interaction with a single TfR-IREb (1xIRE) by EMSA. Saturating amounts of IRP1 and IRP2 were incubated with 5xIRE and 1xIRE RNAs (while maintaining an equal IRE concentration of 2 nM) under standard binding conditions (see "Experimental Procedures"), and complexes were resolved in 6% polyacrylamide gels. IRP2·1xIRE complexes migrated faster then IRP1·1xIRE complexes as previously reported (38, 43, 51, 52). Unbound TfR-IREb is not shown in the figure because it migrated so much faster than the other species. B and C, 5xIRE RNA (0.02 pmol of RNA; 2 nM IRE) binding activity of IRP1 (B) and IRP2 (C) (0.02-6 pmol of IRP) was monitored after RNase T1 digestion (6 units/ng RNA) and filtration through nitrocellulose filters as described under "Experimental Procedures." Data were analyzed as percentage of maximally retained radioactivity (60-80% of total applied radioactivity). Binding was saturable with a linear fit higher than 0.95 at subsaturating IRP·IRE ratio. Representative data from 3 independent experiments with different protein and RNA preparations are shown.

The similarity of IRP1 and IRP2 interaction with the multiple TfR-IRE element was further confirmed by filter-binding assay that enabled us to quantitatively assess and compare the interaction of IRP1 and IRP2 with the RNA. IRP binding was analyzed as the uniformly 32P-labeled 5xIRE RNA retained on a filter following binding and nuclease digestion (6 units/ng RNA). Data are shown in Fig. 3, B and C. The relationship between IRP·IRE ratio and protein binding was linear for both IRPs (the correlation coefficient for the linear plot of the data was >0.95), emphasizing the independence of IRP binding by individual IRE structures in the RNA. Binding was saturable, and both proteins saturated 5xIRE RNA binding at similar IRP·IRE molar ratios (>10). If the binding of IRP to a single IRE changed significantly the binding properties of the remaining IRE sequences in the RNA, the binding versus protein/RNA ratio would be expected to deviate from linearity.

Probing TfR-3'-UTR RNA with Cu-(phen)2 Detected a Specific Structure of TfR-IREs in the Context of the Native Sequence-- The enhanced interaction of IRP2 with TfR-3'-UTR RNA compared with poor binding to a single TfR-IREb suggested a structural change in TfR-IREs when integrated with other TfR-IREs and the native, non-IRE linking sequences. To address this possibility, we tested the reactivity of TfR-IREc with Cu-(phen)2 in the context of 5xIRE RNA. Cu-(phen)2 is a chemical nuclease with specific three-dimensional structure that has been previously shown to recognize, i.e. bind and cleave, RNA helix distortions (50). The reactivity of TfR-IREs in the context of the 5xRNA, annealed the same way as for the IRP binding and for the nuclease resistance experiments, was markedly changed compared with single TfR-IREc (32). Two major cleavage sites, G9 and A25, were detected in TfR-IREc in the context of 5xIRE RNA (Fig. 4, A and B). With respect to the secondary structure of TfR-IREc (Fig. 4C), these residues bracketed the C-bulge of the IRE helix, in a similar manner to the modification by Cu-(phen)2 in the internal loop/bulge of ferritin IRE helix, which is a specific determinant for IRP2 binding (33). Cu-(phen)2 cleavage sites analogous to those observed for TfR-IREc within the context of 5xIRE were also observed for TfR-IREe (data not shown) in the same context, further supporting the hypothesis that positioning of TfR-IREs within native non-IRE sequences modifies TfR-IRE structure around the C-bulge to enable enhanced IRP2 binding.


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  		Fig. 4.   Chemical probing of the native TfR-3'-UTR RNA (5xIRE) revealed a Cu-(phen)2 modification site in the mid-stem of TfR-IREc. A, denaturing 8% polyacrylamide gel showing cDNA products from reverse transcription of full length TfR-3'-UTR RNA incubated with (+) or without (-) Cu-(phen)2. The region of TfR-IREc is boxed. Two major cleavage sites within the region of TfR-IREc, corresponding to cleavage at G9 and A25, are indicated by arrows. B, densitometry scanning of the IREc region shown in panel A. Arrows indicate a >3-fold increased signal at the Cu-(phen)2 cleavage sites (+Cu), compared with background (-Cu). C, arrows denote the location of the Cu-(phen)2 cleavage sites relative to the secondary structure model of TfR-IREc in the context of 5xIRE. Modeling was performed with RNAstructure software version 3.71 (D.H. Mathews, M. Zuker, and D.H. Turner). The experiments were performed 4 times with 2 different RNA preparations, and representative results are shown.

IRP Binding and Nuclease Resistance of the TfR-3'-UTR RNA Are Enhanced with 5xIRE Compared with 2xIRE or 3xIRE RNAs-- To investigate further the question of why five IRE sequences are phylogenetically conserved in the TfR-RNA, we compared the interaction of IRP·IRE complexes formed with 5xIRE, to complexes formed with 3xIRE and 2xIRE RNA constructs (see Fig. 1), using filter-binding assay as described above (Fig. 3). Comparison of IRP interaction with different IRE copy number RNAs was possible by maintaining an equal concentration of IRE sequences in each reaction mixture, employing different total concentrations of each uniformly labeled RNA construct (0.4-120 nM IRP and 2 nM IRE; see "Experimental Procedures"). Retained radioactivity was quantified and normalized to 100% binding of the RNAs by each protein preparation. We first performed filter-binding assays using protein concentrations encompassing both linear and saturating IRP·IRE molar ratios for 5xIRE, and 6 ubits/ng RNA nuclease T1. Lower IRP concentrations per IRE were required to render higher resistance to nuclease T1 digestion in the case of the native 5xIRE RNA sequence, relative to lower IRE copy number RNAs. For example, at equal IRP·IRE ratio, ~75% of the wild-type 5xIRE RNA was retained on the filter compared with only 40% of 3xIRE or 25% of the 2xIRE constructs (data not shown). To confirm differential nuclease sensitivity of RNAs with low IRE copy number (<5), we asked whether the variations between the different IRE copy number RNAs could be observed when all IREs were maximally bound (protected), and RNase concentration was increased to the limit digest (196 units/ng RNA; see "Experimental Procedures"). Under saturating concentrations of IRP1 or IRP2, measured as the IRP amount required for maximum retention of RNA on the filter, and for complete shift of the RNAs in non-denaturing gel, increasing the RNase concentration still displayed differential protection of RNA in the IRP·5xIRE complexes (Fig. 5, A and B). This observation implied that the interaction between IRPs and IREs in the context of 3xIRE and 2xIRE RNAs is less effective in protecting the RNA from nuclease digestion. The differences that we observed between the different IRE copy number RNAs could be attributed to the higher ratio of nucleotides to IREs in the lower IRE copy number RNAs (3xIRE and 2xIRE) compared with the native sequence, 5xIRE. However, the difference in the number of nucleotides per IRE between 3xIRE and 2xIRE (5xIRE, 132 nt/IRE; 3xIRE, 172 nt/IRE; 2xIRE, 238 nt/IRE) would have predicted an increased resistance of the 3xIRE compared with 2xIRE whereas we observed equal sensitivity of 2xIRE and 3xIRE to nuclease. A prediction from the data, that IRP-protected RNA fragments resolved in calibrated denaturing gels will be larger than the IRE itself, was confirmed: when compared with 3xIRE and 2xIRE RNAs, a larger fraction of the 5xIRE RNA, in fragments ranging between 50 and 120 nt, were detected in the limit digest of IRP·5xIRE RNA complexes (data not shown). Furthermore, a nuclease-resistant ~50-nt fragment was also detected when the unbound 5xIRE RNA was limit-digested (data not shown), indicating that native sequences in 5xIRE RNA missing in 3xIRE or 2xIRE RNAs conferred a specific conformation to the full length, unbound native TfR-3'-UTR RNA sequence, which may contribute to IRP binding and/or nuclease resistance.


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  		Fig. 5.   Effect of IRE-copy number on the IRP-dependent nuclease protection. Comparison between the IRP-dependent nuclease resistance of maximally bound RNAs with different IRE copy number (5xIRE, 3xIRE, and 2xIRE). IRP·RNA complexes were formed with saturating IRP concentrations (IRP/IRE ratio = 12), digested with increasing amounts of RNase T1 (3-196 units/ng RNA), and filtered through 0.1-µm nitrocellulose filter as described under "Experimental Procedures." Data were analyzed as percentage of maximally retained radioactivity (without nuclease digestion). A, IRP1-dependent nuclease protection. B, IRP2-dependent nuclease protection. Asterisk denotes significant 5xIRE resistance (p < 0.04, Student's t test; n = 3) compared with 3xIRE and 2xIRE. The experiments were performed 3 times with different RNA preparations, and representative results are shown.

The high percentage of retention of the IRP-bound 5xIRE RNA (~50-70%; see Fig. 5, A and B), suggested that the RNA was "over-protected", i.e. not only were the IREs protected (only ~20% of the RNA sequence), but non-IRE sequences were also protected. To assess which non-IRE sequences were protected, Northern blot analysis of nuclease limit-digested IRP1 and IRP2 complexes with unlabeled 5xIRE, using specific [32P]DNA probes (~20 nt) targeted to non-IRE sequences was performed (see "Experimental Procedures"). The results of a single set of experiments (data not shown) indicated that sequences in the b/c linker close to TfR-IREc, and sequences in the c/d linker near TfR-IREd were relatively inaccessible to the DNA probe in the IRP·5xIRE complexes (10-50% of probe hybridization to undigested IRP·RNA complexes), whereas linking sequences in IRP complexes with 2xIRE were poorly protected (<1-10%). IRP1 protection of non-IRE sequences was previously observed with Pb hydrolysis of IRP1 complexes with TRS-1, a 3xIRE RNA with truncated b/c linker (18). Here, using linker hybridization probes, we extended these observation to the interaction of the native TfR-3'-UTR sequence with both IRP1 and IRP2.

Tissue and Developmental Specificity of IRP2 Expression-- The enhanced IRP2 binding observed in the 5xIRE compared with a single IRE, or RNAs with 2 or 3 IREs would be advantageous if the ratio of IRP1 and IRP2 varied widely in different cell types or during development. Recent studies that show the impact of IRP2 deletion (51) on the central nervous system emphasized the role of IRP2 expression on normal physiology and development. To extend the information on variations in IRP2 expression relative to IRP1 (19, 43, 52) to a broad range of cell types, IRP expression was examined using human MTETM array of poly(A)+ RNA from 76 human tissues (Clontech) and 32P-end labeled DNA probes specific to IRP1 and IRP2.

A wide variation in tissue expression was observed for each mRNA tested and between each mRNA in the same tissue. Large ranges in the amount of IRP1 mRNA, of as much as 10-fold, were observed, with the lowest expression in the brain and the highest in kidney and liver (Fig. 6A), as previously observed for protein levels in rabbit, rat and mouse (19, 20, 43, 52). The highest expression level of IRP2 was in testis, but IRP2 predominated in most tissues. IRP2 expression was also high in HeLa and HL-60 cells and in other cell lines (data not shown), as previously observed (43, 53), and in tumor tissue (lung carcinoma; data not shown), which may relate to up-regulation of IRP2 mRNA expression during cell proliferation and transformation by c-myc (54) or in regenerating liver (22). In contrast, IRP2 protein level was reported to be below the limits of detection in spleen, lung, and lymph node (52), although the mRNA levels were relatively high (Fig. 6A), emphasizing tissue specificity of IRP2 post-translational regulation (22). When IRP2/IRP1 ratios were determined (Fig. 6B), in only 3 tissues (heart apex, liver, and kidney) did the ratio approach 1, and in many tissues was 3-12. Differences between IRP1 and IRP2 expression were also observed in the earlier developmental stage with IRP2/IRP1 ratios of 4-6 in most fetal tissues (Fig. 6B). IRP2-mRNA levels in adult tissues dropped significantly compared with the levels in the same tissue from fetus, with the exception of liver tissue (Fig. 6A).


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  		Fig. 6.   Variable expression of IRP1 and IRP2 mRNAs in human tissues. A, poly(A)+ RNA from multiple human tissues and cell lines was probed with specific 32P-labeled cDNA probes for IRP1 and IRP2 using the MTE array from Clontech. Autoradiograms were scanned with PhosphorImager, quantified with ImageQuant (5.0), and normalized using the specific activity of each probe (cpm/A260). WBC, white blood cells; BM, bone marrow; B, brain; SPLN, spleen; TES, testis; TYM, thymus; SM, skeletal muscle; TG, thyroid gland; LNG, lung; HIP, hippocampus; AG, adrenal gland; STM, stomach; H, heart; APX, apex of the heart; L, liver; K, kidney. Fetal tissues are marked with f, to demonstrate developmental regulation of IRP expression. In addition to the tissues shown, IRP2 expression was 3- to 10-fold higher than IRP1 expression in cultured cells and in tumor tissue (lung carcinoma). B, ratios of IRP2/IRP1 expression. Black bars, IRP2; Striped bars, IRP1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stability elements in mRNA mediate the essential function of post-transcriptional regulation of mRNA decay. Messenger RNAs encoding proteins involved in iron and oxygen metabolism are specifically regulated by IREs, a family of mRNA helix-apical loop structures in the UTR of these mRNAs that display differential binding of the two iron regulatory proteins (IRP1 and IRP2) (12, 48, 55). When IRP binding to single IRE structures was studied, IRP2 was more selective in IRE binding than IRP1 and displayed differential recognition of both native mRNA-specific IRE structures as well as mutated IRE sequences (32, 33, 40). The best characterized difference among native IRE sequences is in the stem of the hairpin loop, which is interrupted either by a C-bulge or by an internal loop/bulge (IL/B) (15-17, 32-34, 40). Whereas IRE binding by IRP1 requires the apical loop with a conserved CG base pair spanning it, IRP2 requires both the apical loop and an IL/B spanned by another conserved CG base pair, as in the structure of ferritin-IRE. Thus, several naturally occurring C-bulge IREs, including a single transferrin receptor IRE (~30 nt) bind IRP2 much more poorly than IRP1 (33, 40).

Our results showed similar IRP1 and IRP2 interactions (binding/nuclease protection) with the C-bulge TfR-IREs when analyzed in the context of the multiple-IRE TfR-3'-UTR RNA that contrasted with the less effective binding of IRP2 to a single TfR-IRE (Figs. 3 and 5). A structural basis for the observed discrepancy was provided by probing the RNA with a shape-selective transition metal complex, Cu-(phen)2, a 1:2 complex of Cu and 1,10-phenanthroline that binds to small bends and loops of tRNA. Redox properties of Cu-(phen)2 yield radicals that cut RNA in the vicinity of the RNA bending site (50). Cu-(phen)2 specifically binds the internal loop/bulge of the ferritin IRE, but appears to be too large to bind the C-bulge in other IREs (3, 50) or in ferritin IRE with U6 deletion in the IL/B, that converts the IL/B in the helix to a bulge (32, 33). The site of Cu-(phen)2 binding in the helix of the ferritin mRNA is the same site associated with metal binding, proton-induced disorder, and IRP2 binding in solution (17, 32). Recent studies with HeLa cells confirm that the specific RNA fold of the ferritin IL/B, recognized by Cu-(phen)2, occurs in vivo as well (56). In the present study, using the wild-type TfR-3'-UTR RNA (5xIRE), Cu-(phen)2 mediated the cleavage of nucleotides in the C-bulge region of TfR-IREc (Fig. 4) and TfR-IREe, displaying a structural change of TfR-IREs in the context of multiple-IRE RNA (5xIRE) compared with the single, Cu-(phen)2-unreactive TfR-IRE. The relationship between IRP2 binding and the mid-helix IRE distortion of different IREs, as detected by Cu-(phen)2 modification, is illustrated in Fig. 7. The relative roles of the multiple IRE sequences and the linking sequences on the change in the TfR-IRE helix induced by the context of the full length TfR-3'-UTR structure is only a matter for conjecture at this time. However, our results provide a clear explanation for the enhanced binding of IRP2 by IREs in the full length TfR-3'-UTR RNA structure.


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  		Fig. 7.   A distortion in the stem of IREs correlates with selective Cu-(phen)2 binding and strong IRP2 binding. Shown are models for secondary structures of TfR-IREc in the context of the full length TfR-3'-UTR regulatory element (5xIRE), ferritin-IRE (30-mer), and single TfR-IREc (29-mer). Specific Cu-(phen)2 binding/cleavage sites, detected in the mid-stem of TfR-IREc within 5xIRE and in the internal loop/bulge region of ferritin-IRE (arrows), correlate with strong IRP2 binding, whereas single TfR-IREc, which is not modified by Cu-(phen)2, binds IRP2 poorly (32). A context-dependent conformational change in TfR-IREs within the multiple-IRE RNA sequence (5xIRE) is suggested (see "Discussion"). S, strong IRP binding; W, weak IRP binding.

The results of IRP binding and nuclease protection confirmed that multiple-IRE RNA, i.e. with 2 or more copies of IREs, is sufficient to mask the differential IRP binding to single TfR-IREs, since IRP1 and IRP2 bound equally well and protected 2xIRE and 3xIRE from nuclease degradation (Fig. 5). This observation provides an explanation for the apparent independent interaction of IRPs with the multiple IRE RNAs (Figs. 2 and 3). Will partially IRP-bound TfR-3'-UTR mRNA protect the TfR mRNA in vivo (e.g. when IRP levels are relatively low and only 2 or 3 IREs are occupied by the proteins (Fig. 2))? Partial binding, resulting in limited nuclease protection, may further expand the combinatorial nature of IRP·IRE interaction in the cell (2) and is in agreement with the observation of iron regulation of a TfR-3'-UTR construct in vivo with 2 wild-type IREs and one mutant TfR-IRE element (42).

When fully saturated (protected) IRP complexes with TfR-3'-UTR RNAs were challenged with high nuclease concentrations (Fig. 5), the IRP-dependent protection of the wild-type TfR-3'-UTR RNA (5xIRE) was more effective than with either 2xIRE or 3xIRE, emphasizing the requirement for the full length native TfR-3'-UTR mRNA regulatory element to generate an efficient interaction with IRPs. The advantage of multiple TfR-IRE RNA, and the importance of the context for TfR-IRE interaction with IRPs, is in agreement with a previous observation, that a single TfR-IRE, engineered between the ferritin promoter and a reporter gene, mediated translational iron regulation poorly compared with ferritin-IRE (13). The full length TfR mRNA regulatory element (5xIRE) contains sequences that are absent in 3xIRE and 2xIRE (IREa, IREe, and linker d/e). The high sequence conservation of IREa and IREe, and of linker d/e, further supports the notion that although the IREs make the major contribution to the IRP interactions, other sequences, essentially linking the IRE elements to each other, contribute to the interaction of the IRPs with the RNA. Thus, the conclusion regarding the requirement for more then 2 copies of IREs for similar & effective binding/protection of TfR-RNA by IRP1 and IRP2 has to be refined to include the native non-IRE linking sequences. The specific role of the linker sequences and the relationship to nuclease resistance of the RNA-protein complex (Fig. 5) are not shown. Several possible roles of the linkers could be, first, interaction with AU-rich element-binding proteins (7, 57), possibly controlled by IRP (exposing or masking AU-rich elements in the linkers). (IRP-dependent changes in nuclease sensitivity of flanking sequences of ferritin mRNA have been reported (46, 58, 59).) Second, there may be an effect of the linkers on the IRE-helix structure itself (Fig. 4).

The physiological significance of efficient IRP2 binding to ferritin-5'-UTR IRE and TfR-3'-UTR IREs is exemplified by iron-dependent changes in ferritin biosynthesis and TfR mRNA levels in a pre-B lymphocyte cell line lacking IRP1 expression (60) and by the distinctive distribution pattern of IRP1 and IRP2 in the normal mouse brain (61), where misregulation of iron homeostasis is associated with neurodegenerative diseases, as in mice with targeted IRP2 deletion or in brains of Alzheimer patients where IRP·IRE interaction is altered (51, 62). In contrast to TfR and ferritin, proteins synthesized from single, C-bulge IRE mRNAs, such as m-aconitase, erythroid-aminolevulinate synthase, DMT1, and ferroportin should be very sensitive to cell-specific differences in IRP1 and IRP2 compared with TfR mRNA or ferritin mRNA, and will probably be mainly regulated by IRP1. An example of an in vivo selective response mediated by different IRE sequences and differential IRP binding was provided when ferritin synthesis in the liver of rats exposed to increased iron concentrations was found to be greatly induced compared with m-aconitase synthesis (48). Accordingly, in contrast to effective translational inhibition of ferritin after overnight exposure of RAW 264.5 cells to NO, m-aconitase levels did not change (63). These observations could be related to more stable IRP1 and IRP2 binding to ferritin IRE (33, 48). Here we show that the range of IRP1 and IRP2 expression ratio varied among different cell types, and in most cells IRP2-mRNA predominated (Fig. 6). In these cells, the synthesis of proteins encoded in mRNAs with a single C-bulge IRE will display much lower sensitivity to changes in iron or other IRP-activating signals because of the poor binding by IRP2. Full repression of these proteins would likely not occur even at full IRP-binding activity, unless the expression of IRP was very high, e.g. in testis tissue. Superimposed on cell-specific variations in IRP1 and IRP2 mRNA concentrations will be post-translational modification, such as phosphorylation or formation of the [4Fe-4S] cluster in the case of IRP1, or regulated degradation in the case of IRP2 (2, 22, 52), that further expand the possible RNA/protein combinations in any specific cell type. It is clear that the abundant expression of both IRPs will ensure critical regulation of cellular iron levels by TfR and ferritin as a result of the greater impact of IRP binding on the single ferritin IRE and multi-IRE element in the TfR-3'-UTR RNA, which places these messages at the maximum range of regulation by iron and oxygen.

The possible interrelationships of the IRE and the linkers in the TfR-3'-UTR mRNA emphasize the importance of context for RNA structure/function relationships as demonstrated by Cu-(phen)2 probing and IRP2 binding. Unraveling further the influence of TfR-IREs and the linkers on each other, and the possible interaction between IRP1 and IRP2 in vitro and in vivo, to control mRNA stability, should yield important insights for understanding iron homeostasis and mRNA stability.

    ACKNOWLEDGEMENT

We are grateful to Dr. H.E. Johansson for insightful scientific discussions.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant DK-20251.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Children's Hospital Oakland Research Institute, 5700 Martin Luther King, Jr. Way, Oakland, CA 94609-1672. Tel.: 510-450-7670; Fax: 510-597-7131; E-mail: etheil@chori.org.

Published, JBC Papers in Press, August 27, 2002, DOI 10.1074/jbc.M207918200

2 R. Erlitzki, H. E. Johansson, and E. C. Theil, unpublished results.

    ABBREVIATIONS

The abbreviations used are: UTR, untranslated region; TfR, transferrin receptor; IRP, iron-responsive protein; IRE, iron-responsive element; m-aconitase, mitochondrial aconitase; DMT1, divalent metal iron transporter-1; IL/B, internal-loop/bulge; EMSA, electrophoretic mobility-shift analysis; Cu-(phen)2, copper 1,10-phenanthroline; nt, nucleotide.

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ABSTRACT
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RESULTS
DISCUSSION
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