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Nucleic Acids Research, Vol. 18, No. 5

The mRNA-binding protein which controls ferritin and transferrin receptor expression is conserved during evolution Sylvia Rothenberger, Ernst W.MuLllner and Lukas C.Kuhn Swiss Institute for Experimental Cancer Research, Genetics Unit, CH-1066 Epalinges, Switzerland Received November 24, 1989; Revised and Accepted February 6, 1990

ABSTRACT A post-transcriptional regulatory protein, termed iron regulatory factor (IRF), that binds specifically to the iron-responsive elements of ferritin and transferrin receptor mRNA, has recently been identified in the cytoplasm of human and mouse cells. Activation of this factor by low intracellular iron levels leads to inhibition of ferritin translation and an increase of TR mRNA stability. To investigate whether these feedback regulatory mechanisms are conserved during evolution, we analysed cytoplasmic extracts from 12 different species for a specific IRE-binding activity. We found mRNA-binding proteins in chicken, frog, fish and fly, which are equivalent to human and mouse IRF in gel-retardation assays with radiolabeled RNA transcripts. Competition experiments, molecular weight determinations, and modulation of the mRNA-binding activity in response to intracellular iron levels or reduction by j-mercaptoethanol indicate that IRF has similar structural and functional properties in these different species. INTRODUCTION Iron-responsive elements (IREs) are short palindromic sequences that have been found as single copies within the 5' untranslated region (UTR) of human, rat, chicken, and frog ferritin mRNAs (1-4) and as five repeats in the 3' UTR of human and chicken transferrin receptor (TR) mRNA (5-7). These elements are essential to the iron-dependent regulation of ferritin (2,3,8-10) and human TR expression (5,6,11,12). A cytoplasmic protein, termed iron regulatory factor (IRF), IRE-binding protein (IREBP), or ferritin repressor protein (FRP), that binds specifically the IREs from ferritin (13-16) and TR mRNA (6,17) has recently been identified in human, rabbit and rodent cells. Through its interaction with IREs, IRF inhibits ferritin mRNA translation (15,18) and increases the stability of TR mRNA (6,12). The mRNA-binding activity of IRF is induced by low intracellular iron levels (6,13,14), indicating that IRF plays a central role in cellular iron metabolism. Concerning the mechanism by which iron modulates IRF activity, it has been observed that iron salts or chelators have no direct effect on the binding of IRF to RNA in vitro (6,19). Instead it was found that the in vitro RNA-protein interaction depends on the reduction

of intramolecular sulfhydryl groups by 3-mercaptoethanol (19). Since IRF can not be further activated in extracts from cells that have been exposed to desferrioxamine, this mechanism of activation appears to be physiologically important. Iron transport, uptake, and storage by transferrin, TR and ferritin, respectively, are well documented in vertebrate cells. However, much less is known about the existence of homologous proteins in the iron metabolism of lower species (20-22). The conservation of IRE sequences among mammals, birds and amphibians suggested a common post-transcriptional mechanism regulating iron homeostasis. It was of interest therefore to test directly whether IRF is present among the different vertebrate species, and possibly beyond among invertebrates, lower eukaryotes and prokaryotes. The results of the present study show that conservation of a non-coding RNA structure can indeed serve in predicting post-transcriptional RNA-protein interactions.

MATERIALS AND METHODS Preparation of tissue and cell extracts The following cell lines were used in this study: human HL-60 (23), mouse Ltk- (24), chicken DU249 (25,26), Xenopus laevis A6 (27), and Drosophila melanogaster Schneider's cell line 3 (28). For treatment with iron salts or iron chelator, cells were incubated for 24 h with medium containing 20 Atg/ml ferric ammonium citrate or 100 AM desferrioxamine (Desferal, gift from Ciba-Geigy, Basel, Switzerland), respectively. Cytoplasmic extracts were prepared as described (6) in extraction buffer containing 10 mM HEPES (pH 7.6), 3 mM MgCl2, 40 mM KCl, 5% glycerol, 2 mM dithiothreitol and 0.5 % Nonidet P40. Trout (Salmo gairdneri) liver and total worms (Tubifex tubifex) were homogenized directly in the extraction buffer. Yeast cells (Saccharomyces cerevisiae and Saccharomyces pombe) were broken using glass beads in extraction buffer, and plant cells (Beta vulgaris, Physcomitrella patens) were pulverized under liquid nitrogen and extraction buffer added to the homogenates. Escherichia coli bacteria were sonicated in extraction buffer. All extracts were prepared in the presence of 1 mM phenylmethylsulfonyl fluoride and 50 Ag/ml aprotinin. The lysates were cleared by two centrifugations at 10,000xg for 5 min. Protein concentrations were determined by the Bio-Rad (Bio-Rad, Richmond, CA) protein assay.

1176 Nucleic Acids Research

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Figure 1. Detection of IRF in cytoplasmic extracts from various species. Radiolabeled in vitro transcripts (0. 1 ng) containing either the human TR palindrome E (pSPT-TR34) or the human ferritin heavy-chain IRE (pSPT-fer) were incubated with various concentrations of protein extracts from indicated species. RNA-protein complexes were treated with ribonuclease T1 and heparin and analysed in 6% non-denaturing polyacrylamide gels.

In vitro transcription The plasmid constructions used for in vitro transcription of probes and competitor RNAs have been described previously (6). Transcripts from pSPIT-TR12 and pSPT-TR34 contain the human TR palindromes A or E (5), respectively, those from pSPT-TR13 correspond to the 3' UTR of human TR mRNA in inverse orientation, and those from pSPT-fer have the human ferritin heavy-chain IRE. Transcription reactions were performed with 1 pig of linearized plasmid in the presence of 100 ,tCi of [ae-32P]CTP (800 Ci/mM)(Amersham, Buckinghamshire, UK), 2.5 mM ATP, GTP, UTP, and 20 units of T7 RNA polymerase (Boehringer Mannheim, FRG) in a final volume of 20 pd. Samples were incubated for 1 h at 37°C. The specific activity of the transcripts was 1.3 x 109 dpm/Ag. Unlabeled RNAs were transcribed with all four rNTPs at 2.5 mM. Full length transcripts were purified on a Sephacryl S-200 column (Pharmacia, Uppsala, Sweden).

Gel-retardation assay RNA-binding reactions were carried out as described previously (6,13). Cytoplasmic proteins (0.4 to 50 ,tg) and 0. 1 ng radiolabeled RNA (1.3 x 109 dpmn/g) were incubated for 30 min at room temperature in a final volume of 20 Al extraction buffer. Subsequently, unprotected RNA was digested for 10 min with 1 unit of ribonuclease TI (Calbiochem, San Dieggo, CA). To compete for unspecific RNA-protein interactions, heparin (Sigma, St.Louis, MO) was added at a final concentration of 5 mg/ml and the samples further incubated for 10 min at room temperature. The complexes were resolved in 6 % nondenaturing polyacrylamide gels. Gels were dried on a nitrocellulose filter and autoradiographed. Quantitation was performed by densitometric scanning of appropriately exposed autoradiographs. Molecular weight determination of IRF For UV crosslinking, ribonuclease T, resistant RNA-protein complexes were irradiated under a UV lamp (Philips, TUV 15 W, 10 mW/cm2) for 30 min as described previously (6,13). The samples were analysed in 10% SDS-polyacrylamide gels under reducing conditions. For gel filtration, 100 jig cytoplasmic

proteins were separated on a Superose 12 column by FPLC (Pharmacia). Fractions of 200 y1 were collected and tested for RNA-binding activity by the gel-retardation assay. The column was calibrated with catalase (232 kDa), immunoglobulin G (160 kDa), transferrin (80 kDa), and ovalbumin (43 kDa) as standards.

RESULTS AND DISCUSSION Detection of IRF in cytoplasmic extracts from different species To detect specific interactions between IRE-containing RNA and IRF in cellular extracts from various species, we applied a gelretardation assay (6,13). Crude cytoplasmic proteins from tissues or cultured cells were incubated with radiolabeled RNA transcripts containing either the human TR palindrome E (pSPTTR34) or the human ferritin heavy-chain IRE (pSPT-fer). After ribonuclease T, digestion the complexes were resolved in 6% non-denaturing polyacrylamide gels. RNA-binding activity was clearly detectable in extracts from human, mouse, chicken, frog, fish, and fly (Fig. 1), suggesting that IRF has evolved prior to the evolutionary divergence of vertebrates and insects. Both human TR or ferritin IRE were equally well recognized. Cell lines from distantly related species such as mouse, frog and fly, had the same relative concentration of the IRE-binding protein, as judged by densitometric scanning of the autoradiographs. The lower signal obtained with fish liver extract is probably due to a high proportion of serum proteins. With the exception of two distinct RNA-protein complexes with mouse extracts (Fig. 1 and refs. 6,13,17), there is no apparent heterogeneity in the gelretardation pattern with extracts from other species. The two bands observed in the mouse could not be attributed to differential RNA digestion, since the pattern was unchanged if the binding assays were performed without ribonuclease T, treatment (data not shown). It remains unknown whether the two complexes are due to a heterogeneity of IRF, or an additional component that binds the RNA-protein complex. The possibility of minor charge or size differences in the IRF of the other species can not be excluded despite a homogeneous migration of the RNA-protein complexes in non-denaturing gels. Besides the species presented in Fig. 1, we found IRF to be

Nucleic Acids Research 1177 pretreatment of cells

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Figure 2. Specificity of the RNA-protein interaction. Five ,ug of cytoplasmic protein extract from human or fly were incubated with 0.05 ng radiolabeled probes containing either the human TR palindrome E (pSPT-TR34) or the human ferritin heavy-chain IRE (pSPT-fer) and increasing amounts of unlabeled RNA containing either the human ferritin heavy-chain IRE (pSPT-fer), human TR palindrome E (pSPT-TR34), human TR palindrome A (pSPT-TR12) or an antisense strand from the 3' UTR of human TR mRNA (pSPT-TR13).

also present in the annelid worm Tubifex tubifex. However, no IRE-binding protein could be detected in extracts from fission and budding yeast, plants or bacteria (data not shown). Identical results were obtained if the stringency of the assay was lowered by omitting the ribonuclease T1 digestion, or in the presence of the ribonuclease inhibitor RNasin. The addition of a 10-fold excess of yeast extract to human IRF did not affect the formation of human RNA-protein complex, thus excluding protein degradation by a protease activity in the yeast extract (data not shown). Therefore, it is likely that IRF is either not present in lower eucaryotes and procaryotes, or that its divergence in evolution precludes recognition of the human IRE probes. The characterization of human IRF binding to the various IREs from human TR mRNA has revealed that palindromes B and E are stronger binding sites than palindromes A, C and D (17). We analysed the conservation of IRF in human and fly extracts by competition experiments with increasing amounts of various unlabeled RNAs (Fig.2). In both cases, the formation of the RNA-protein complexes was inhibited by unlabeled human ferritin and TR IREs, but not with an antisense RNA from 3' UTR of human TR mRNA (pSPT-TR13). Invariantly, ferritin IRE (pSPIT-fer) was the strongest competitor, human TR

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Figure 3. Iron-dependent activation of IRF. Cell lines from indicated species were grown for 24 h in presence of 20 jig/mn ferric ammonium citrate or 100 tcM desferrioxamine. Five gg of cytoplasmic protein extract were incubated with radiolabeled RNA (0.05 ng) containing human ferritin heavy-chain IRE (pSPTfer) and increasing amounts of unlabeled RNA containing either the human ferritin heavy-chain IRE (pSPT-fer) or human TR palindrome A (pSPT-TR12).

palindrome E (pSPT-TR34) slightly less effective, and human TR palindrome A (pSPT-TR12) a weak competitor. These results indicate that fly cells contain an RNA-binding factor that exibits the same specificity and relative affinity for IRE sequences as human IRF.

Modulation of the RNA-binding activity of IRF Previous experiments indicated that the RNA-binding activity of IRF in human and mouse cells is induced by low intracellular iron levels (6,13,14). We compared the IRE-binding activity in the different species by cultivating the respective cell lines for 24 h in the presence of either ferric ammonium citrate or the iron-chelator desferrioxamine. With the exception of mouse Ltk- cells, the treatment with ferric iron generated a 2- to 3-fold reduction of the IRE-binding activity in the extracts from the various cell lines tested (Fig.3). No further effect was observed if the cells were treated for 48 or 72 h with iron (data not shown). Human, mouse, chicken and frog cells that had been exposed to desferrioxamine showed a clear 6- to 8-fold increase in their

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1178 Nucleic Acids Research

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Figure 4. Induction of the RNA-binding activity by ,B-mercaptoethanol. One 14g protein from human, mouse, frog and fly extracts, or 5 jig from chicken and fish liver extracts were pre-treated for 30 min at room temperature with the indicated concentration of,-mercaptoethanol. The gel-retardation assay was performed with an excess of radiolabeled RNA (0.6 ng) containing human TR palindrome E (pSPT-TR34).

Figure 5. Molecular weight determination of IRF after UV crosslinking. RNAprotein complexes formed with radiolabeled RNA containing the human ferritin heavy-chain IRE and 50 Ag of cytoplasmic protein extract were crosslinked by UV irradiation after ribonuclease T1 digestion and subsequently separated in 10% SDS-polyacrylamide gels under reducing conditions. Binding specificity was tested by competition with a 100-fold molar excess of the same unlabeled RNA.

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IRE-binding activity when compared to control cells (Fig.3). Although such an induction by desferrioxamine was not apparent in Drosophila melanogaster cells, the decrease in the binding activity after treatment with ferric iron could nevertheless be observed. These data suggest that the mechanism of IRF activation is conserved among vertebrates and insects. Reduction of an intramolecular disulfide bridge appears to be crucial for the interaction between mRNA sequences and IRF, since its binding activity can be enhanced in vitro by exposure to reducing agents (19). We therefore tested whether the IREbinding factor in the different species could be activated by incubating extracts with various concentrations of B-mercaptoethanol. This was indeed the case, as shown in Fig.4. The treatment with 2% f3-mercaptoethanol resulted in a 5- to 10-fold induction of IRE-binding activity in mammalian, chicken, and insect extracts. The activity was completely lost at 8% 3-mercaptoethanol, probably by protein denaturation. The RNAbinding factors from frog and fish, although activated by 3-mercaptoethanol, were more sensitive to reduction. Loss of their binding activity was observed at f3-mercaptoethanol concentrations below 2%. Molecular weight determination of IRF in different species Additional support for homologous IRFs in different species was obtained from molecular weight determinations. Radiolabeled RNA-protein complexes were UV irradiated to induce covalent crosslinks and resolved in 10% SDS-polyacrylamide gel under reducing conditions. The RNA-protein complexes from all the species tested migrated at approximately 110 kDa (Fig.5). Since the RNA fragment protected by IRF after ribonuclease T, digestion is about 40 bases (6,13), the molecular weight of the binding factor can be estimated as 95 to 100 kDa. This conclusion was directly confirmed by measuring the size of IRF by gel filtration on a Superose 12 column (Fig.6). The present

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determinations are in agreement with those obtained from affinitypurified human and rabbit IRF (15,16,29). The remarkable similarity in molecular weight of IRF among distantly related species suggests structural conservation. More pronounced variations in migration were observed for RNA-protein complexes in non-denaturing gels (Fig. 1). These differences are best explained by a heterogeneity in the net charge of IRF among the species analysed.

CONCLUSIONS In the present study

we confirm our initial premise that IRF is conserved both in its structural and functional properties among

Nucleic Acids Research 1179 all vertebrate species. This indicates co-evolution between a transacting regulatory protein and a cis-acting RNA structure. According to this view, the binding of IRF may explain the high degree of conservation of IREs in the 5' UTR of human, rat, chicken and frog ferritin mRNAs, as well as in the 3' UTR of human and chicken TR mRNA. The detection of a specific IREbinding protein in cells from Drosophila melanogaster suggests that the regulatory feedback mechanisms controlling iron homeostasis in vertebrates have actually emerged prior to the divergence of insects and vertebrates. It is remarkable that even subtle differences in the relative affinity for various human IREs are conserved between Drosophila and human IRF. Although the natural targets for insect IRF have not been characterized, it is likely that IRE-containing mRNAs are also present in Drosophila cells. The close similarity in the way vertebrate and insect IRF activity are modulated in response to intracellular iron levels or reducing agent support the idea that IRF has as well a regulatory function in insects. Our negative results in attempting to detect IRF in lower eukaryotes or prokaryotes may reflect a fundamental difference in the regulation of iron metabolism. Although iron storage proteins exist in yeast, plants and bacteria, the primary sequences of these 'ferritins' have not been shown to be related to those of vertebrates (22). Moreover, iron uptake at least in plants and bacteria involves the absorption of chelated iron and thus diverges markedly from the mode of iron uptake in mammalian cells (30). We conclude that the selective pressure preventing the divergence of a specific RNA structure within a non-coding region is best explained by its functional importance. In the case of IREs, we show that conserved structural elements serve the specific interaction with a post-transcriptional regulatory factor that is itself maintained through evolution. As there are a large number of mRNAs with high sequence homologies in untranslated regions (31), it will be of interest to investigate whether such conservations have also a predictive value for RNAprotein interactions.

ACKNOWLEDGEMENTS The present work was supported by the Swiss National Science foundation, the Swiss Ligue for Cancer Research and the Fores Foundation. We thank Dr. A. Conzelmann, Dr. M. Nabholz and A. Emery-Goodman for critical reading of this manuscript.

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