There is accumulating evidence that genes are controlled not only at the stage of transcription initiation but that several post-transcriptional events may limit or modulate gene expression. One of the best characterized model systems for the study of post-transcriptional regulation is reflected in the maintenance of cellular iron homeostasis by an RNA-binding protein, iron regulatory protein-1 (IRP-1). ()This cytoplasmic protein binds with high affinity and specificity to mRNA stem-loop structures known as iron-responsive elements (IREs) (for review, see Klausner et al.(1993) and Kühn(1994)). IRP-1 was formerly referred to as IRE-binding protein (Rouault et al., 1988), iron regulatory factor (Müllner et al., 1989), or ferritin repressor protein (Walden et al., 1989). This protein has been positively identified as the cytosolic aconitase (Hentze and Argos, 1991; Rouault et al., 1991; Kaptain et al., 1991; Haile et al., 1992a; Kennedy et al., 1992) and is now viewed as a bifunctional regulator with two mutually exclusive functions: RNA-binding and enzymatic activity (Haile et al., 1992a, 1992b; Constable et al., 1992; Emery-Goodman et al., 1993). While its role as an enzyme remains enigmatic, IRP-1 is clearly functional as a trans-regulator of mRNA. Under low iron conditions, IRP-1 actively binds IREs in the 5`- untranslated regions of ferritin and erythroid 5-aminolevulinic acid synthase mRNAs, thus inhibiting their translation (Aziz and Munro 1987; Hentze et al., 1987; Bhasker et al., 1993; Melefors et al., 1993; Gray and Hentze, 1994). Transferrin receptor mRNA contains five IREs in its 3`-untranslated region, and in this case, binding by IRP-1 results in mRNA stabilization (Casey et al., 1988; Müllner and Kühn, 1988; Müllner et al., 1989; Koeller et al., 1989). The net outcome is increased iron uptake and availability. When iron is high, the reverse situation ensues due to poor RNA-binding by IRP-1 (for review, see Kühn(1994)).

Recently, we characterized a second IRE-binding protein in rodents, named IRP (Henderson et al., 1993). This cytoplasmic protein was first detected as a rodent RNA band-shift complex (Leibold and Munro, 1988; Müllner et al., 1989) and was found to have a mass of 105 kDa and to bind different mRNA IREs with equally high affinity as IRP-1 (Henderson et al., 1993, 1994). Rat IRP has been purified (Guo et al., 1994), and antibody cross-reactivity experiments indicate that IRP is the homologue of a second IRE-binding protein recently cloned and expressed in human cells (Rouault et al., 1992; Samaniego et al., 1994); each protein will now be referred to as IRP-2. Human IRP-2 is 57% identical and 79% similar to IRP-1 in amino acid sequence (Rouault et al., 1992). IRP-2 has conserved the active site cysteine residues shown by mutagenesis to bind a [4Fe-4S] cluster in IRP-1 (Philpott et al., 1993; Hirling et al., 1994); however, the ability of IRP-2 to ligate such a cluster is unknown, and the protein does not appear to be enzymatically active (Guo et al., 1994). The ability of IRP-2 to inhibit translation in vitro (Guo et al., 1994; Kim et al., 1995) and the correlation between changes in IRP-2 activity and transferrin receptor mRNA levels in vivo (Cairo and Pietrangelo, 1994) together suggest that both IRPs may function as mRNA regulators.

The RNA-binding activity of the two IRPs is regulated in response to iron levels (Henderson et al., 1993). In contrast to IRP-1, IRP-2 does not appear to be post-translationally converted between active and inactive forms. Immunoblot analyses of different cell lines revealed that IRP-2 protein levels change in response to iron availability (Guo et al., 1994; Samaniego et al., 1994). Moreover, pulse-chase experiments using human RD4 cells showed directly that IRP-2 is much less stable in iron-treated cells (t 6 h) than in cells deprived of iron (t, >24 h) (Samaniego et al., 1994). In this study, we have investigated further the differences between the iron-regulatory pathways of IRP-1 and IRP-2. We first analyzed the expression and in vitro modulation of IRP RNA-binding activities. Translation inhibitors were then utilized to provide supportive evidence that iron depletion increases cellular IRP-2 activity by permitting the stable accumulation of newly synthesized IRP-2 apoprotein. We further show that unlike IRP-1, IRP-2 degradation/inactivation requires both the presence of iron and the translation of an independent protein. Possible models to account for the multi-step control of IRP-2 protein levels are discussed.

MATERIALS AND METHODS

Cell Culture and Treatments

RNA-Protein Band-shift Analysis

Modifying IRPs by in Vitro Alkylation and Oxidation

Immunoblot Analysis

RESULTS

Differential Expression of IRP-1 and IRP-2 in Cell Lines

Figure 1: IRE-binding activities in different cell lines. Upper panel, RNA band-shift assay was performed using 2 µg of cytoplasmic protein and an excess of P-labeled IRE probe in the absence(-) or presence (+) of 2% 2-ME. Extracts were prepared from log-phase cells, either untreated (L), treated for 20 h with 100 µM desferrioxamine (D), or treated 20 h with desferrioxamine, then washed and exposed to 60 µg/ml ferric ammonium citrate for 4 h (Fe). Complexes corresponding to IRE-bound IRP-1 and IRP-2 are indicated. The gels were quantitated, and relative band-shift signals are shown plotted in arbitrary units (lower panel). IRP-1 (blackbars) and IRP-2 (hatched) signals are at the same scale and from the same gel. The data are typical of at least two independent experiments.

RNA-binding Activities of IRP-1 and IRP-2 Are Modulated in Vitro by Alkylation and Oxidation/Reduction

Figure 2: Inhibition of IRP RNA-binding activity by alkylation and oxidation. IRP-1 and IRP-2 activities were detected by band-shift assay using a P-labeled IRE (RNA) and 2 µg of cytoplasmic extract from Ltk and B16 cells, as described under ``Materials and Methods.'' To test the effects of alkylation on RNA-binding, 1 mM NEM was added to the binding reaction either 5 min before or 5 min after the addition of P-labeled RNA. Alternatively, to test the effect of in vitro oxidation, 1.5 mM diamide (DIA) was likewise added to the binding reactions. In both cases, reversibility of the treatment was tested by 5-min incubation with 2% 2-ME, added 5 min after probe (lanes3 and 7). Quantitation of the data is presented (see boxes), showing mean values from two different experiments, given in arbitrary units where the control is assigned a value of one.

It is intriguing that while NEM did not affect IRP-1 when added after the RNA-protein complex was formed, NEM treatment appeared to ``split'' the preformed IRP-2 complex into two bands. This is most clearly demonstrated for B16 cells in Fig. 2. The effect was immediate, was specific to IRP-2-enriched fractions, and was observed consistently in several different rodent cell lines (data not shown). The additional cysteines present in IRP-2 may make this protein more susceptible than IRP-1 to alkylation once bound to the IRE. The two split IRP-2IRE complexes might thus represent different forms of the same protein, one being more readily alkylated and thereby altered in its charge and subsequent migration on the nondenaturing band-shift gel.

The effects of oxidation were also examined, and 1.5 mM diamide was shown to inhibit the activity of both IRPs (Fig. 2). The effect was reversible upon addition of 2% 2-mercaptoethanol. We conclude that each IRP is indeed susceptible to the effects of oxidation and alkylation of free sulfhydryl groups.

Desferrioxamine-mediated Activation of IRP-2 Is Prevented by Inhibitors of Transcription and Translation in LtkCells

Figure 3: Translation and transcription inhibitors block induction of IRP-2 RNA-binding activity. Upper panel, P-labeled IRE-bound IRP-1 and IRP-2 was detected in Ltk cell extracts by band-shift assay as shown. Ltk cells were left untreated (CON), or treated for 8 or 20 h with 100 µMDES to induce IRP activity. Cells were similarly treated with 10 µg/ml cycloheximide (CHX), 15 µg/ml anisomycin (ANI), and 5 µg/ml actinomycin D (AD) for 8 or 20 h, alone or in combination with DES. Binding reactions were performed in the absence(-) or presence (+) of 2% 2-ME. Lower panel, IRP-1 and IRP-2 band-shift signals were quantitated, and values for samples (without reductant) are plotted in arbitrary units, where the untreated sample is set a value of one. This experiment was performed at least twice with similar results, and the effect of cycloheximide was confirmed in five independent experiments.

In contrast to the temporary effect on IRP-1, the translation inhibitors completely prevented activation of IRP-2, even up to 20 h (see Fig. 3). The same observation was made several times over a 2-year period, using at least three different passages of Ltk cells, and was also observed for NIH 3T3 fibroblasts (data not shown). Furthermore, we observed that 5 µg/ml of the transcription inhibitor, actinomycin D, also selectively blocked the induction of IRP-2 by desferrioxamine in Ltk cells (Fig. 3). The activity of IRP-1, but not of IRP-2, was regained by 2-mercaptoethanol treatment. Our findings are compatible with recent immunoblot experiments observing iron-mediated changes in IRP-2 protein levels (Guo et al., 1994; Samaniego et al., 1994). We suggest that prolonged iron starvation increases IRP-2 activity in Ltk cells by allowing the stable accumulation of IRP-2 mRNA and protein following de novo synthesis.

Iron-mediated Repression of IRP-2, but Not IRP-1, Is Translation-dependent

Figure 4: Translation inhibitors prevent iron-mediated inactivation of IRP-2. Upper panel, P-labeled IRE complexes with IRP-1 and IRP-2 were detected by band-shift assay. Extracts were prepared directly from Ltk cells untreated (CON), or induced for 20 h with 100 µMDES, and then washed and incubated for 1, 2, or 4 h with 60 µg/ml ferric ammonium citrate (Fe). In some samples, 10 µg/ml cycloheximide (CHX) or 15 µg/ml anisomycin (ANI) were included 30 min prior to iron treatment. NIH 3T3 fibroblasts were also tested, using 4-h treatments with ferric ammonium citrate ± inhibitors. 2% 2-ME was added as indicated, normalizing IRP-1 activity but not that of IRP-2. Lower panel, bandshift signals were quantitated, and data from Ltk cell experiments was plotted in arbitrary units, where time zero (0 h) indicates -fold induction by DES treatment relative to control samples, and prior to iron treatment. The relative values shown are means (<25% variation) from two independent experiments. Curves are plotted for(-) and (+) 2-mercaptoethanol treatments to highlight the exclusive effect of translation inhibitors on IRP-2. The scale is arbitrary for each IRP but was increased about 6-fold for IRP-1 (+ 2-ME) samples for ease of comparison (all 2-ME-treated IRP-1 values were similar to control).

Iron Regulation of IRP-1 Activity Occurs Post-translationally

DISCUSSION

We previously characterized a second IRE-binding protein in rodents, now named IRP-2, which shares certain features in common with IRP-1 (Henderson et al., 1993, 1994). IRP-2 was recently purified (Guo et al., 1994) and expressed from a full-length cDNA (Rouault et al., 1992; Samaniego et al., 1994) and was shown to inhibit the in vitro translation of IRE-containing mRNAs as effectively as IRP-1 (Guo et al., 1994, Kim et al., 1995). These studies, when considered together, suggest that both IRPs may act as trans-regulators of mRNA stability and/or translation. In this report, we have focussed on how RNA-binding ability is regulated for these two proteins. We show that not only can IRP-1 be inactivated in vitro but that RNA-binding by IRP-2 is likewise inhibited by chemicals that modify free sulfhydryl groups. We confirmed in our system that manipulation of cellular iron levels modulates IRP-1 RNA binding activity by predominantly post-translational mechanisms. By contrast, the stimulation and down-regulation of IRP-2 activity were completely translation-dependent events. Our data suggest that removal of iron increases cellular IRP-2 activity by permitting the stable accumulation of newly synthesized IRP-2 apoprotein. Subsequent exposure to iron then leads to the inactivation and/or degradation of IRP-2, but only after another protein is newly translated. These findings fit well with recent evidence showing that iron promotes specifically the degradation of IRP-2, but not IRP-1 (Guo et al., 1994; Samaniego et al., 1994), and lead us to propose that an independent regulatory protein may prove integral to this process.

According to cDNA sequence information, human IRP-1 and IRP-2 are predicted to be 57% identical and 79% similar in amino acid sequence (Rouault et al., 1992). There is indirect evidence that these proteins might in addition share similarities in their RNA-binding domains. For instance, both proteins protect similar sized IRE fragments from RNase T1 digestion (Leibold et al., 1990) and bind IREs from different mRNAs with comparable affinity (Henderson et al., 1993; Guo et al., 1994; Samaniego et al., 1994). The RNA-binding site of IRP-1 is predicted to lie within the active site cleft, as suggested by mapping of the IRE UV cross-link site (Basilion et al., 1994) and the fact that alkylation of the [4Fe-4S] cluster-ligating residue, Cys-437, prevented IRE binding (Philpott et al., 1993; Hirling et al., 1994). It was proposed that the alkylation of Cys-437 by NEM, but not by the smaller iodoacetamide group, interfered sterically with IRE binding (Philpott et al., 1993; Hirling et al., 1994). In this study, RNA-binding by IRP-2 was likewise inhibited following alkylation with 1 mM NEM. Furthermore, partial inhibition of IRP-2 activity was achieved by 5 mM iodoacetamide (data not shown). Therefore, we predict that if alkylation targets the conserved IRP-2 residue homologous to Cys-437, then the IRE may bind in close proximity to this IRP-2 cysteine (Cys-512).

Surprisingly, a recent paper by Kim et al.(1995) observed no effect of 1 mM NEM on IRE-binding by recombinant human IRP-2. It is not yet clear whether this discrepancy in results can be ascribed to a species difference or to the physical form of IRP-2 tested. The latter notion is intriguing, as it raises the possibility that the cellular form of IRP-2 may differ in some way from the purified recombinant protein.

The two IRPs are certainly susceptible to other forms of protein modification. In this study, we showed that the RNA-binding activity of each IRP can be reversibly inhibited in vitro by oxidation of free sulfhydryl groups. In IRP-1, Cys-437 is the critical residue affected by oxidation (Philpott et al., 1993; Hirling et al., 1994). It is possible that IRP-2 is readily inhibited by oxidation at the same site, although susceptibility at additional sites cannot be excluded, as this protein contains 9 more cysteine residues than IRP-1 (Rouault et al., 1992). We conjecture that some IRP-2 molecules may in addition undergo a preferential modification, based on our finding that IRE-bound IRP-2, but not IRP-1, was split into two distinct band-shift complexes following alkylation with NEM. The nature of the IRP-2 modification, which in this instance did not abolish RNA-binding, awaits further definition.

The existence of two related IRPs prompts one to consider how these proteins are expressed and differentially regulated by cellular iron. Expression of the IRP proteins is known to differ. In those studies that examined IRP RNA-binding activities (Henderson et al., 1993) and mRNA levels (Samaniego et al., 1994) in whole tissue extracts, IRP-1 expression was generally much higher than that of IRP-2, although the IRP activity profiles were quite distinct (Henderson et al., 1993; Samaniego et al., 1994). In this study, we found that IRP-2 activity can vary quite dramatically relative to IRP-1 when comparing different cell lines. IRP-2 is thus clearly expressed in a cell-type specific manner. Furthermore, with the exception of NMuMG mammary epithelial cells, both IRP proteins were responsive to changes in intracellular iron levels. Given these observations, we cannot exclude that factors in addition to iron-dependent mechanisms may contribute to the steady-state RNA-binding ability of these proteins.

The iron-dependent modulation of each IRP was examined for sensitivity to translation inhibition. Our inhibitor and immunoblot data for IRP-1 expression in Ltk cells are compatible with some previous reports (Tang et al., 1992; Samaniego et al., 1994). We conclude that the increase and decrease in IRP-1 RNA-binding activity can occur without changes in protein levels, suggesting a post-translational conversion between apo- and holo- forms of the protein (outlined in upperpanel of Fig. 5). Cluster removal in the presence of desferrioxamine was slow and may reflect a biphasic process that initially depends solely on de novo synthesis, as IRP-1 activation by desferrioxamine was translation-dependent in Ltk cells during the first 8 h (Fig. 3). Inactivation of IRP-1 by iron was rapid and may involve the spontaneous insertion of a [4Fe-4S] cluster, as proposed elsewhere (Haile et al., 1992a, 1992b; Constable et al., 1992; Emery-Goodman et al., 1993). We point out that translation-independent degradation of IRP-1 by heme and iron salts has been observed in rabbit RAB-9 cells (Goessling et al., 1994), cautioning that alternate regulatory pathways may exist in different species or cell types.

Figure 5: Models to illustrate the iron-dependent regulatory pathways of IRP-1 and IRP-2. Upper panel, schematic outline showing the post-translational conversion of IRP-1 between active (apoprotein) and inactive ([4Fe-4S] cluster-containing protein) RNA-binding forms. Our data largely concur with other studies (Tang et al., 1992; Samaniego et al., 1994), although we find that some de novo synthesis accounts for the early induction (up to 8 h) of IRP-1 RNA-binding activity in Ltk cells, following DES treatment. Lower panel, two working models are presented to describe the pathways that regulate IRP-2 in response to changes in iron levels. Both cases incorporate our current findings, which support the view that activation of IRP-2 reflects the stable accumulation of newly synthesized IRP-2 protein (Samaniego et al., 1994). The two models account also for the recent finding that iron promotes the degradation of IRP-2 (Guo et al., 1994; Samaniego et al., 1994), and our observation that IRP-2 inactivation/degradation requires new translation. In Model1 (Fedirect), newly made apo-IRP-2 is stable until iron is inserted in the form of a [4Fe-4S] cluster. This insertion could be mediated by a labile accessory protein (X), and the cluster-loaded IRP-2 readily degraded. Model2 predicts an indirect role of iron. By analogy to its role in translation induction of ferritin, iron may induce an unstable protein (Y) that directly modifies IRP-2. The modified IRP-2 would then be targeted for degradation. Further details are discussed in the text. Note, desferrioxamine (DES) is not implied to directly induce synthesis of either IRP; it acts only indirectly by chelating available intracellular iron. IRP-1 is depicted as two globular domains coupled by a linker region (for review, see Kühn(1994)). IRP-2 is drawn similarly based only upon its amino acid relatedness to IRP-1. Arrow thickness indicates the relative contribution each process plays in IRP regulation.

In striking contrast to IRP-1, both the induction and inactivation of IRP-2 were found to be translation-dependent processes. IRP-2 activation following iron starvation of Ltk cells was completely blocked by inhibiting translation and transcription. This finding fits well with recent studies using IRP-2 specific antibodies, that observed an increase in IRP-2 protein levels following desferrioxamine treatment of different rodent and human cell lines (Guo et al., 1994; Samaniego et al., 1994). Our results provide an alternate line of evidence that the increased activity of IRP-2 reflects simply the stable accumulation of newly translated IRP-2 protein (Fig. 5, lower panel). The ability of actinomycin D to prevent induction of IRP-2 (but not IRP-1) might well be explained if IRP-2 induction required continued transcription of the IRP-2 gene. It is therefore possible that IRP-2 mRNA is highly unstable in Ltk and other cell lines, providing an additional control point for IRP-2 expression.

The fact that IRP-2 accumulates following iron chelation suggests that the protein is unstable in the presence of iron. It thus follows that iron removal leads to the synthesis of stable IRP-2 apoprotein. This view is supported by recent pulse-chase experiments in human RD4 cells, in which the half-life of IRP-2 was observed to decrease from >24 h in iron-starved cells to 6 h in iron-treated cells (Samaniego et al., 1994). Furthermore, we observed that newly synthesized IRP-2 RNA-binding activity was stable for >12 h in Ltk cells treated with translation inhibitors, either alone or in combination with desferrioxamine (data not shown). How then, does iron promote degradation of IRP-2? To address this question, several different pieces of evidence must be considered. First, we have shown that IRP-2 inactivation (and presumably degradation) requires on-going translation. It is unlikely that iron affects IRP-2 co-translationally (e.g. by co-translational insertion of a [Fe-S] cluster), as this does not explain how the large preexisting pool of stable IRP-2 is so rapidly diminished (Fig. 4). Second, indirect effects of iron on cellular redox potential do not account for how 2-mercaptoethanol treatment reactivates IRP-1, but not IRP-2, in extracts from iron-treated cells.

We propose two models that best account for the dual requirement of both iron and translation for inactivation of IRP-2 (outlined in Fig. 5). The first is the ``Fe direct'' model, in which translation is necessary but secondary to the action of iron. The conservation of appropriate cysteines in IRP-2 (Rouault et al., 1992) leaves open the possibility for direct insertion of a [4Fe-4S] cluster. Unlike IRP-1, IRP-2 in this case would require new synthesis of a protein X, perhaps an enzyme, to mediate or chaperone cluster ligation (see Model1, Fig. 5). Considering that IRP-2 is degraded in response to iron in several different cell lines (Guo et al., 1994; Samaniego et al., 1994), then if this model were correct, it may be the cluster-loaded form of IRP-2 that is highly susceptible to degradation. This could be tested by expressing IRP-2 mutants in which the cluster-ligating cysteines were altered and asking whether such mutants are also degraded in response to iron.

The second working model is the ``Fe indirect'' scenario. In this situation, iron acts indirectly by stimulating the translation of a repressor protein Y (Model2, Fig. 5). Here, it is the repressor Y that determines IRP-2 turnover. How might this undefined protein act? An important clue is provided by the special case of human HeLa cells; iron treatment of HeLa cells does require new translation for inactivation of IRP-2, ()but apparently it does not lead to IRP-2 degradation (Samaniego et al., 1994). These findings suggest that the degradation machinery is likely to be cell-type specific, and independent of protein Y. We favor the view that IRP-2 is somehow modified, and that the modified IRP-2 is then a target for degradation in most, but not all, cell types (Samaniego et al., 1994). In model 2, protein Y would cause the undefined modification, thereby tagging IRP-2 for degradation. The two models described above are not mutually exclusive, and help to reconcile published data on IRP-2 with our finding that IRP-2 inactivation in several different cell lines is translation-dependent. More importantly, they predict several steps of the IRP-2 degradation pathway that are readily amenable to experimental verification.

FOOTNOTES

*

This work was supported by a grant from the Swiss National Fund (to L. K. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. Tel.: 01223-402-384; Fax: 01223-412-178.

¶

To whom reprint requests should be addressed: Swiss Inst. for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges s/Lausanne, Switzerland.

()

The abbreviations used are: IRP, iron regulatory protein; IRE, iron-responsive element; NEM, N-ethylmaleimide; 2-ME, 2-mercaptoethanol; DES, desferrioxamine.

()

B. R. Henderson, unpublished data.

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