(Received for publication, November 28, 1994; and in revised form, December 16, 1994)
From the
Iron regulatory proteins (IRPs) bind to specific RNA stem-loop structures known as iron-responsive elements (IREs) which mediate the post-transcriptional regulation of many genes of iron metabolism. Most studies have focused on the role of IRP1, which has previously been shown to bind with high affinity to IREs and mediate repression of in vitro translation of ferritin mRNAs. More recently, a second IRP has been identified that is expressed in all tissues and that binds IREs (Rouault, T. A., Haile, D. H., Downey, W. E., Philpott, C. C., Tang, C., Samaniego, F., Chin, J., Paul, I., Orloff, D., Harford, J. B., and Klausner, R. D.(1992) BioMetals 5, 131-140; Henderson, B. R., Seiser, C., and Kuhn, L. C.(1993) J. Biol. Chem. 268, 27327-27334; Guo, B., Yu, Y., and Leibold, E. A.(1994) J. Biol. Chem. 269, 24252-24260; Samaniego, F., Chin, J., Iwai, K., Rouault, T. A., and Klausner, R. D.(1994) J. Biol. Chem. 269, 30904-30910). Here we report that purified recombinant IRP2 inhibits translation of ferritin mRNAs with a molar efficacy equal to that of recombinant IRP1. There is a quantitative correlation between binding to isolated RNA target motifs, as judged by gel retardation assays, and translational repressor function as assayed in an in vitro translation system. In contrast to IRP1, IRP2 is not inactivated for RNA binding by alkylation with N-ethylmaleimide or phenylmaleimide, and as we would therefore predict, IRP2 treated with N-ethylmaleimide remains an effective repressor of ferritin translation. As IRP1 and IRP2 clearly have equal capability of mediating translational repression in vitro, the contributions of both IRPs to overall regulation must be considered in describing the pathways of iron regulated gene expression in individual cells.
The expression of several genes involved in iron metabolism are
regulated at a post-transcriptional level by the interaction of an
iron-sensing protein with iron-responsive elements (IREs) ()present in target transcripts. IREs are stem-loop motifs,
which have been identified in the 5` UTR of ferritin H- and L-chain
transcripts, the 5` UTR of the erythrocyte form of ALA synthase mRNA,
and the 3` UTR of the transferrin receptor mRNA (reviewed by Klausner et al.(1993)). An iron-sensing protein binds to IREs when
cells are iron-depleted. This protein has been previously referred to
as the iron-responsive element-binding protein (IRE-BP), ferritin
repressor protein, iron regulatory factor, and p90; more recently, the
term iron regulatory protein, IRP, has been chosen. IRP1 was originally
identified by characterization in gel retardation assays (Leibold and
Munro, 1988; Rouault et al., 1988) and by its capacity to
repress translation of ferritin mRNA in vitro (Walden et
al., 1988). The initial identification of a gene for a second
IRE-binding protein accompanied our initial cloning of the IRE-BP
(Rouault et al., 1990). By probing a cDNA library with a
degenerate oligonucleotide corresponding to peptide sequence obtained
from purified IRE-BP, a second clone was isolated, which was 57%
identical in amino acid sequence to the purified IRP1. Recombinant
protein expressed from a complete cDNA of this clone, initially termed
IRE-BP2 and now referred to as IRP2, also bound IREs (Rouault et
al., 1992). In most tissues, with the exception of brain, the
level of mRNA for IRP2 was much lower than that of IRP1, and the
endogenous form of IRP2 was difficult to detect in some tissues because
of low abundance, and/or co-migration with the gel retardation complex
produced by IRP1 (Samaniego et al., 1994). Endogenous IRP2 has
been detected in murine cells using an antibody raised to a peptide
contained within a 79-amino acid insertion unique to IRP2 (Samaniego et al., 1994). Other evidence for the existence of a second
IRE-binding protein activity in cells has been accumulated in several
laboratories. A second IRP distinct from IRP1 has been described in
rodent cells and assigned a molecular mass of 105 kDa, the size of
IRP2, making it quite likely that the authors were describing the
murine homologue of IRP2 (Henderson et al., 1993).
Interestingly, this protein showed the highest levels of binding
activity in intestine and brain. In another report of a second specific
IRE binding activity in rodent cells, the second complex was detectable
in regenerating liver cells, whereas it was barely detectable in
lysates of non-proliferating liver cells, suggesting that expression of
the second protein, which bound to IREs in gel shift assays, was
preferentially modulated during cell proliferation (Cairo and
Pietrangelo, 1994). Finally, a second IRE-binding protein from rat
liver has been recently purified and partially cloned, and shown to
correspond in sequence to human IRP2 by peptide sequencing (Guo et
al., 1994). Antibodies raised against the IRP2-specific 79-amino
acid insertion shifted the IRP2-IRE complex to a higher position in the
gel, resulting in a super-shifted complex. Western blot analysis
revealed that levels of rat IRP2 decreased to undetectable levels after
iron treatment, in contrast to IRP1, where binding activity decreased
markedly, but levels of protein did not change significantly (Guo et al., 1994). We have also observed marked changes in total
levels of IRP2 after iron manipulations in numerous cell types,
although not in HeLa cells (Samaniego et al., 1994). Iron
treatment does not affect the biosynthetic rate of IRP2, but rather
regulates the level of IRP2 by increasing the rate of degradation of
IRP2 in iron-replete cells. The half-life of IRP2 changes from
immeasurably long when cells are iron-depleted to 6 h or less when
cells are iron-replete (Samaniego et al., 1994).
The existence of two IRE-binding proteins which are regulated by different mechanisms has increased the complexity of the IRP regulatory system. Thus far, there have been no descriptions of cell types in which one form of IRP is expressed while the other is not. It remains unclear why there are two IRE-binding proteins. One possibility would be that the two IRPs respond to different stimuli, as was suggested by the authors of the study on regenerating liver cells (Cairo and Pietrangelo, 1994). Another possibility is that the targets of the two proteins differ. Although gel retardation studies have shown that the two IRPs bind with equal affinity to isolated IRE motifs derived from ferritin, eALA synthase, and the transferrin receptor mRNAs (Guo et al., 1994; Samaniego et al., 1994), these studies do not necessarily mean that in the setting of the intact transcript that the two proteins bind with equal affinity to target IREs, as context and the potential for protein-protein interactions may modify binding affinity in vivo.
In this study, we utilize purified recombinant IRP1 and IRP2 and compare the ability of each to repress translation of ferritin in vitro. We show that equal molar amounts of IRP1 and IRP2 bind isolated IREs equally and, furthermore, that equal binding in the gel retardation assay predicts equal impact on translation of a target mRNA.
Reticulocyte lysates were used to study the in vitro translation of capped transcripts. As has been previously observed, endogenous repressor activity in reticulocyte lysate is present and results from the presence of IRP(s) in the lysate (Dickey et al., 1988; Walden et al., 1988, 1989). In the experiments reported here, titration curves revealed that the translation of exogenous transcripts containing IREs was fully repressed when added to the reticulocyte lysates at RNA concentrations of less than 5 µg/ml (16 nM RNA). Repression of translation was not observed for an identical transcript that lacked a functional IRE. Transcripts that contained a functional IRE, when added at concentrations in excess of 16 nM, were translated as efficiently as transcripts that lacked a functional IRE (Fig. 1). Studies of translational repression were subsequently conducted at concentrations of RNA of 20 µg/ml (64 nM), so that translational repression could be attributed primarily to the effect of purified IRPs that were added to the reaction.
Figure 1: Reticulocyte lysates contain an endogenous repressor of translation of mRNAs that contain IREs. Two different mRNAs encoding ferritin H-chain subunits were transcribed. In one transcript (+IRE), the transcript was comparable to the native ferritin mRNA with the IRE located 26 nucleotides from the cap. In the other transcript (-IRE), the sequence of the IRE is 5` to 3` reversed with the positioning of the stem-loop unchanged in the transcript. SDS-polyacrylamide gel electrophoresis analysis was performed on the in vitro translation reaction products, radiolabeled ferritin was quantitated, and a value of 100% for +IRE versus -IRE was assigned at 40 µg/ml added RNA (128 nM RNA final concentration). The remaining points were represented as percentages of the ferritin synthesized at 40 µg/ml.
Equal
quantities of purified recombinant IRP1 and IRP2 were titrated against
a fixed amount of ferritin IRE probe (Haile et al., 1989), and
binding activities of the two recombinant purified IRP preparations
were equivalent over the 0-200 nM protein range (Fig. 2a). Previously, we have performed formal binding
curves and Scatchard analyses of the interaction of IRP2 with IREs and
compared the values with those obtained on IRP1 binding to IREs. A K of 60 pM was calculated for the
IRP2-IRE interaction, which was indistinguishable from the K
calculated for IRP1 (Samaniego et al.,
1994). When equal molar amounts of IRP1 or IRP2 were added to the
reticulocyte lysate, equal translational repression was observed (Fig. 2b). In these assays, utilizing 64 nM transcript, 50% inhibition of translation was achieved when 80 ng
of either IRP1 or IRP2 was added to the reaction (32 nM IRP
final concentration). As shown in Fig. 1, there is enough
endogenous repressor to fully repress translation of 5 µg/ml (25%)
of this IRE-containing transcript. Therefore, the addition of IRE
containing transcript in excess of that which can be fully repressed by
endogenous repressor (15 µg/ml or 48 nM) will be actively
translated unless exogenous repressor is added. If we assume that at
50% translation inhibition, 50% of the IRE-containing transcripts are
sequestered by IRP, we calculate that 0.6 pmol of transcript are
translationally repressed by 0.8 pmol of added IRP. Thus, IRP-mediated
translational repression occurs at a stoichiometry of approximately one
IRP per transcript, under these binding conditions, which directly
reflects the binding efficacy of these high affinity repressors to
isolated IREs.
Figure 2: a, graphic representation of quantitated gel retardation assays of IRP1 versus IRP2. Increasing concentrations (0, 20, 40, 80, and 200 nM) of recombinant purified IRP1 or IRP2 were added to 100 nM radiolabeled IRE probe. The RNA-protein complex was quantitated for IRP1 versus IRP2 at the indicated protein concentrations, and the amount was comparable for IRP1 versus IRP2. b, binding to IREs quantitatively predicts translational repressor activity. Equal amounts of IRP1 versus IRP2 were added to reactions containing 64 nM IRE containing RNA transcript (lanes 2-7) or no transcript (lane1). Amounts of added IRP for lanes 1-7 were 0, 200, 0, 20, 40, 80, and 200 nM, and in lane 8, 200 nM IRP1 or IRP2 was added to an in vitro translation reaction that contained transcript containing the reverse IRE. Biosynthetically labeled proteins were resolved on a 15% SDS-polyacrylamide gel electrophoresis. The bands depicted were approximately 20 kDa in size and correspond to ferritin synthesized over the course of the in vitro translation. c, graphic representation of translational inhibition produced by IRP1 versus IRP2 on equal amounts of IRE containing ferritin transcript as depicted in panel a. The point at which no IRP was added (lane3) was taken to represent 100% ferritin mRNA translation, and percentage of ferritin mRNA translation was subsequently determined by quantitation, revealing that translational inhibition produced by each IRP is equivalent. IRPs did not repress translation of transcripts containing the reverse IRE.
IRP1 binding activity is known to be inactivated by NEM due to the alkylation of cysteine 437 in the active site cleft of IRP1 (Philpott et al., 1993; Hirling et al., 1994). In contrast to IRP1, IRP2 binding activity is not inactivated by treatment with NEM (Fig. 3a). When IRP1 alkylated with NEM was assayed in the in vitro translation system, translational repressor activity decreased proportionally to the loss of RNA binding activity, as assessed by gel retardation assays. The difference in the impact of NEM treatment on RNA binding activity of the two IRPs was profound. This is reflected in the inability of NEM to impair the capacity of IRP2 to mediate translational repression (Fig. 3b).
Figure 3: a, IRP2 binding activity for IREs is not abrogated by treatment with the alkylating reagent NEM. IRPs (150 ng) were treated with NEM at concentrations of 0, 0.2, 0.5, or 1 mM NEM and band shift activity was assessed and quantitated. b, inactivation of RNA binding quantitatively predicts loss of translational repressor function.
NEM is thought to inhibit IRE binding by steric hindrance. Mutagenesis has demonstrated that the cysteine residue at position 437 in IRP1 is not itself required for high affinity IRE binding. Furthermore, an alkylating reagent that adds a smaller alkyl group, iodoacetamide, does not interfere with IRE binding, though clearly it also binds to C437, because pretreatment with iodoacetamide protects against the NEM (molecular mass = 125 kDa) effect (Philpott et al., 1993). We therefore thought it possible that the NEM was failing to inhibit RNA binding of IRP2 because a small difference in the RNA binding site permitted the accommodation of a larger alkylating agent. We therefore treated IRP2 with a larger alkylating agent, phenylmaleimide (molecular mass = 174 kDa). Once again, IRP2 binding of RNA was not inhibited by treatment with this alkylating reagent, whereas IRP1 binding was eliminated by treatment with 0.2 mM phenylmaleimide.
The IRP/IRE system provides a clear example of a regulatory system in which the fates of mRNAs are mediated by the binding of a specific high affinity RNA-binding protein. The mechanism by which binding alters the ability of the cell to either translate or degrade IRE-containing mRNAs is not known. We have proposed that in both cases, binding of the IRP interferes with the local function of the transcript (Klausner and Harford, 1989). This proposal is supported by observations on the requirements for IRE-mediated translational inhibition in which the IRE must be placed within 50 nucleotides of the cap site (Goossen et al., 1990). Binding of the IRP appears to inhibit association of the 43 S ribosomal subunit with the mRNA (Gray and Hentze, 1994). While a nonspecific steric hindrance model can explain these results (Stripecke et al., 1994), the possibility that the bound IRP has some specific interaction with one or more components of the translational initiation machinery (Merrick, 1992) has not been ruled out. The identification of a second cellular IRP led us to quantitatively compare the ability of these two proteins to inhibit translation and to correlate this inhibition with their demonstrated in vitro binding to isolated RNA target IRE structures.
In this study we demonstrate that purified recombinant IRP1 and IRP2 bind to isolated IREs with similar affinities, and we show that this binding is predictive of efficacy in translational repression of full-length ferritin mRNA, which contains an IRE within 26 nucleotides of the cap site and which is a likely in vitro target for binding by one or both IRPs. The use of a transcript that is capped and full-length increases the likelihood that the results obtained will reflect in vivo regulatory events. Although these two IRPs likely share binding targets, it is also possible that there are as yet unidentified targets of each protein which are unique. Recent experiments selecting random variations of the IRE loop sequences capable of binding to IRP1 have revealed the interesting result that a loop sequence different from the previously described loop consensus sequence recognized by IRP1 is bound with high affinity by IRP1, but is apparently recognized poorly by IRP2 (Henderson et al., 1994). This suggests that there are differences in the binding sites which remain to be characterized in greater detail. Our study reveals another difference between the two proteins in that IRP1 is inactivated both for IRE binding and in vitro translation repression by alkylation, whereas IRP2 is not inactivated for either activity by the same treatment. Reagents such as NEM or phenylmaleimide can be used to distinguish IRP1 from IRP2 under the experimental conditions described here. This difference may prove to be a useful tool for distinguishing the two proteins under other experimental conditions.
Observations showing that IRP2 can function as a translational repressor of ferritin in a wheat germ extract system were reported (Guo et al., 1994) as we were completing these comparisons of IRP1 and IRP2. Importantly, that study utilized protein preparations that were purified partially on the basis of their ability to bind to ferritin IREs in affinity chromatography. In our study, protein overexpressed from full-length IRP1 and IRP2 clones is purified, and the protocol does not include an affinity purification by binding to IREs. The fact that purification on the basis of ability to bind to IREs is not used to select material for the in vitro translation assay is important in that there is not a selection bias toward IRE binding introduced within the population to be assayed. Most importantly, the possibility of low level cross-contamination of the two IRP species is avoided since only one IRP is overexpressed at a time.
The use of purified recombinant proteins and RNAs in this study permits us to reach quantitative conclusions. We show that there is a strong correlation between IRE binding activity in gel retardation assays and capacity to mediate translational repression. Furthermore, the stoichiometry of translational repression is approximately 1:1 at the concentrations of reactants described here, and the 1:1 stoichiometry is consistent with the high in vitro binding affinity of the IRP-IRE interaction (Haile et al., 1989; Barton et al., 1990). Finally, the translational repressor activity of IRP1 for a ferritin transcript is indistinguishable from the repressor activity of IRP2, implying that both IRP1 and IRP2 repress ferritin translation when activated in vivo. Although there is clearly overlap in the targets of these two proteins, it is also possible that there will be other unique interactions in which one IRP binds with high affinity to a specific RNA structure/sequence while the other does not. We have previously established that IRP2 differs in its mode of regulation from IRP1 in that the rate of protein degradation is regulated by iron levels (Samaniego et al., 1994); in addition, a differential stimulus for expression has been described in the increased binding activity of IRP2 after partial hepatectomy, although there is no apparent change in the binding activity of IRP1 (Cairo and Pietrangelo, 1994). However, even in this setting IRP1 accounts for the major part of the IRE binding activity, so that the increased RNA binding activity contributed by IRP2 would not have a significant impact unless there is an IRP ligand that is specifically bound by IRP2. The amount of IRP2 in the hepatoma cell line FTOB is up to 50% of the amount of IRP1, whereas there is 30 times as much IRP1 as IRP2 in normal rat liver lysates (Guo et al., 1994), observations that again correlate increased IRP2 expression with proliferation status. We demonstrate here that both IRPs can act on a common target and repress translation, and therefore that differential expression of the two proteins may be important in cellular iron metabolism. The possibility also remains that there will be unique in vivo target specificities for each protein, which may clarify why two IRPs are maintained in cells.