Translational Regulation of mRNAs with Distinct IRE Sequences by Iron Regulatory Proteins 1 and 2*

Eric Menotti, Beric R. HendersonDagger , and Lukas C. Kühn§

From the Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges s/Lausanne, Switzerland

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Iron regulatory proteins 1 and 2 (IRP-1, IRP-2) interact with iron-responsive elements (IREs) present in the 5'- or 3'-untranslated regions (UTR) of several mRNAs coding for proteins in iron metabolism. Whereas binding of IRP-1 and -2 to an IRE in the 5'-UTR inhibits mRNA translation in vitro, it has remained unknown whether either endogenous protein is sufficient to control translation in mammalian cells. We analyzed this question by taking advantage of published mutant IREs that are exclusively recognized by either IRP-1 or IRP-2 in vitro. These IREs were inserted into the 5'-UTR of a human growth hormone reporter mRNA, and translational regulation was measured in stably transfected mouse L cells. Cells cultured in iron-rich or -depleted medium were labeled with [35S]methionine, and secreted growth hormone was immunoprecipitated. IREs with loop sequences specific for IRP-1 (UAGUAC), IRP-2 (CCGAGC), or both proteins (GAGUCG and the wild-type CAGUGC sequence) all mediated translational regulation, in contrast to a control sequence (GCUCCG) that binds neither IRP-1 nor IRP-2. Control experiments excluded IRP-1 binding to the IRP-2-specific sequence in vivo. The present data demonstrate that IRP-1 and IRP-2 can independently function as translational repressors in living cells.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Post-transcriptional mechanisms controlling protein synthesis are generally mediated by specific RNA-protein interactions (1). Coordinate regulation of ferritin mRNA translation and transferrin receptor (TfR)1 mRNA stability constitutes one of the best studied examples of such a control (2-4). H- and L-chain ferritin mRNAs contain cis-acting elements in their 5'-UTR which form a stem-loop structure known as the iron-responsive element (IRE) (5-7). Several IRE copies are also present in the 3'-UTR of TfR mRNA (8, 9). IREs are specifically recognized by the cytoplasmic iron regulatory protein 1 (IRP-1) (10-12), and these RNA-protein complexes inhibit ferritin mRNA translation and TfR mRNA degradation (2-4, 13) and thus controlling iron storage and uptake, respectively. Several other mRNAs contain also an IRE in the 5'-UTR and are translationally regulated: those of erythroid 5-aminolevulinic acid synthase (14-17), mammalian mitochondrial aconitase (15, 18), and succinate dehydrogenase subunit b of Drosophila melanogaster (18-20).

IRP-1 modulates its IRE-binding activity in response to available cytoplasmic iron (8, 10, 11). In normal iron supply, due to the insertion of a [4Fe-4S] cluster, IRP-1 converts from an IRE-binding apoprotein to an enzymatic holoprotein with aconitase activity (21-24), whereas in iron deprivation, the apoprotein accumulates (3, 4). While the two functions are mutually exclusive, the conversion does not affect the total amount of IRP-1. Full RNA-binding activity is also obtained upon in vitro reduction with 2% 2-mercaptoethanol. Furthermore, induction by nitric oxide (NO) (25-27) or exposure of cells to hydrogen peroxide (H2O2) (28, 29) stimulate the RNA-binding activity of IRP-1. Whereas NO exerts a slow effect, similar to an iron chelator, activation by H2O2 is rapid (30).

A second cytoplasmic protein, IRP-2, shows extensive sequence homology to IRP-1 and an equally high affinity for ferritin IREs (31-35). IRP-2 activity is similarly induced by iron deprivation and decreased in iron-replete cells. However, IRP-2 is less abundant than IRP-1 in most tissues (32, 35) and differs in several other aspects. No associated aconitase activity could be found, and it remains uncertain whether IRP-2 incorporates an iron-sulfur cluster (33, 34). In extracts of iron-replete cells, IRP-2 cannot be reactivated by 2-mercaptoethanol (32, 33, 36), and this reflects a decrease in the amount of IRP-2 protein (33, 34). A unique 73 amino acid-region, absent in IRP-1, is thought to target IRP-2 for protein degradation by the proteasome complex (37, 38).

In the present study, we analyzed whether IRP-2 can also function as a trans-acting repressor of translation in cells. In vitro mutagenesis has revealed alternative IRE stem-loop sequences with specificity for either IRP-1 (39) or IRP-2 (40, 41). Here, we inserted several specific IRE variants into the 5'-UTR of a human growth hormone (hGH) expression vector and analyzed the translational repression in stably transfected L cells. Translational inhibition through both IRP-1- and IRP-2-specific IREs was observed, thus providing evidence for a regulatory role of IRP-2 in cells.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results & Discussion
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Cell Culture-- Mouse thymidine kinase-deficient L cells (Ltk-) were cultured in alpha -minimal essential medium (Life Technologies Inc.) with 10% fetal calf serum. Stably transfected Ltk+ cells were selected in alpha -minimal essential medium containing 100 µM hypoxanthine, 0.4 µM aminopterin, and 16 µM thymidine (42). Iron was modulated by culturing cells for 20 h in medium with 100 µM desferrioxamine (Desferal; a gift from Ciba-Geigy, Basel, Switzerland) or for an additional 4 h with 60 µg/ml of ferric ammonium citrate (Sigma). In some experiments, L cells were incubated for 45 min in medium with 100 µM H2O2 (Fluka, Switzerland) without fetal calf serum (essentially iron-free) to prevent the toxic effect of iron combined with H2O2. Where indicated, 1 h before the addition of iron-rich medium, 100 µM MG132 (peptide aldehyde) (ProScript, Cambridge, MA) was added and maintained for 4 h.

Plasmid Constructions and Stable ;transfections-- IRE mutants with different loop sequences were inserted as oligonucleotides into the 5'-UTR of the vector pL5-GH containing a ferritin promoter and the hGH gene (43). Double-stranded oligonucleotides with the sequence GATCCTGCTTCAANNNNNNTTGGACGGACGGATCTT (where N represents different loop sequences) were inserted between the BamHI and XbaI sites of pL5-GH, nine nucleotides downstream of the RNA cap site. Constructs with the following mutations in the IRE loop sequence were made2: G1AGUC5G (GC6 in Ref. 40), U1AGUA5C (UA34 in Ref. 39), CC2GA4GC (CG125 in Ref. 40), and GCUCCG (GC1 in Ref.40), a construct also referred to as 213-GH (19). All constructs were verified by DNA sequencing. Plasmid pFer-GH (15) was kindly provided by Dr. M. Hentze, EMBL, Heidelberg. Sub-confluent Ltk- cells were co-transfected with a plasmid containing the herpes simplex thymidine kinase gene using the calcium phosphate method (44).

Metabolic Labeling and Immunoprecipitation-- Cells were washed twice in phosphate-buffered saline, incubated in methionine-free medium for 90 min, and then labeled with 40 µCi/ml of [35S]methionine (>1000 Ci/mmol, Amersham Corp.) for 2-4 h. hGH was immunoprecipitated from equal amounts of secreted 35S-labeled protein (105 cpm, determined by trichloroacetic acid precipitation) with a specific rabbit polyclonal antibody (Dako, Carpinteria, CA), followed by protein A-Sepharose. Samples were analyzed on 15% SDS-polyacrylamide gels, and 35S-labeled hGH was quantitated with a Compaq PhosphorImager equipped with Molecular Dynamics Image software.

Gel Retardation Assay-- Cytoplasmic extracts were prepared by cell lysis of 35S-labeled cell populations with 0.3% Nonidet P-40 (8). Extracts were mixed with specific IRE probes to detect IRP-1 and/or IRP-2. Cytoplasmic protein (2 µg) was incubated for 10 min in lysis buffer with 2 × 104 cpm of gel-purified 32P-labeled IRE (specific activity: 1.3 × 109 dpm/µg). The 32P-labeled IRE probes were prepared by in vitro transcription of linearized plasmids pGEM-3zf containing IRE inserts in the presence of [alpha -32P]CTP (800 Ci/mmol; Amersham Corp.) (39, 40). Nonspecific binding was reduced by adding 2.5 mg/ml heparin. Where specified, 2% 2-mercaptoethanol was added to activate IRP-1 in vitro. RNA-protein complexes were analyzed in 6% non-denaturing polyacrylamide gels (8).

RNA Isolation and Northern Blot Hybridization-- Total RNA was prepared from 3 × 107 cells using the RNeasy Kit of Qiagen Inc. (Chatsworth, CA). Equal amounts of RNA (8 µg) were separated in 1.2% agarose gels in the presence of formaldehyde, transferred to GeneScreen Plus nylon membranes (NEN Life Science Products), and cross-linked with ultraviolet light. Equal loading was assessed from the ethidium bromide staining of ribosomal RNA prior to the transfer. Filters were hybridized with a random-primed 32P-labeled probe comprising the 600-bp SmaI fragment of the hGH gene (45).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Alternative IRE Stem-loops Discriminate between IRP-1 and IRP-2-- The iron-responsive element, first identified by phylogenetic comparison in the 5'-UTR of ferritin mRNA (5, 46), is highly conserved in evolution. The "consensus" IRE has a loop sequence CAGUGN and an unpaired cytosine immediately 5' of 5 paired nucleotides forming the "upper stem" (reviewed in Refs. 3 and 47). By employing in vitro screening procedures, we have recently identified alternative IRE loop sequences that preferentially bind to IRP-1 (39) or IRP-2 (40). In this study, we analyzed different IRE mutants for their ability to control translation when placed in the 5'-UTR of a hGH reporter construct (Fig. 1). The mutant IRE with the loop sequence G1AGUC5G (mutant GC6) was chosen as it binds to both IRP-1 and IRP-2 in vitro (40). In contrast, the loop sequences U1AGUA5C (mutant UA34) and CC2GA4GC (mutant CG125) were tested because of their exclusive interaction with IRP-1 or IRP-2, respectively (39, 40). As a positive control for translation regulation, we analyzed pFer-GH, a construct which carries the ferritin H-chain wild-type IRE (15). A mutant IRE containing the loop sequence GCUCCG (mutant GC1) served as a negative control since it binds to neither IRP-1 nor IRP-2 in vitro (40).


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Fig. 1.   IRE mutants with different loop sequences function as cis-elements for translational repression in cells. The wild-type ferritin IRE or mutants GC6, UA34, CG125, and GC1, showing different specificity for IRP-1 and IRP-2 in vitro (top panel; Refs. 39 and 40), were inserted into the 5'-UTR of the hGH expression vector L5-GH (43). Each construct was stably transfected into mouse L cells. Transfected cell populations were incubated either for 24 h with the iron chelator desferrioxamine (Des) or for an additional 4 h with 60 µg/ml ferric ammonium citrate (Fe) to modulate RNA-binding activities of IRP-1 and IRP-2. Proteins were labeled with [35S]methionine, and hGH was immunoprecipitated from the culture supernatant (panel B). To increase the contrast, lanes 1 and 2 of panel B are 4-fold longer exposed than the other lanes of the same experiment. In parallel, cytoplasmic extracts of each cell population were tested for RNA-binding activities by gel retardation assays with the wild-type or mutant [32P]IREs present in the respective transfectants (panel A). Binding reactions were done in the absence or presence of 2% 2-mercaptoethanol (2-ME). The hGH mRNA expression was analyzed on Northern blots (panel C).

The hGH constructs were stably transfected into mouse L cells and proteins labeled with [35S]methionine in iron-deprived (Des) or iron-rich (Fe) cell cultures. Following 35S-labeling, cytoplasmic extracts were prepared to confirm the activation and specificity of IRP-1 and IRP-2 by gel retardation analysis (Fig. 1A). Both proteins were activated in iron-deficient cells and bound strongly to the 32P-labeled wild-type ferritin IRE (probe CG42 of Ref. 39) as well as to the mutant IRE GC6, as expected (40). However, the IRE UA34 detected only IRP-1, while the CG125 probe identified IRP-2 specifically, as previously observed (40). The negative control, GC1, revealed no binding to either protein. In cell extracts of iron-replete cells, inactive IRP-1 was fully activated by pre-incubation with 2% 2-mercaptoethanol (2-ME) (Fig. 1A, lower panel), whereas RNA-binding activity of IRP-2 could be re-activated 3-4-fold by 2-ME only in extracts of iron-deprived, but not iron-replete cells. This finding is consistent with the degradation of IRP-2 in iron-replete cells (33, 34), and suggests that some IRP-2 molecules are inactivated by oxidation in the iron-deprived cell extract, as previously observed (36, 40).

IRP-specific IRE Stem-loops Regulate hGH Translation in Transfected L Cells-- Translation assays were performed by immunoprecipitation of secreted 35S-labeled hGH (Fig. 1B). In iron-deprived cells, diminished translation was observed for all IRE mutants, except for GC1, without a concomitant change in hGH mRNA levels (Fig. 1C). These results indicate that IRP-1 and/or IRP-2 activated by desferrioxamine bind to the IRE mutants and thereby diminish hGH translation as compared with the situation in iron-replete cells. In agreement with previous data (48), the control ferritin IRE-hGH construct was 10-fold repressed in translation after desferrioxamine treatment (Table I). Translational inhibition mediated by the IRE mutants GC6, UA34, and CG125 was somewhat weaker (Table I) and seemed to correlate with lower in vitro binding affinities of mutant IREs in competition assays (39, 40). The GC6 mutant, which binds both IRPs 2.5 times less efficiently than a wild-type IRE, mediated a translation inhibition of only 40%. The mutants UA34 and CG125, despite being almost equivalent to the wild-type IRE as competitors, but very specific only for one or the other IRP, mediated only a 4- or 2.5-fold reduction of hGH translation, respectively. Thus, one has to be cautious in predicting the precise effect of a given mutant on the basis of in vitro measurements. Differences in the absolute mRNA levels in different cell populations probably did not affect the extent of translational regulation. In all cell populations, an excess of unbound IRP could be measured in gel retardation assays (Fig. 1A). This excess was not strongly affected in cells with higher mRNA levels. One cannot argue, therefore, that the trans-acting IRPs were titrated out by the transfected mRNA.

                              
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Table I
Correlation between relative binding affinities and translation inhibition

Another study has reported iron-independent enhancement of translation by a 5'-IRE but showed that this effect was insensitive to a mutation in the IRE loop (49). Our present results are in agreement since translation efficiencies in iron-rich medium varied less than 1.4-fold between different cell populations excluding a positive effect on translation due to mutations in the loop.

Most importantly, the present data show that alternative IRE loop sequences with a sub-optimal binding to IRP-1 or IRP-2 are effective as cis-acting regulatory elements in cells. Moreover, an IRE that binds exclusively to IRP-2 in vitro (CG125) appears to be sufficient to confer iron-dependent translational regulation, indicating that IRP-2 itself acts as a genuine translation regulator.

IRP-2 Is Sufficient to Repress Translation of IRE-containing mRNAs in Cells-- The results of Fig. 1 do not exclude the possibility that activated IRP-1 in cells may cross-react with the IRP-2-specific IRE. Therefore, experiments were performed under conditions in which only IRP-1 was activated. Transfected L cells were pre-treated for 2 h with ferric ammonium citrate to reduce IRP RNA-binding activities and then incubated for 45 min with 100 µM H2O2, which is known to specifically and rapidly induce IRP-1 (28, 30). Indeed, in gel retardation assays with extracts from H2O2-treated cells, we detected only complexes of IRP-1 with the wild-type ferritin and the UA34 IRE probes (Fig. 2A). No complexes were visible for IRP-2, either with the wild-type IRE or with the IRP-2-specific probe CG125 (Fig. 2A, lane 6), indicating that IRP-2 was not activated by H2O2. Immunoprecipitation of the secreted 35S-labeled hGH revealed that H2O2-activated IRP-1 prevents translation when bound to the ferritin IRE (Fig. 2B). This agrees with findings by others (30). A clear inhibition was also observed with the IRP-1-specific UA34 construct (Fig. 2B), indicating that IRP-1 activation alone is sufficient to repress translation. However, despite a strongly induced IRP-1 activity, no regulation was observed with the IRP-2-specific IRE CG125 and the negative mutant IRE GC1. These findings indicate that IRP-1 does not bind to the IRP-2-specific IRE in cells, and confirms that the regulation mediated by CG125 in iron-deficient cells (Fig. 1) cannot be attributed to IRP-1, but depends only on IRP-2.


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Fig. 2.   IRP-1 induced by H2O2 regulates only IRP-1-specific IRE-hGH constructs. IRP-1 in transfected L cell populations (Fig. 1) was selectively induced by 100 µM hydrogen peroxide (H2O2) or inactivated with 60 µg/ml ferric ammonium citrate (Fe). IRP-1 activity was measured in extracts, without or with pre-incubation in 2% 2-ME, by performing gel retardation assays with the corresponding 32P-labeled IRE mutants as probes (panel A). In parallel, translational regulation was analyzed by immunoprecipitation of [35S]methionine-labeled secreted hGH in the culture medium (panel B). Results are shown for constructs with a ferritin IRE (Fer-GH) and mutant IREs UA34, CG125, and GC1.

Attempts were also made to study the question by selectively inactivating cellular IRP-1 but not IRP-2. Ltk- cells were pre-incubated with 100 µM MG132, a proteasome inhibitor thought to prevent IRP-2 degradation specifically (37, 38), and then exposed to 60 µg/ml ferric ammonium citrate to fully inactivate IRP-1. We found that not only was IRP-2 degradation inhibited, but the inactivation of IRP-1 was also partly prevented. Its RNA-binding activity in the presence of MG132 for 4 h remained 1.6-fold higher than without MG132 (data not shown). It was not possible, therefore, to obtain cells with active IRP-2 alone.

Conclusions-- In this study, we demonstrate for the first time that both endogenous IRP-1 and IRP-2 function as translation repressors in mammalian cells. This finding extends those of previous studies demonstrating that exogenously expressed IRP-1 acts as a translational repressor (50, 51) and that recombinant IRP-1 and IRP-2 repress ferritin mRNA translation in reticulocyte lysates (52) and wheat germ extracts (33) in vitro. We can conclude, therefore, that both IRPs, when simultaneously activated by iron-deprivation or NO-synthase induction, exert a combined effect in cells. As the two proteins have a relatively early evolutionary origin and were detected both in insects and mammals (53), it remains to be explained why, during evolution, organisms have acquired and maintained such a duplication of function. One possible reason could relate to a potentially broader and more differentiated response to distinct signals. Hydrogen peroxide was reported to induce only IRP-1 activity, whereas IRP-2 could be detected with preference in certain cells or tissues (32, 35) and under certain inductive conditions in erythroid cells (54) and rat liver (55). Another reason for the maintenance of two IRE-binding proteins may relate to the potential of specific targets. Although no naturally occurring IRP-1- or IRP-2-specific IRE sequences have yet been identified, there is a clear potential for the existence of mRNAs with such sequences. The results of the present study, which show that sub-optimal affinities of IRE-IRP interactions are sufficient for translational control, therefore suggest these RNA-binding proteins may mediate other novel functions within cells.

    ACKNOWLEDGEMENT

We thank Dr. Matthias Hentze for the gift of plasmids L5-GH and Fer-GH.

    FOOTNOTES

* This work was supported by the Swiss National Foundation.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 Present address: Westmead Institute for Cancer Research, University of Sydney, Westmead Centre, Westmead, New South Wales 2145, Australia.

§ To whom correspondence should be addressed: Genetics Unit, Swiss Institute for Experimental Cancer Research, 155, Ch. des Boveresses, CH-1066 Epalinges s/Lausanne, Switzerland. Tel.: 41-21-692-58-36; Fax: 41-21/652-69-33; E-mail: lukas.kuehn{at}isrec.unil.ch.

1 The abbreviations used are: TfR, transferrin receptor; hGH, human growth hormone; IRE, iron-responsive element; IRP, iron regulatory protein; UTR, untranslated region; Ltk-, thymidine kinase-deficient L cells; 2-ME, 2-mercaptoethanol.

2 Bold letters indicate mutated nucleotides and superscript numbers indicate position in the loop.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

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