(Received for publication, September 19, 1995; and in revised form, November 28, 1995)
From the
Iron regulatory proteins (IRPs) 1 and 2 bind with equally high affinity to iron-responsive element (IRE) RNA stem-loops located in mRNA untranslated regions and, thereby, post-transcriptionally regulate several genes of iron metabolism. In this study we define the RNA-binding specificities of mouse IRP-1 and IRP-2. By screening loop mutations of the ferritin H-chain IRE, we show that both IRPs bind well to a large number of IRE-like sequences. More significantly, each IRP was found to recognize a unique subset of IRE-like targets. These IRP-specific groups of IREs are distinct from one another and are characterized by changes in certain paired (IRP-1) or unpaired (IRP-2) loop nucleotides. We further demonstrate the application of such sequences as unique probes to detect and distinguish IRP-1 from IRP-2 in human cells, and observe that the IRPs are regulated similarly by iron and reducing agents in human and rodent cells. Importantly, the ability of each IRP to recognize an exclusive subset of IREs was conserved between species. These findings suggest that IRP-1 and IRP-2 may each regulate unique mRNA targets in vivo, possibly extending their function beyond the regulation of intracellular iron homeostasis.
The regulation of cellular iron homeostasis is under the
post-transcriptional control of iron regulatory protein-1 (IRP-1), ()a cytoplasmic RNA-binding protein with specificity for
mRNA stem-loop structures known as iron-responsive elements (IREs)
(reviewed by Klausner et al. (1993) and
Kühn(1994)). IRP-1, formerly referred to as
IRE-binding protein (Rouault et al., 1988; Leibold and Munro,
1988), iron regulatory factor (IRF; Müllner et
al., 1989), or ferritin repressor protein (Walden et al.,
1988), has been identified as the cytosolic counterpart of the citric
acid cycle enzyme, aconitase (Hentze and Argos, 1991; Rouault et
al., 1991; Kaptain et al., 1991; Haile et al.,
1992a; Kennedy et al., 1992). IRP-1 is now regarded as a
bi-functional ``sensor'' of iron, switching between RNA
binding and enzymatic activities depending on cellular iron status
(Haile et al., 1992a, 1992b; Constable et al., 1992;
Emery-Goodman et al., 1993; Basilion et al., 1994).
In iron-depleted cells, IRP-1 inhibits translation of ferritin and
erythroid 5-aminolevulinic acid synthase mRNAs by binding to IREs
located in their 5`-untranslated region (UTR) (Aziz and Munro, 1987;
Hentze et al., 1987; Bhasker et al., 1993; Melefors et al., 1993). Binding of IRP-1 to a cluster of five IREs in
the 3`-UTR of transferrin receptor mRNA stabilizes this transcript
(Casey et al., 1988; Müllner and
Kühn, 1988; Müllner et
al., 1989; Koeller et al., 1989). The net result of this
RNA-protein interaction is thus increased cellular iron uptake and
availability. The inverse effect ensues when iron is high, as IRP-1 no
longer binds well to the IRE hairpins (reviewed by
Kühn(1994)).
A second IRE-binding protein has been characterized in rodents (Henderson et al., 1993; Guo et al., 1994) and in human cells (Samaniego et al., 1994), and is now commonly referred to as IRP-2. IRP-2 is a 105-kDa protein that binds specifically to all known mRNA IREs with an affinity equally as high as that of IRP-1 (Henderson et al., 1993; Guo et al., 1994). The two proteins are encoded by separate genes (Rouault et al., 1990), and their cDNA sequences (Rouault et al., 1992; Guo et al., 1995a) reveal conservation of cysteine residues known to ligate a [4Fe-4S] cluster in IRP-1 (Philpott et al., 1993; Hirling et al., 1994), although IRP-2 is apparently enzymatically inactive (Guo et al., 1994). The IRPs both respond to iron, but via different pathways. IRP-1 is post-translationally converted between active and inactive RNA-binding forms (reviewed by Kühn(1994)). IRP-2, however, is induced following iron starvation through renewed synthesis of stable IRP-2 protein (Henderson and Kühn, 1995; Pantopoulos et al., 1995), and its inactivation by iron reflects degradation of IRP-2 protein (Guo et al., 1994; Samaniego et al., 1994) by a translation-dependent mechanism (Henderson and Kühn, 1995). The IRPs are able to inhibit the translation of IRE-containing mRNAs in vitro (Guo et al., 1994; Kim et al., 1995), and therefore both proteins are potential mRNA regulators in vivo.
The possibility that the IRPs may bind additional IRE-like hairpins, and thus regulate an extended repertoire of mRNAs, is supported by a recent study in which a wide range of suboptimal IRP-1 binding sequences were selected from a partially degenerate pool of ferritin IRE RNAs (Henderson et al., 1994). Interestingly, several of these bound exclusively to IRP-1 and not to IRP-2. These findings suggest that the RNA-binding specificities of these two related proteins are overlapping, but different. We postulated that IRP-2 might also bind not only to wild-type IREs, but exclusively to a unique set of RNA targets. In this study we investigated this premise, using the ferritin IRE as a model for mutagenesis, as this sequence is the optimal IRP target and a range of well characterized mutants is already established for comparison (Henderson et al., 1994). By focusing on sequential mutagenesis of different IRE loop bases, we directly define and compare the RNA-binding specificities of mouse IRP-1 and IRP-2. Our new findings demonstrate the favorable effect that a structured IRE loop has on RNA binding by both IRPs and identify a novel set of four RNAs that bind exclusively to IRP-2. Furthermore, an assay is described for the discrimination of human IRP-1 from IRP-2, using IRP-specific RNA probes. Finally, we show that several features that distinguish the regulation of rodent IRP-1 and IRP-2 are well conserved in humans.
Fractions were
then analyzed by band-shift assay using P-labeled
degenerate IRE stem-loops. Two sets of four partly degenerate IREs were
designed, which essentially represent variations of a previously
described hairpin sequence, identical to the wild-type ferritin IRE
except for randomization of the 7 conserved bases in the loop and bulge
(for details of sequence, see Henderson et al.(1994)). The
first group of 4 IRE hairpins (Series 1) was made partly degenerate
only in the loop. Each Series 1 pool maintained a fixed base at loop
position 1, while the remaining 5 bases were completely randomized
except for the base at position 6, which could not pair with the first
loop base. Series 2 RNAs differed from those of Series 1 only in that
the base at loop position 5 was constrained to pair with that of
position 1. The loop sequence and degeneracy of these RNAs is shown in Fig. 2. The RNAs were labeled and used to probe B16 fractions.
In each case, protein was in excess, and the detection of IRP complexes
was limited by the number of actual IRP-binding sequences in each RNA
mixture. The detection limit of our assay was about 100 cpm/complex,
and mixed RNA probes were used at levels (5
10
to
10
cpm) sufficiently high to detect a protein binding to a
single sequence with high affinity. While certain degenerate IRE pools
detected IRP-1 or IRP-2, all RNA mixtures detected some additional
proteins, none of which were sequence-specific as judged by band-shift
competition assays.
Figure 2:
Screening mouse B16 cell fractions with
degenerate IRE probes. A, sequence of the human ferritin
H-chain IRE (used in Fig. 1) is shown at top, adjacent
to a degenerate IRE made by randomizing the 7 conserved nucleotides of
the IRE bulge (cytosine) and loop (top right; numbering of
loop bases is indicated). Two series of RNA pools (degenerate only in
the loop) were then prepared and tested for binding to the IRPs (see
``Materials and Methods''). The degeneracy of each randomized
IRE pool is given in brackets; Roman N is any base,
and italic N is any base that does not pair with the first
loop position. Equivalent limiting amounts (5 10
cpm) of each
P-labeled, gel-purified RNA was used to
probe the mouse B16 fractions shown in Fig. 1. The ability of
Series 2 RNAs to bind IRPs is shown relative to that of Series 1 RNAs (lower panel, box). B, examples of
band-shift assay gels are shown, comparing the C
G
and G
C
RNAs of Series 2 with their Series
1 counterparts. Fractions were preincubated with 200 ng of yeast tRNA,
and additional nonspecific complexes are visible in each sample above
the IRP complexes.
Figure 1:
Detection of IRE-binding proteins in
mouse extract. Mouse B16 melanoma cytoplasmic extract was fractionated
by linear gradient over a Mono-Q column, and 1 µl of selected
fractions analyzed by band-shift assay. Samples were probed with 5
10
cpm of
P-labeled IRE, and
IRE-protein complexes corresponding to IRP-1 and IRP-2 are clearly
indicated. A third IRE-binding protein was detected in fractions
16-19; however, this RNA-binding protein (RBP) was not
specific for IRE stem-loops. The gel was scanned with a Molecular
Dynamics PhosphorImager, and relative signal strength is plotted in the lower panel. The graph does not accurately portray the total
relative amounts of IRP, as while each fraction volume was 1 ml, most
IRP-1 eluted in the flow-through (24 ml volume). Cross-contamination of
IRP fractions was minimal; < 0.3% of total IRP-2 activity eluted in
the first wash, and in the major IRP-2 peak fraction (175 mM KCl), IRP-1 comprised <0.2% of total IRE binding
activity.
Figure 3:
Selection of IRP-binding sequences. A, degenerate DNA fragments corresponding to the Series 2
CG
and G
C
RNA pools
were cloned, transcribed, and individual IRP-1 or IRP-2 binding
sequences identified by band-shift assay using the selection procedure
outlined. B, equal amounts (2
10
cpm) of
selected RNAs were labeled with [
P]CTP,
gel-purified, and compared for binding to IRP-1 or IRP-2 by band-shift
assay. The IRPs were in excess in total or enriched B16 fractions. The
wild-type human ferritin IRE (clone 42) is included as positive
control; a randomly selected non-binding sequence (mutant GC-1) is a
negative control.
More specific conditions were
required to detect individual human IRPs using selected IRP-specific
probes. Importantly, human IRP-2 appears more susceptible to oxidation
than rodent IRP-2, and thus extracts were prepared fresh and processed
quickly for experiments to be performed in the absence of reductant. Up
to 4 µg of human cytoplasmic extract was preincubated with 300 ng
of yeast tRNA, and then mixed with 5 10
cpm of IRE
probe (mutant GG1 or CG125) for 10 min in the presence or absence of 1%
2-ME (higher concentrations of 2-ME can decrease human IRP-2 activity),
and in the presence of 5 mg/ml heparin. Gels were scanned and bands
quantitated using a Compaq PhosphorImager equipped with Molecular
Dynamics Image software.
In
addition to competition assays, which were performed in the absence of
heparin, each selected RNA was P-labeled and gel-purified,
and then tested for binding to IRP-1 or IRP-2 (protein in excess) by
band-shift assay. The amount of IRE
IRP complex formed by each
probe (2
10
cpm; 0.04 ng of RNA/reaction) was
determined relative to the wild-type IRE, and a ``binding
ratio'' then assigned to each sequence.
As expected, the above analysis identified
IRP-1 and IRP-2 as the only sequence-specific IRE-binding proteins in
B16 cells. To test whether IRP-related proteins may exist that bind RNA
hairpins of similar structure but different sequence to the IRE, we
probed B16 fractions with a P-labeled ferritin IRE probe
made degenerate at the 7 conserved loop and bulge nucleotide positions
(see Henderson et al.(1994) and Fig. 2A), and
detected several additional RNA-protein complexes (data not shown). In
order to assess the specificity of the extra band-shift complexes, a
set of four less degenerate RNA mixtures were prepared, in which bases
at the bulge and the first loop position were fixed (see Series 1 RNA
pools in Fig. 2A). Band-shift analysis of the mouse B16
fractions revealed binding of IRP-1 and IRP-2 to certain
[
P]RNA pools (e.g. C
N
and G
N
probes in Fig. 2B); however, following detailed examination and a
series of competition assays, we identified no other proteins with
specificity for IRE-like sequences. We therefore have no evidence to
suggest the existence of other IRPs.
Figure 4:
Sequence and binding affinities of
selected RNAs. The loop sequence of selected RNAs is shown relative to
the wild-type sequence (clone 42: 5`-CAGUGC-3`). Bases capable of
pairing (positions 1 and 5) are in large type, and
non-wild-type nucleotides are underlined. The relative binding
affinities of each RNA were determined by competition assay (K values), or by
P-labeling each
RNA and comparing IRP binding of each sequence with a wild-type IRE
probe (see ``Binding Ratios''; see Fig. 3B for an example). Both approaches utilized the band-shift assay
(see ``Materials and Methods'' for details), and all binding
affinities are relative to the wild-type IRE, set a value of 1. Binding
assays were performed in the presence of 2% 2-ME. ND, not
determined. Mutant GC-1 (loop: 5`-GCUCCG-3`) is a negative
control.
The IRE mutant
CG125 (loop: 5`-CCGAGC-3`) showed the greatest specificity for IRP-2,
as judged by comparison of K values and binding
ratios (Fig. 4, Table 1). We tested further the
specificity of this IRE variant by band-shift analysis (Fig. 5).
Binding of the mutant CG125 probe to mouse IRP-1 and IRP-2 was compared
to a wild-type IRE, in the presence or absence of heparin. Remarkably,
in the absence of heparin, the CG125 probe bound mouse IRP-2 50% as
well as the wild-type IRE, but it showed no detectable binding to IRP-1
(see Fig. 5). Increasing the stringency of the reaction, by
adding heparin, reduced binding of the CG125 probe to IRP-2 as expected
(binding decreased to about 12% of wild type). This experiment confirms
mutant CG125 as an IRP-2-specific RNA target.
Figure 5:
Identification of an IRP-2-specific RNA
sequence. P-Labeled mutant CG125 RNA was compared to
wild-type IRE for binding to mouse IRP-1 or IRP-2. Equal amounts (2
10
cpm) of each probe was incubated with enriched
IRP fractions in the presence (+) or absence(-) of 5 mg/ml
heparin, after addition of 2% 2-ME. The band-shift gel was scanned by
phosphorimager, and the relative band intensities graphed (lower
panel). The mutant CG125 probe bound exclusively to IRP-2,
independent of heparin treatment.
We next tested the ability of each
IRP-specific probe to bind different forms of bacterially expressed,
purified recombinant human IRP-1 (Fig. 6A). The
IRP-2-specific probe (mutant CG125) bound to saturating amounts of
histidine-tagged IRP-1 fusion protein less than 1% as effectively as
the IRP-1-specific (mutant GG1) probe. The slightly better binding (5%
of wild type) of the IRP-2-specific probe to a glutathione S-transferase-IRP-1 fusion protein reflected nonspecific
binding, as determined by comparison with other RNA stem-loops (data
not shown). This result confirms the difference in affinity of these
probes for human IRP-1. We then assessed the ability of mutant GG1 and
mutant CG125 P-labeled RNAs to form UV-cross-linked
covalently bound complexes with mouse and human cytoplasmic proteins.
Conditions of partial RNase T1 digestion were attained, which
demonstrated clear UV cross-links to proteins of
100 kDa (Fig. 6B). The wild-type and mutant IRE probes
cross-linked specifically to mouse IRP-1 and/or IRP-2 proteins from
enriched mouse B16 cell fractions. It was not possible under these
conditions to distinguish between the two human IRPs, which are of
similar size (IRP-1,
98 kDa; IRP-2,
105 kDa; Rouault et
al.(1992)). However, UV cross-linking of human Molt-4 extracts
showed strong binding of all probes to a
100-kDa protein. We
expect, given the poor affinity of the CG125 probe for recombinant
human IRP-1, that the
100-kDa human protein cross-linked by this
probe is most likely IRP-2.
Figure 6:
Specificity of different probes for human
IRP-1 and IRP-2. A, 4 10
cpm of wild-type,
IRP-2-specific (mutant CG125) and IRP-1-specific (mutant GG1) IRE
probes were compared for binding to different forms of recombinant
human IRP-1 by band-shift assay. The probes were incubated with
saturating amounts of a glutathione S-transferase-IRP-1 fusion
protein (GST fusion) or histidine-tagged IRP-1. The data were
quantified and plotted relative to wild-type IRE, which is set at 100%. B, the above probes (2
10
cpm) were
UV-cross-linked to proteins in mouse IRP-1 or IRP-2 fractions, and in
human Molt-4 cytoplasmic extract (see ``Materials and
Methods''). Cross-linked RNA-protein complexes were separated on a
denaturing 8% SDS-polyacrylamide gel. The probes covalently bound to
97-kDa proteins corresponding to mouse IRP-1 and/or IRP-2 as
expected. All probes, including mutant CG125, cross-linked to a similar
sized
97-kDa protein in human Molt-4 extracts, in addition to some
smaller sized proteins that may include IRP degradation products. The
autoradiographs shown represent 4-day exposures of x-ray film to the
gels.
Figure 7:
Detection of IRP-1 and IRP-2 RNA binding
activities in human cells. A, cytoplasmic extract (4 µg)
from different human cell lines was probed with gel-purified wild-type
and IRP-specific mutant RNAs (5 10
cpm) in the
presence of 1% 2-ME and 5 mg/ml heparin, and analyzed by band-shift
assay. 2 µg of total mouse B16 extract was included as a control.
All probes detected human IRE-protein complexes migrating at the same
position as mouse IRP-2 (mIRP-2); however, IRP-1 and
IRP-2-specific probes generated different patterns, corresponding to
variations in expression of the human IRPs (hIRPs). The
autoradiographs shown represent different exposures (e.g. ratio of exposure times was 1:2:9 for wild-type, mutant GG1, and
mutant CG125 probes, respectively) to compensate for differences in
binding affinities of the probes. This scaling was made according to
the relative affinities of these probes for the mouse IRPs, as evident
by comparison of the mouse IRP signals. B, gels were
quantitated and the relative signal intensities plotted as shown, after
normalizing the values for IRP-1 (mutant GG1;
2) and IRP-2
(mutant CG125;
9) probes against the wild-type IRE. To aid
comparison, the IRP-2 activity profile is shown superimposed over the
wild-type expression profile.
The IRP-1-specific probe generated a single human band-shift complex with a pattern very similar to that of the wild-type ferritin IRE probe; IRP-1 activity was lowest in human TK6 lymphoblasts and HL60 promyelocytic leukemia cells. By contrast, the IRP-2-specific probe formed a band-shift complex migrating at the same position as that of IRP-1 in human cells; however, the pattern was markedly different. IRP-2 activity was strongest in Molt-4 T-cell leukemia cells and markedly lower in fibroblasts (HFF, FEK) and different tumor-derived cell lines. The same expression pattern was observed independently using a different IRP-2-specific probe (mutant GC157; loop sequence: 5`-GCGCCG-3`), although the background was somewhat higher (data not shown).
These results confirm that the exclusive binding specificities of IRP-1 and IRP-2 are at least partly conserved between rodents and humans. Furthermore, the IRP-specific probes now provide a useful assay for the differential detection of the human proteins, and indicate that their expression is cell type-specific.
Figure 8:
Iron regulation of human IRP-1 and IRP-2.
Cytoplasmic extract was prepared from Molt-4, HL60, and HeLa cells,
untreated in log-phase (L) or treated for 20 h with 100
µM desferrioxamine (D), or 20 h of
desferrioxamine followed by washing and 4 h of treatment with 60
µg/ml ferric ammonium citrate (Fe). The effect of
pretreating HeLa cells with 10 µg/ml cycloheximide 15 min before
iron treatment was also tested (Fe/C). 2 µg of each
extract was mixed with 6 10
cpm of gel-purified
mutant GG1 (IRP-1-specific) and mutant CG125 (IRP-2-specific) RNA
probes in the presence of 300 ng yeast tRNA, 5 mg/ml heparin, and the
presence (+) or absence(-) of 1% 2-ME, and complexes
analyzed by band-shift. Autoradiographs were exposed for 24 h (IRP-1
gel) or 48 h (IRP-2 gel). Gel bands were quantitated and the relative
intensities plotted in the lower panel, after normalizing
values to account for differences in binding affinity. This experiment
was performed twice with similar results.
This experiment reveals that endogenous human IRP-1 and IRP-2 activities are iron-regulated. In addition, we observed that pretreatment of HeLa cells with cycloheximide blocked the inactivation of IRP-2 by iron, but not the inactivation of IRP-1 (see Fig. 8, lower panel, for quantitation). The same result was observed with the translation inhibitor anisomycin (data not shown) and correlates perfectly with recent data on rodent IRP-2 (Henderson and Kühn, 1995). When considered together, our findings strongly suggest that our probes do indeed detect and discriminate between the two human IRPs. Furthermore, the features that distinguish binding activity of IRP-1 from IRP-2 are well conserved between human and rodent cells.
The IRPIRE interaction represents a unique cellular
mechanism underlying the post-transcriptional regulation of several
genes. Recent evidence supports the view that IRP-1 and IRP-2 each
function as mRNA trans-regulators involved in maintaining
intracellular iron homeostasis, as both proteins (i) are regulated by
iron levels (Henderson et al., 1993), (ii) inhibit
IRE-containing mRNA translation in vitro (Guo et al.
1994; Kim et al., 1995), and (iii) bind ferritin and
transferrin receptor mRNA IREs with equally high affinity (Henderson et al., 1993; Guo et al., 1994). In this study, we
further show that the two IRPs have conserved the ability to
individually recognize exclusive and distinct sets of RNAs. This novel
finding suggests the possibility that the IRPs may regulate additional
mRNA targets. We also describe features of the IRE hairpin that permit
binding by either or both IRPs and have used this information to
establish an assay for the detection of individual IRP RNA binding
activities in human cells.
IRP-1 was generally
more tolerant of IRE base changes than was IRP-2, particularly of
specific base alterations in the bulge and loop (1:5) base pairs. Only
IRP-1 recognized IRE loops containing a UA
pair, and some less stable non-Watson-Crick base pairs (Henderson et al., 1994). While none of the 600 randomly selected
C
G
or G
C
-loop RNAs were
specific for IRP-1, we identified four such sequences that
preferentially bound to IRP-2. Each contained a double mutation of two
loop bases (positions 2 and 4) predicted to be unpaired and accessible
for protein contact. These findings are not restricted to the ferritin
IRE, as fusion of an IRP-2-specific loop (mutant CG125: 5`-CCGAGC-3`)
to the erythroid 5-aminolevulinic acid synthase IRE stem resulted in
comparable selectivity for IRP-2, although the binding affinity was
greatly reduced (data not shown). We have thus dissected the wild-type
IRE loop and identified specific changes in paired or unpaired
nucleotides that are tolerated by only IRP-1 or IRP-2. These findings
reveal that the IRPs bind overlapping, but quite distinct, sets of RNA
targets.
The list of regulatory RNA-binding proteins is growing rapidly, and several of these bind with specificity to defined RNA hairpin structures (reviewed in Varani and Pardi(1994), Burd and Dreyfuss (1994), and McCarthy and Kollmus(1995)). Aside from the IRPs and tRNA synthetases, there are relatively few sets of related proteins known to discriminate between similar RNA hairpin targets via differences in loop sequence. The eukaryotic splicing snRNP proteins U1A and U2B" are one example; they share strong sequence similarity and bind closely related U1/U2 small nuclear RNA stem-loops (reviewed by Nagai and Mattaj(1994)). Furthermore, the bacteriophage MS2 and GA coat proteins are 62% identical in sequence and act as translation repressors that recognize similar RNA hairpins differing primarily in the 4-base loop sequence (see Lim et al.(1994)). Discernment of the different RNA loop sequences by MS2 and GA coat proteins was attributed to a single amino acid residue (Lim et al., 1994). Thus, while the relatedness of IRP-1 and IRP-2 (Rouault et al., 1992) may account for their shared capacity to bind a wild-type IRE, it should prove at least equally as interesting to learn the amino acid differences which decide the individual specificities of the two IRPs.
In our view, intraloop base pairing induces a
conformational change within the IRE loop, such that the intervening
three bases (positions 2, 3, and 4; see Fig. 2A for
numbering) are made accessible for contact with IRP-1 and IRP-2 (model
outlined by Henderson et al.(1994)). We propose that the 3
unpaired loop bases splay outward to form hydrogen bonds with specific
IRP amino acids, as was shown previously for other RNAprotein
interactions including that between the tRNA
and
tRNA
anticodon loops and their respective glutaminyl- and
aspartyl-tRNA synthetases (Rould et al., 1991; Cavarelli et al., 1993), and recently for the U1 small nuclear RNA
stem-loop and U1A spliceosomal protein (Oubridge et al.,
1994). The distinction in binding by the IRPs arises following base
substitution at IRE loop positions 2 and 4, which is clearly far better
tolerated by IRP-2. It is interesting that IRP-1 is more tolerant of
different 1:5 base pair combinations in the IRE loop. Perhaps after
initial binding, IRP-1 is better able to fold the IRE into a
conformation favoring the 1:5 loop interaction, thereby enabling base
pairs with lower energy to form. This explains the ability of IRP-1,
but not IRP-2, to bind IRE-like hairpins with less stable
non-Watson-Crick base pairs previously observed in other RNAs (Gutell,
1993; Henderson et al., 1994). Similar reasoning predicts RNA
hairpins with C
G
and
G
C
-type loops to be the most stable, possibly
even forming spontaneously in solution, and thus facilitating easier
recognition by IRP-2. Indeed, the wild-type IRE loop may form a
C
:G
base pair in solution (Sierzputowska-Gracz et al., 1994), and it will be interesting to learn if
alternate loop base pairs also form prior to RNA interaction with the
IRPs. Ultimately, refined structural analyses of the IRP
RNA
complexes are required to confirm these proposals.
In this study, we demonstrated that IRE-like hairpins specific for mouse IRP-1 or IRP-2 showed similar selectivity for the human IRPs. Despite an increase in background caused by a lower binding affinity of the IRP-2-specific probes, the availability of different IRP-2-specific and non-binding sequences enables a controlled assay for human IRP-2 activity. A survey of human cell lines revealed different IRP expression patterns, and as in most rodent cell lines (Henderson and Kühn, 1995), IRP-1 activity was always highest. In fact, the human IRPs displayed most of the previously defined characteristics that distinguish rodent IRP-1 and IRP-2. Both of the human proteins were regulated by iron, but only IRP-1 activity was recovered in extracts from iron-treated cells by in vitro reduction with 2-ME. We recently reported that iron-mediated inactivation of mouse IRP-2, but not IRP-1, was translation-dependent (Henderson and Kühn, 1995). This difference in the iron regulatory pathways of the two IRPs also applies in human cells. These findings establish several aspects of IRP regulation that are well conserved and describe a general assay to detect individual human IRP RNA binding activities.