(Received for publication, August 28, 1995)
From the
Iron-responsive elements (IREs) are cis-acting mRNA stem-loop structures that specifically bind cytoplasmic iron regulatory proteins (IRPs). IRP-IRE interactions mediate the coordinate post-transcriptional regulation of key proteins in iron metabolism, such as ferritin, transferrin receptor, and erythroid 5-aminolevulinic acid synthase. Depending on whether the IRE is located in the 5`- or 3`-untranslated region (UTR), binding of IRP will inhibit mRNA translation or degradation, respectively. Here we describe a new IRE in the 5`-UTR of succinate dehydrogenase subunit b (SDHb) mRNA of Drosophila melanogaster. The SDHb IRE binds in vitro to vertebrate and insect IRPs with a high affinity equal to that of human ferritin H chain IRE. Under conditions of iron deprivation, SDHb mRNA of Drosophila SL-2 cells shifts to a non-polysome-bound pool. Moreover, translation of a human growth hormone mRNA with the SDHb IRE in its 5`-UTR is iron-dependent in stably transfected L cells. We conclude that the SDHb IRE mediates translational inhibition both in insect and vertebrate cells. This constitutes the first identification of a functional IRE in insects. Furthermore, Drosophila SDHb represents the second example, after porcine mitochondrial aconitase, of an enzyme of the citric acid cycle whose mRNA possesses all necessary features for translational regulation by cellular iron levels.
Cytoplasmic control of mRNA translation and stability is well
documented for transcripts that encode proteins in iron metabolism (1, 2, 3) . As a shared feature, the
regulated mRNAs contain specific RNA stem-loop structures, the
iron-responsive elements (IREs), ()which are binding sites
for iron regulatory proteins (IRP-1 and
IRP-2)(4, 5, 6, 7, 8, 9) .
The RNA binding activity of these trans-acting proteins is induced in
the cytoplasm under conditions of iron deprivation. Ferritin H and L
chain mRNA, erythroid 5-aminolevulinic acid synthase mRNA and
mitochondrial aconitase mRNA, have a single IRE in their
5`-untranslated region (UTR)(10, 11, 12) . As
documented for the first three of these mRNAs, interaction with an IRP
prevents ribosomes from initiating translation (13, 14, 15) . Unlike the translationally
regulated mRNAs, transferrin receptor (TfR) mRNA contains five IREs
located in the 3`-UTR(5, 6) . Here, IRP binding causes
an inhibition of TfR mRNA degradation and, consequently, an increase
rather than a decrease in protein expression. This coordinate
regulation of ferritins and TfR is thought to act as a feedback
mechanism to maintain cellular iron homeostasis, since iron scarcity
diminishes cellular iron storage but enhances the potential of iron
uptake. In contrast, under the inverse conditions of ample iron supply,
IRPs are inactivated, permitting ferritin mRNA translation and TfR mRNA
degradation. These conditions are likely to prevent iron overload.
Similarly, translational inhibition of the key enzyme in erythroid
porphyrin biosynthesis, 5-aminolevulinic acid synthase, has been viewed
as a mechanism to match this pathway with iron
availability(11, 12, 16) .
The functional importance of the IRP-IRE interactions is also supported by their phylogenetic conservation(17, 18) . IRE-binding proteins have been identified in vertebrates, insects, and annelids but seem to be missing in yeasts, plants, and bacteria. IREs are similarly conserved in the respective mRNAs of vertebrate and some invertebrate species (19, 20) . However, it remains unknown how many more hitherto unidentified mRNAs are regulated through IRP-IRE interactions. In fact, a certain variability in the IRE sequences does not permit the isolation of new members of the family by hybridization or PCR methods, and their discovery relies at present mainly on the analysis of newly acquired sequences in data bases. For this purpose, Dandekar et al.(12, 21) have designed a computer-aided screening program.
On the basis of phylogenetic comparisons and in vitro mutagenesis, the IRE has been defined as a strongly
conserved mRNA structure(19, 22) . Invariably, it
consists of an upper and a lower stem of paired ribonucleotides with a
G value for base interactions of approximately -5
kcal/mol. The upper stem comprises always 5 complementary bases, which
need to be paired for IRP binding. The sequences of these stems vary
among mRNAs from different genes and do not seem to be crucial in the
RNA-protein interaction. However, they are well conserved during
evolution for any given mRNA species (19) . Below the upper
stem there is invariably a small 5`-bulge consisting either of a single
unpaired C or a C preceded by two additional nucleotides opposite to a
single unpaired nucleotide (ferritin IRE). The loop at the tip consists
of 6 nucleotides with a consensus CAGUGN sequence. This sequence
together with the bulge C confers the binding specificity(23) .
The consensus loop sequence exhibits the highest affinity for either
IRP-1 or IRP-2, but we have recently defined a spectrum of suboptimal
sequences with slightly lower affinities that are still able to bind to
IRPs in vitro. Because of the existence of such alternative
sequences, we have carried out computer searches of the EMBL data base
to identify possible new candidate IREs in mRNAs. In the course of this
search, the recently entered succinate dehydrogenase subunit b (SDHb)
gene of Drosophila melanogaster(24) revealed a
classical IRE in the 5`-UTR. This enzyme subunit of the citric acid
cycle is translated in the cytoplasm prior to its import into the
mitochondrial matrix. It contains three different iron-sulfur
clusters(25) , which are essential for the transfer of
electrons to the respiratory chain(26) . In the present study
we show the functional importance of this SDHb IRE.
Figure 1:
Drosophila SDHb mRNA
contains an IRE-like sequence. A, the sequence and position of
the IRE in the SDHb mRNA of D. melanogaster is depicted. The
IRE is printed in boldface letters with the loop sequence framed, and the transcription start-codon is indicated by an arrow. The structure of the insect SDHb IRE, as predicted by
the GCG ``Fold'' program(43) , is shown in B (top) in comparison with the IREs of human ferritin H
chain and erythroid 5-aminolevulinic acid synthase (eALAS)
mRNAs (bottom). In these structures we have included the
recent finding of a C-G
base pairing in
the loop(23, 30) . C, the transcription start
site of the SDHb gene was mapped by primer extension as described under
``Materials and Methods,'' and the product is indicated by an arrow. A sequencing reaction of the plasmid pGEM-3Zf(-)
(Promega) using an oligonucleotide complementary to the T7 promoter is
shown as a size marker.
Figure 2:
The SDHb IRE forms distinct complexes with
a protein in Drosophila cell extracts and with purified
recombinant human IRP-1. Radiolabeled human ferritin H chain IRE (Fer), SDHb IRE (SDH), and the entire 5`-UTR of SDHb
mRNA (5`) form RNA-protein complexes with Drosophila and human IRPs. The gel retardation assay was carried out with 2
µg of protein from Drosophila SL-3 cell extract or 250 ng
of purified recombinant human IRP-1 as described under ``Materials
and Methods.'' 2 10
cpm of the IREs or 3
10
cpm of 5`-UTR were used as probes. After
binding, RNase T1 was added to samples containing the 5`-UTR-probe and,
after 5 min of incubation at room temperature, heparin was added to all
samples at 5 mg/ml. The incubation was allowed to proceed for 10 min
before loading of samples on a 6% nondenaturing polyacrylamide
gel.
Further evidence for a direct interaction
between Drosophila SDHb IRE and IRP was obtained by a
UV-cross-linking experiment. Extracts from either SL-3 cells or
iron-deprived murine B16.F1 cells were UV-irradiated in the presence of
an excess gel-purified radiolabeled SDHb IRE (Fig. 3). Where
indicated, unlabeled ferritin H chain IRE was mixed at 100-fold molar
excess with the [P]RNA probe before the addition
of extract. Two specifically cross-linked proteins could be detected in
B16.F1 cells, presumably IRP-1 (97 kDa) and IRP-2 (105 kDa). One
specific band of about 100-kDa size is visible in Drosophila extracts. These results confirm the phylogenetic conservation of
the IRP-IRE interaction as observed
previously(17, 18) .
Figure 3:
Drosophila cytoplasmic extracts contain a
specific IRE-binding protein of approximately 100 kDa. Cytoplasmic
protein extracts were prepared from untreated Drosophila SL-3
cells or mouse B16.F1 cells that had been deprived of iron by
incubation with 100 µM desferrioxamine for 24 h to induce
IRP-1 and IRP-2. Extracts (15 µg) were incubated with 8
10
cpm (
1.6 ng) of radiolabeled SDHb IRE and
UV-irradiated. Where indicated, a 100-fold molar excess of unlabeled
ferritin H chain competitor IRE was premixed with the
[
P]RNA probe (Comp.). Samples were
treated with RNase T1 prior to addition of heparin to 5 mg/ml. Proteins
were separated on a 8% SDS-polyacrylamide gel, blotted onto a
nitrocellulose membrane, and
autoradiographed.
Since functional IREs show a strong affinity for IRPs, we measured the relative affinity of the SDHb probe compared with the human ferritin H chain probe. A fixed amount of radiolabeled IRE (about 50 pg) was analyzed in cross-competition assays with various concentrations of unlabeled RNA for the binding to IRP from man, fruit fly, or mouse (Fig. 4). The results revealed that the Drosophila SDHb IRE is an almost as good a competitor as the ferritin H chain IRE in any of the settings, whereas tRNA was unable to compete even at 200-fold molar concentration. Noteworthy, both IREs showed similarly high affinity for IRP-2 in extracts of iron-deprived mouse B16.F1 cells (data not shown).
Figure 4:
The SDHb IRE has a similar affinity for Drosophila IRP and human IRP-1 as the ferritin H chain IRE.
Radiolabeled ferritin H chain IRE (left panels) or labeled
SDHb IRE (right panels) was incubated with IRPs in the
presence of various concentrations of unlabeled competitor RNA (SDHb
IRE, ferritin H chain IRE, or yeast tRNA (expressed as mol of
unlabeled/mol of labeled RNA)). Premixed radiolabeled probe (2.5
10
cpm 50 pg) and x-fold competitor were incubated
with a constant amount (750 ng of protein) of Drosophila SL-3
cell extract (panel A) or 1 ng of purified recombinant human
IRP-1 (panel B). To the reactions containing the SL-3 cell
extract, heparin was added at 5 mg/ml after binding. To allow direct
comparison for each set of competitors, the gels were exposed for the
same period of time.
Figure 5:
Drosophila SDHb IRE is an active
translational regulatory element in mouse L cells. The 5`-UTR of the
insect SDHb cDNA was cloned into the 5`-UTR of the human growth hormone
expression vector L5-GH(28) . This plasmid DNA (SDH-GH) was stably co-transfected into Ltk cells by the calcium phosphate method(31) . Control
plasmids had either a human ferritin H chain IRE (Fer-GH) or a
mutated H chain IRE with a more than 100-fold lower IRP binding
affinity (213-GH). Pools of transfected cells were cultured
for 24 h in medium supplemented with either 100 µM desferrioxamine (Des) or 60 µg/ml ferric ammonium
citrate (Fe). Cells were then labeled in methionine-free
medium with 40 µCi/ml [
S]methionine, and
secreted human growth hormone was quantitatively immunoprecipitated
from the culture medium.
Finally, we wanted to obtain direct evidence that iron deprivation regulates SDHb mRNA translation in insect cells. This was analyzed by the mRNA distribution on polysome gradients. Drosophila SL-2 cells were incubated in the presence of 150 µM desferrioxamine for 48 h and then either harvested or further incubated for another 4 h with 60 µg/ml ferric amonium citrate. Cytoplasmic extracts were prepared and separated on a 15-60% sucrose gradient as described under ``Materials and Methods.'' RNA from every odd gradient fraction was extracted and analyzed by Northern blotting. Methylene blue staining of the membrane revealed the distribution of ribosomal RNAs (Fig. 6). The membrane was then successively hybridized with probes specific for SDHb and the Drosophila ribosomal protein A1 (rpA1). Whereas the rpA1 mRNA could be detected in the same gradient fractions regardless of cellular iron status, SDHb mRNA redistributed from a free RNA-pool (fractions 5-7) under low iron conditions to heavier polysomes (fractions 9-17) upon addition of iron. We conclude that in insect cells translation of the SDHb mRNA is regulated by cellular iron levels.
Figure 6:
Redistribution of insect SDHb mRNA between
a polysome-bound and a nontranslated pool as a function of cellular
iron. SL-2 cells of D. melanogaster were either cultured for
48 h in medium with 150 µM desferrioxamine (Des)
or after the desferrioxamine treatment for an additional 4 h in medium
with 60 µg/ml ferric ammonium citrate (Des/Fe). Cells (2.5
10
) were then lysed and centrifuged at 13,000
g for 10 min. The supernatant was separated on a
15-60% sucrose gradient. RNA from sucrose gradient fractions was
analyzed by electrophoresis on a 1.5% agarose/formaldehyde gel and
Northern blotting. Blots were hybridized with
P-labeled
probes of the Drosophila SDHb cDNA and the rpA1 cDNA. A sample
corresponding to the total RNA that was loaded onto the gradient is
shown in the first lane (T).
We have identified a new IRE in the 5`-UTR of the D.
melanogaster SDHb mRNA. This IRE is single-C bulge type and
contains the consensus loop sequence CAGUGN, thus resembling the IREs
present in TfR and erythroid 5-aminolevulinic acid
synthase(5, 34) . Its energy of folding (G = -5.9 kcal/mol) lies in the normal range of
functional IREs. Using gel retardation assays and UV-cross-linking, we
demonstrate that in vitro the new IRE binds with an equally
high affinity as a human ferritin H chain IRE to IRP in Drosophila cell extracts, mouse IRP-1 and IRP-2, and recombinant human IRP-1.
These results confirm the earlier notion that the IRP-IRE interaction
is phylogenetically conserved between human and
fly(17, 18) . The SDHb IRE represents a functional cis-acting translational regulatory element in vivo since it confers iron regulation to an hGH reporter construct
transfected into L cells. However, regulation appeared to be slightly
less efficient than with the Fer-GH construct (Fig. 5). This
might be due to the higher base line of expression in the
SDH-GH-transfected cell line. Another reason could be the relatively
great distance of the SDHb IRE from the 5`-cap structure (71
nucleotides). The efficiency of IREs as translational control elements
was shown to depend on their position with respect to the cap
site(15, 35) . In mouse B6 fibroblasts, regulation was
lost with an IRE located 67 or more nucleotides from the
cap(35) . However, the authors noted that the relative effect
of distance on the efficiency of regulation varied between different
cell types.
Direct proof for the iron-dependent translation of SDHb in insect cells would require a specific antibody that recognizes the native protein for immunoprecipitation. Since no such antibody was available to us, we analyzed the association with ribosomes of SDHb mRNA from iron-depleted and iron-replenished Drosophila cells on a polysome gradient. In the case of ferritin mRNA, translational inhibition in iron-deprived rodent cells promotes a substantial shift of the mRNA from the polysome-bound mRNA to lighter, nonbound mRNA fraction(15, 36) . Here we show that association of the SDHb mRNA with polysomes could only be detected when iron was abundant, indicating that under conditions of low iron, translation of the mRNA was inhibited. This pattern is indicative of a block in the ribosome association with the SDHb mRNA in iron-deprived Drosophila cells.
Besides porcine mitochondrial aconitase(12, 37) , Drosophila SDHb is the second enzyme of the citric acid cycle, which seems to be subject to translational regulation by iron. This finding is presently difficult to interpret in terms of its physiological meaning. Whereas regulation of ferritins and transferrin receptor corresponds to a compensatory feedback loop in the control of iron homeostasis adjusting iron storage and uptake according to iron levels(4, 5, 6, 7, 8, 9) , it is less evident why enzymes in cellular energy production should be coupled to iron availability. One possible explanation relates to the fact that both enzymes are iron-sulfur proteins. Mitochondrial aconitase contains one [4Fe-4S] cluster, which is essential for aconitase activity(38) , and the SDHb subunit appears to contain three different clusters: one [4Fe-4S], one [3Fe-4S], and one [2Fe-2S] cluster(25) , which are needed to deliver electrons to the electron transport chain(26) . It seems possible in the case of such vital enzymes that the synthesis of apo-protein lacking the iron-sulfur cluster might be detrimental to enzyme subunit assembly and mitochondrial function. Such a hypothesis implies that expression of other mitochondrial iron-sulfur proteins should also be subject to iron regulation. It will therefore be of interest to learn whether this is indeed the case. Another connection previously proposed for the putative translational control of mitochondrial aconitase is the idea that cytoplasmic citrate might be needed for transport of iron into mitochondria and that under conditions of iron scarcity a certain level of citrate needs to be preserved to ensure this transport(39) . However, it remains unexplained why SDHb subunit should be controlled in the same way.
Thus far mRNAs that contain an IRE were found to be regulated in all
vertebrates, indicating the functional importance of a regulated
expression of these proteins. However, despite the strong phylogenetic
conservation of the IRP-IRE regulatory system between insects and
mammals, the SDHb IRE does not seem to be conserved in humans; IRE-like
sequences are absent from the recently published human genomic SDHb
sequence(40) . Consistent with this we found that, in contrast
to ferritin H chain and transferrin receptor mRNA, SDHb mRNA of human
HL-60 cells is not retained by an IRE affinity column consisting of
immobilized IRP. ()However, down-regulation of SDH activity
in skeletal and heart muscle of iron-deprived rats has been
reported(41, 42) , but the level at which regulation
occurs has not been determined. These notions suggest that SDHb
synthesis, for a specific reason, may be regulated in insects but not
in humans. Thus, the present study is the first one to identify a
functional IRE in insects and demonstrates that iron-dependent
translational control by IRP-IRE interaction is conserved in insects.
This opens the possibility of applying genetic approaches to the
further investigation of this post-transcriptional regulatory system.