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RED CELLS, IRON, AND ERYTHROPOIESIS
e-Blood
Iron regulatory protein-1 and -2: transcriptome-wide definition of binding
mRNAs and shaping of the cellular proteome by iron regulatory proteins
Mayka Sanchez,1-3 Bruno Galy,1 Bjoern Schwanhaeusser,4 Jonathon Blake,1 Tomi Bähr-Ivacevic,1 Vladimir Benes,1
Matthias Selbach,4 *Martina U. Muckenthaler,2,5 and *Matthias W. Hentze1,2
1European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; 2Molecular Medicine Partnership Unit (MMPU), EMBL, University of Heidelberg,
Heidelberg, Germany; 3Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Barcelona, Spain; 4Max Delbrück Center for Molecular Medicine,
Berlin, Germany; and 5Department of Pediatric Oncology, Haematology, and Immunology, University Hospital of Heidelberg, Heidelberg, Germany
Iron regulatory proteins (IRPs) 1 and 2 are
RNA-binding proteins that control cellular
iron metabolism by binding to conserved
RNA motifs called iron-responsive elements (IREs). The currently known IRPbinding mRNAs encode proteins involved
in iron uptake, storage, and release as
well as heme synthesis. To systematically
define the IRE/IRP regulatory network on
a transcriptome-wide scale, IRP1/IRE and
IRP2/IRE messenger ribonucleoprotein
complexes were immunoselected, and the
mRNA composition was determined using microarrays. We identify 35 novel
mRNAs that bind both IRP1 and IRP2, and
we also report for the first time cellular
mRNAs with exclusive specificity for IRP1
or IRP2. To further explore cellular iron
metabolism at a system-wide level, we
undertook proteomic analysis by pulsed
stable isotope labeling by amino acids in
cell culture in an iron-modulated mouse
hepatic cell line and in bone marrowderived macrophages from IRP1- and
IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with irondependent regulation, IRP-dependent
regulation, or both. (Blood. 2011;118(22):
e168-e179)
Introduction
Iron homeostasis in mammalian cells is maintained through
posttranscriptional regulation by the IRP/IRE regulatory system.1,2
In iron-deficient cells, active iron regulatory protein (IRP) 1 and
IRP2 recognize iron-responsive elements (IREs), conserved RNA
structures located in the untranslated regions (UTRs) of mRNAs
that encode proteins involved in iron metabolism.3 IRP/IREregulated mRNAs include those encoding proteins for iron acquisition (transferrin receptor 1 [Tfrc], divalent metal transporter
1 [Slc11a2]), storage (ferritin H [Fth1] and ferritin L [Ftl]), use
(erythroid 5-aminolevulinic acid synthase [ALAS2], mitochondrial
aconitase [Aco2], Drosophila succinate dehydrogenase [Sdh]), and
export (ferroportin [Slc40a1]). A typical IRE is composed of a
6-nucleotide apical loop (5⬘-CAGWGH-3⬘, where W stands for A
or U and H for A, C, or U) on a stem of 5 paired nucleotides, an
unpaired asymmetrical cytosine bulge on the 5⬘strand of the stem,
and an additional lower stem of variable length (depicted in
Figure 3A); the nucleotide composition forming the 2 stem
segments may vary considerably.4,5 The mRNAs of Fth1, Ftl,
Alas2, Aco2, dSdh, and Slc40a1 contain one single IRE in their
5⬘UTRs, whereas the Slc11a2 mRNA harbors a single IRE in its
3⬘UTR; Tfrc mRNA is the only currently known mRNA with
multiple (5) IREs, and all of them are located in its 3⬘UTR.
Depending on the location of the IRE, IRP binding regulates gene
expression by different mechanisms. Both IRPs inhibit translation
initiation when bound to 5⬘UTR IREs (eg, Fth1 and Ftl mRNAs),
whereas their association with the 3⬘UTR IREs of the Tfrc mRNA
mediates mRNA stabilization by preventing endonucleolytic
cleavage.6-8
IRP binding activity to IREs is differentially regulated by
intracellular iron levels and other stimuli, including nitric oxide,
oxidative stress, and hypoxia. IRP1 and IRP2 share 60% to 70%
overall amino acid identity, depending on the studied species.
Notably, not all amino acids of IRP1 that directly contact the IRE in
the IRP1/H-ferritin IRE crystal structure9 are conserved in IRP2,
which may be relevant in the context of IRP-specific mRNAs. In
response to the cellular labile iron pool, distinct mechanisms
control the activities of IRP1 and IRP2, which is high in irondeficient cells and low in iron-replete cells. Under iron-replete
conditions, an iron-sulfur cluster (4Fe-4S) assembles in IRP1,
preventing IRE binding and converting it into the cytosolic
aconitase. In iron deficiency, IRP1 undergoes conformational
changes that allow it to bind to IREs as an apoprotein.9-11 In
contrast, IRP2 does not contain an Fe-S cluster and is regulated by
ubiquitination and degradation mediated by the iron-regulated
ubiquitin ligase FBXL5.12,13 The IRP/IRE regulatory system is
essential, as demonstrated by the embryonic lethality of mice
lacking both IRPs.14,15 From a cell biology perspective, the IRPs
are critical for securing sufficient iron supplies to mitochondria.16
Overall, the regulation of the IRE-binding activities of IRP1 and
IRP2 ensures the appropriate expression of IRP target genes and
cellular iron balance.
Over the past 25 years, IREs have been identified successively
in less than a dozen mRNAs by coincidence or after diverse
Submitted April 1, 2011; accepted September 1, 2011. Prepublished online as Blood
First Edition paper, September 22, 2011; DOI 10.1182/blood-2011-04-343541.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
*M.U.M. and M.W.H. contributed equally to this study.
This article contains a data supplement.
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© 2011 by The American Society of Hematology
BLOOD, 24 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 22
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BLOOD, 24 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 22
directed approaches (eg, study of iron-regulated genes, specific
bioinformatics searches). To systematically define the IRP/IRE
regulatory network on a genome-wide and proteomic scale, we
followed 2 approaches. First, we isolated complexes formed
between IRP1 or IRP2 and mRNAs isolated from 5 different
murine tissues, and then we identified their mRNA constituents
using genome-wide microarrays. As a proof of concept for this
method, we recently identified 2 novel IRE-containing mRNAs in
the oxygen-sensing transcription factor Epas1 (Hif2␣) and in the
human cell cycle phosphatase Cdc14A using a small, custom-made
cDNA microarray, the IronChip.17,18 Second, we used bone marrowderived macrophages from control mice or animals lacking both
macrophage IRPs as well as an iron-perturbed hepatocytic cell line,
and then we analyzed the impact of IRP expression, activity, and
iron-induced changes on cellular protein synthesis by pulsed stable
isotope labeling by amino acids in cell culture (pSILAC).19
Methods
Mice and RNA extraction
Brain and bone marrow (BM) samples were obtained from 8- to 10-week-old
C57BL6 mice fed with a standard chow. BM cells were flushed out from the
femur with ice-cold Hanks balanced salt solution (HBSS) and pelleted at
300g for 10 minutes at 4°C for RNA extraction. Duodenum, liver, and
spleen tissues were obtained from C57BL6 mice fed with a low iron
(⬍ 10-mg/kg) diet (C1000; Altromin) for 3 weeks, starting at weaning age;
iron deficiency was functionally validated by decreased hematocrit and
hemoglobin values compared with animals that received a control diet.
Mice with selective ablation of both IRP1 and IRP2 in macrophages
were generated using Cre/Lox technology. Mice homozygous for floxed
Aco1 (Irp1) and Ireb2 (Irp2) alleles (Aco1flox/flox, Ireb2flox/flox)15 were bred to
a knockin strain (LysM⫹/Cre) with an insertion of the Cre recombinase
cDNA into the LysozymeM locus.20 Aco1flox/flox, Ireb2flox/flox, LysM⫹/Cre
animals [designated IrpLysM::Cre(⫹)] were born at mendelian ratios and were
physically indistinguishable from Aco1flox/flox, Ireb2flox/flox, LysM⫹/⫹ control
littermates [IrpLysM::Cre(⫺); Ferring-Appel et al, manuscript in preparation].
Animals were housed under a constant light/dark cycle in the European
Molecular Biology Laboratory specific-pathogen-free mouse barrier unit
and had access to food and water ad libitum. They were killed by CO2
inhalation. Animal handling was in accordance with institutional guidelines.
Total RNA used for immunoprecipitations (IPs) was extracted from
mouse tissues using TRIzol reagent (Invitrogen) following the manufacturer’s protocol.
Immunoprecipitations
The IP experiments were performed as described previously.18 In brief,
50 ␮g of total RNA was combined with purified, His6-tagged recombinant
IRP1 or His-tagged IRP2 produced in Escherichia coli and a rabbit
polyclonal anti-IRP1 antibody (for IRP1 IPs) or a mouse monoclonal
anti-His tag antibody (for IRP2 IPs). A control reaction in which the
recombinant IRP was omitted was performed in parallel (mock IP). The IPs
were done in duplicate using 2 independent pools of total RNA, each one
composed of a pool of total RNA extracted from 4 to 6 mice. Coimmunoprecipitated RNAs from the messenger ribonucleoproteins (mRNPs) were
isolated by proteinase K digestion and ethanol precipitation.
Quantitative real-time PCR
Quantitative (q)PCR was performed in an ABI PRISM 7500 Real Time
PCR system (Applied Biosystems) with SYBR Green and ROX as a passive
reference dye. Seventy nanograms of RNA recovered from the immunoprecipitation reactions was used for qPCR analysis. Genes analyzed were as
follows: Fth1, Tfrc, Slc11a2-IRE, Slc11a2-nonIRE, Slc40a1, Epas1, Gapdh,
and ACtb. Levels of Actb, Gapdh, and Slc11a2-nonIRE were used as
SYSTEMS ANALYSIS OF THE MURINE IRE/IRP NETWORK
e169
negative controls, for normalization, or both. PCR product quality was
monitored by post-PCR melt curve analysis. Fold enrichments were
calculated using the relative expression software tool.21 Primer sequences
are available on request.
Microarray experiments
In total, 40 Affymetrix GeneChip Mouse Genome 430 2.0 arrays were used
to determine the mRNA composition of mRNPs obtained by immunoprecipitation with the recombinant IRPs. Immunopreciptated RNA (120 ng) was
used as input for a 2-step amplification procedure to generate biotin-labeled
RNA fragments for hybridization to the Affymetrix microarray according to
the Standard Affymetrix 2 Cycle protocol (Eukaryotic Sample and Array
Processing manual 701024 Rev.3). The amplified material was verified for
specificity by qPCR before labeling and hybridization (data not shown).
Intensity values for the hybridizations were obtained either using robust
multichip average (RMA), with calculations done in bioconductor (www.
bioconductor.org) or Affymetrix Microarray Suite 5 (MAS5), with calculations done using the Affymetrix GCOS package. MAS5-calculated intensities were further quantile normalized using bioconductor. Both methods are
complementary and commonly used normalization procedures in the
context of established algorithms for microarray evaluation. Ratios between
the intensities from immunoprecipitate and mock reactions were calculated
to obtain the fold enrichment level for each probe. Genes were considered
“positive” if both independent biologic replicas yielded significant enrichment values above the cut-off threshold, based in the lowest log2 ratio
obtained from probes of known IRE-containing mRNAs or a 0.6 log2 ratio
value as threshold (1.5-fold enrichment). Microarray data reported here
have been deposited within Gene Expression Omnibus (National Center for
Biotechnology Information; GSE17096, GSE17097).
Plasmids
Mouse full-length cDNA clones for Fth1, Slc40a1, Ppp1r1b, Gyg, Gstm6,
Cxcl16, Pfn2, and Pdcl3 were obtained from Origene Technologies or
Riken FATOM 3 clone collections. Clones containing the H-ferritin
wild-type and mutant IRE followed by the chloramphenicol-acetyltransferase
(CAT) mRNA (wt and mut in this paper, originally pI-12.CAT and
pI-19.CAT clones)10 and the Renilla luciferase control plasmid were
described previously.22 All plasmids were verified by DNA sequencing.
Competitive EMSAs
Competitive EMSAs were done using from 15 000 to 30 000 cpm of
32P-radiolabeled H-ferritin IRE probe mixed with appropriate molar excess
of trace-labeled competitor (1⫻, 2⫻, 5⫻, 10⫻, and 40⫻ fold molar excess)
and 10 to 60 ng of recombinant IRP1 in cell lysis buffer as described
previously.17 The H-ferritin mutant with a deletion (␦-C, N14) in the IRE
loop was used as a negative control. An extensive description of competitive EMSAs with H-ferritin IRE mutants and with full-length transcripts is
provided in supplemental Methods (available on the Blood Web site; see the
Supplemental Materials link at the top of the online article).
Tissue culture, pSILAC, and protein detection
Cells were grown at 37°C in a 5% CO2 atmosphere.
The murine hepatocellular carcinoma cell line Hepa 1-6 was purchased
from DSMZ and grown in Dulbecco modified Eagle medium with 4.5 g/L
glucose supplemented with 10% of heat-inactivated FCS (HyClone Laboratories), and 1% penicillin ⫹ streptomycin (Invitrogen). Hepa 1-6 cells were
adapted to SILAC light medium that contains dialyzed FCS for 1 week. On
the day of the experiment, cells were incubated with 100␮M hemin (Leiras
Oy) or 200␮M desferrioxamine (DFO; Sigma-Aldrich) or left untreated for
2 hours, followed by 1 hour of amino acid starvation to enhance the
incorporation of isotopes, and 6 hours of labeling with M SILAC medium
for control cells and H SILAC medium for hemin- or DFO-treated cells;
total treatment time was therefore 9 hours. Cells were washed twice,
harvested on ice with cold PBS, and then centrifuged; the pellets were
frozen in liquid nitrogen.
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SANCHEZ et al
Time and concentration of the hemin or DFO treatments for pSILAC
experiments were optimized in [35S]methionine/cysteine metabolic labeling
studies monitoring ferritin L and H protein levels and transferrin receptor
1 mRNA levels (data not shown). Cell toxicity of the treatments was
assessed by the colorimetric MTT assay, and no adverse effects were
observed under the chosen conditions (data not shown).
Bone marrow–derived macrophages (BMDMs) were recovered from
the femur of IrpLysM::Cre(⫹) and IrpLysM::Cre(⫺) animals using ice-cold HBSS.
The cell suspension was filtered through an 80-␮m cell strainer (Falcon, BD
Biosciences Discovery Labware), and cells were seeded at a density of
⬃ 5 ⫻ 104 cells/cm2 in RPMI 1640 ⫹ GlutaMAX (Invitrogen) supplemented with 20% of heat-inactivated FCS (HyClone Laboratories),
1% penicillin ⫹ streptomycin (Invitrogen), and 100 ng/mL of macrophage
colony-stimulating factor (M-CSF; Sigma-Aldrich). After 4 days, nonadherent cells were removed by washing with HBSS, and the medium was
subsequently replaced daily with light SILAC medium containing M-CSF
for 2 days. Cells were further incubated in the presence of medium (control
BMDM) versus heavy (IRP-deficient cells) SILAC medium containing
M-CSF for 24 hours. After 3 washes with ice-cold HBSS, cells were
harvested using a rubber policeman and pelleted by centrifugation at 300g
for 10 minutes at 4°C, and pellets were frozen in liquid nitrogen. The
proportion of macrophages typically exceeds 90% as assessed by
labeling with an Alexa Fluor 488–coupled rat monoclonal antibody
against the F4/80 macrophage-specific marker (Serotec); consistent with
its reported efficiency,20 the LysM⫹/Cre deletor strain yields 70% to 80%
recombination of the floxed Aco1 and Ireb2 alleles as assessed by Southern
blotting (data not shown).
Cell pellets were lysed in SDS-sample buffer, and then appropriate
samples were combined, separated by SDS-PAGE, processed, and
analyzed by liquid chromatography-tandem mass spectrometry.19 A
detailed description is fully provided in supplemental Methods. Data
analysis for mass spectrometry was performed as described previously19; a
cut-off for protein regulation of 1.4-fold was chosen based on results with
the control protein Tfrc.
BLOOD, 24 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 22
The searching for iron-responsive elements (SIREs) web-server tool for
prediction of iron-responsive elements has been described previously.23
ground was detected for Gapdh mRNA and for the non-IRE form of
Slc11a2 mRNA (Figure 1A-B). Thus, IRP1- and IRP2-binding
mRNAs are consistently and specifically detected by this procedure.
Genome-wide identification of the immunoprecipitated mRNAs
within the IRP mRNP complexes was achieved using Affymetrix
microarrays (GeneChip Mouse Genome 430 2.0) that cover
⬎39 000 mRNAs and RNA variants. Affymetrix array data were
normalized and analyzed using 2 independent and commonly
used statistical algorithms, MAS5 and RMA, to perform stringent data analysis. The total number of IRP1- or IRP2-binding
mRNAs clustered by tissues is shown below the heatmap in
Figure 1C (RMA analysis), and the total number of mRNAs
detected in each particular tissue is shown in Figure 1D (RMA
analysis). A similar analysis was done with the MAS5 data
(supplemental Figure 1A-B). The majority of IRP-associated
mRNAs is detected in a tissue-specific way (for IRP1, 67.4% or
70.3% and for IRP2, 73.3% or 80.3%, respectively, depending on
the microarray algorithm used).
The RMA microarray data analysis revealed that 64 mRNAs
bind to both IRPs in at least one tissue; 61 mRNAs were detected
using the MAS5 analysis method (Figure 2A). Combining both
analyses, we identified 44 mRNAs that are significantly enriched in
both IRP1 and IRP2 mRNPs (Figure 2A-B). Importantly, all
9 previously known murine IRE-containing mRNAs (Ftl1, Ftl2,
Fth1, Tfrc, Slc40a1, Slc11a2, Alas2, Aco2, and Epas1; marked in
green in Figure 2B) were reidentified by our experiments. Therefore, the genome-wide search for IRP-associated mRNAs reveals
35 novel mRNAs able to bind both IRP1 and IRP2 in at least 1 of
the 5 tested tissues (Figure 2B).
In addition to these mRNAs that are bound by both IRPs, the
microarray data (combining MAS5 and RMA analysis methods)
detect 101 mRNAs as exclusive interactors of IRP1 and 113 IRP2binding mRNAs in at least one tissue (supplemental Tables 1-2). To
the best of our knowledge, this is the first time that exclusive IRP1or IRP2-interacting cellular mRNAs are identified.
Statistics
Experimental refinement of and bioinformatic analysis for IRE
motifs in novel IRP target mRNAs
Bioinformatic analysis
The data are reported as mean ⫾ SEM. All statistical analyses were
performed using Prism version 5.0 (GraphPad Software). Student t tests
were used to compare results between groups of 2. A value of P ⬍ .05 is
considered statistically significant.
Results
Isolation and identification of mRNAs associated with IRP1 and
IRP2
To systematically elucidate the IRP regulatory network, we developed a strategy to specifically immunoprecipitate IRP-containing
mRNP particles and to identify the copurified mRNAs by microarray analysis. Total RNA from 5 different mouse tissues (duodenum,
liver, brain, spleen, and bone marrow) was incubated with recombinant IRP1 or IRP2 and suitable antibodies (see “Methods”). In
parallel, a reaction omitting the recombinant protein was analyzed
(mock IP) to assess the background level of the system.
The IPs were tested for specificity by analyzing the enrichment
of known IRE-containing mRNAs in the IP versus the mock IP
reactions by qPCR. The mRNAs of Fth1, Tfrc, Slc11a2, Slc40a1,
and Epas1 are all strongly enriched (from 3- to 97-fold) in the IP
fractions with recombinant IRP1 or IRP2 from RNA samples of all
5 analyzed tissues (Figure 1A-B). No enrichment over the back-
Earlier bioinformatic searches of mammalian transcriptomes for
IRE motifs24,25 had not identified any of the novel mRNAs that we
found to bind IRP1 and IRP2 in the experiments described so far. In
part, such failure probably results from limitations of the definition
of an “IRE motif” that was used for these searches. To address
some of these limitations experimentally, we first considered
previous systematic evolution of ligands by exponential enrichment experiments that reported that the 6-nucleotide apical loop of
an IRE can differ from the canonical CAG(U/A)GN sequence26-28
or that the C-bulge can be replaced by a G-bulge28,29 (supplemental
Table 3). In addition, we also accommodated atypical IRE structures that are present in the validated Slc11a2, Epas1, and Hao1
mRNAs.30-32
On the basis of these published data, we designed a series of
additional experiments to further define IRP1-binding RNA motifs
by competitive EMSA (Figure 3A; supplemental Table 3). Specifically, we subjected the upper stem and the position below the
bulged cytosine (C8) of the H-ferritin IRE to further analysis,
testing 24 new variants (Figure 3C) together with the H-ferritin
IRE controls (Figure 3B) in competitive EMSAs with recombinant IRP1.
Fifteen of the 24 H-ferritin IRE mutants were used to test
whether the number and position of G.U or U.G wobble base pairs
in the upper stem are relevant for IRP binding. The results show
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BLOOD, 24 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 22
SYSTEMS ANALYSIS OF THE MURINE IRE/IRP NETWORK
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Figure 1. Transcriptomic identification of IRP1- and
IRP2-binding mRNAs. Fold enrichment of known IREcontaining mRNAs (Fth1, Tfrc, Slc11a2, Slc40a1, and
Epas1) in IRP1 (A) or IRP2 (B) IPs versus mock controls
was determined by qPCR. Gapdh and the non-IRE form
of Slc11a2 were used as negative controls. Data show
the values obtained with the first biologic replica used in
microarray analysis; similar data were obtained with the
second biologic replica (data not shown). (C) Heatmap
visualization of microarray data (RMA analysis) for
mRNAs copurified with IRP1 or IRP2. Fold-change
values from 2 independent replicates for each tissue
tested are given for probe sets that were detectable
above background showing at least a 1.5-fold enrichment (log2 ratio ⬎ 0.6). Red and black lines indicate
positive and negative IP enrichment, respectively, relative to mock IPs. The color scales to the right indicate the
magnitude of the fold change (base 2 logarithm) for a
particular transcript. Number of mRNAs clustered by
tissues is shown below each heatmap. (D) Number of
mRNAs detected in each particular tissue bound by
IRP1, IRP2, or IRP1 ⫹ IRP2.
that the presence of more than 2 G.U or U.G base pairs in the IRE
motif dramatically impairs its ability to compete with the wild-type
probe (Figure 3C mutants U, V, W, and Y), whereas variants
bearing 1 (which is naturally present in the H-ferritin IRE) or 2 G.U
or U.G base pairs compete well with the wild-type probe (Figure
3C mutants A, B, C, D, F, G, H, I, M, O). Mutants R and Q are
unable to significantly compete with the wild-type IRE probe,
probably because both of these variants contain a G at position
25 that may interact with the critical and unpaired C8 nucleotide.
Seven additional mutants were used to study the effect of a base
mismatch in the IRE structure. In the context of an H-ferritin
IRE-backbone, one single AxC mismatch at positions other than
n13-n20 (mutant S) or n07-n25 (mutant T) is well tolerated, as
demonstrated by mutants E, J, N, and P. The mismatch at position
n13-n20 (mutant S) was not tolerated, because it will probably
disturb the physiologic definition of the apical loop. Mutant K
(CxA mismatch at position n07-n25), but not mutant T (AxC
position n07-n25), is able to efficiently compete with the
wild-type IRE for binding to IRP1. We suspect that A25 in mutant
K may base pair with the bulged uridine present in the H-ferritin
IRE (Figure 3B U6), which would be not possible for mutant T.
This explanation was not evaluated further. These experimental
results were interpreted into our newly developed SIREs algorithm23 (Figure 3A).
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SANCHEZ et al
BLOOD, 24 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 22
Figure 2. Novel mRNA targets for IRP1 and IRP2.
(A) Number of mRNAs positively detected as enriched in
IRP1, IRP2, or both IPs by microarrays with MAS5,
RMA, or a combined (MAS5 ⫹ RMA) mathematical data
analysis. (B) Virtual heatmap representing the 44 identified mRNAs that are bound by both IRPs in at least one
of the 5 studied tissues. Affymetrix-positive probes were
grouped by gene and reported in each of the rows.
Known IRE-containing mRNAs are shown in green. Red
and black squares indicate positive or lacking IP enrichment, respectively, relative to mock IPs in 2 independent
biologic replicas. Predicted IREs by SIREs program in
mouse (Mus musculus, Mm.) or human (Homo sapiens,
Hs.) databases/species and their positions also are
indicated. N.d. denotes that an IRE motif was not
detected using the SIREs bioinformatic program; n.a.
denotes nonavailable information; and 3⬘ or 5⬘ denotes
the 3⬘ or 5⬘UTR.
Applying SIREs to identify IREs within the mRNAs that
coimmunoprecipitate with IRPs, we not only identify all known
IREs but also at least 1 IRE-like motif in 24 of the 35 (68.6%) novel
IRP1 and IRP2 murine target mRNAs or its human orthologs
(Table 1; Figure 4A). Moreover, the prediction of IRE-like motifs is
significantly enriched in the pool of 35 novel IRP target genes
(P 6 ⫻ 10ˆ-13, Fisher exact test) compared with 150 mouse mRNA
sequences that were arbitrarily selected and randomly shuffled to
avoid the selection of true positive hits by chance. In total, 29 IRE
motifs were detected by SIREs in these 24 mRNAs (Table 1). The
motif distribution of these predicted IREs shows that motif 1 and
2 together with motif 8 (supplemental Table 3) constitute more than
65% of newly detected motifs (27.6%, 24.1%, and 13.8%, respectively; Figure 4B). These new IRE-like motifs are located in all
parts of the mRNAs, the 5⬘UTR (16%), 3⬘UTR (42%), and coding
sequence region (CDS, 39%; Figure 4A; Table 1).
SIREs also detects IRE motifs in 41% (41 mRNAs) and 38%
(43 mRNAs) of the IRP1- or IRP2-specific targets, respectively,
when searching mouse and human databases (Figure 4A; supplemental Tables 1-2). The motif distribution and localization of these
predicted IREs also are indicated (Figure 4A-B).
We observed that the 3 different groups of mRNAs identified as
IRP targets—group 1, IRP1 ⫹ IRP2-binding mRNAs; group 2,
IRP1-specific mRNAs; and group 3, IRP2-specific mRNAs—
display slightly different IRE motif preference (Figure 4C). Group
1 shows a predominance of the canonical IRE sequence without
mismatches or bulges in the upper stem and with the classic apical loop
nucleotide sequence CAGUGN (motif 1; P ⬍ .0001) or CAGAGN
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BLOOD, 24 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 22
SYSTEMS ANALYSIS OF THE MURINE IRE/IRP NETWORK
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Figure 3. Experimental definition of IRP1-binding sites using EMSA. (A) Schematic representation of an IRE motif. Squared region indicates the IRE core region predicted
by SIREs software. (B) Schematic representation of the H-ferritin wild-type and mutant IRE used for electrophoretic mobility shift assays. Deletion of C14 in the IRE is indicated
by a red cross. (C) Competitive EMSA analyses with 24 different H-ferritin IRE variants (mutants A to Y). Values and standard errors for competition with the H-ferritin wild-type
and the ␦-C mutant IRE are highlighted in gray. In the table, mutated positions are indicated according to the nomenclature shown in panel A, and mutated nucleotides are
underlined. Values for fold difference between the different variants versus the H-ferritin IRE wild-type (IRE wt) at 40⫻ fold molar excess are reported. P values comparing each
variant with the H-ferritin IRE mutant, (IRE mut) are reported (***P ⬍ .001, **P ⬍ .01, *P ⬍ .05; unpaired 2-tailed Student t test). Mutants that do not reach statistical
significance are filled in red. The red dashed line indicates the value for Aco2 5⬘IRE. Graph shows percentage of raw signal for competitive EMSAs at 40⫻ molar excess of the
indicated competitor. Data are presented as mean ⫾ SEM from a minimum of 3 experiments.
Description
Glutathione S-transferase, theta 3
Glycogenin
Glycogenin-1
Glycogenin-1
Hydroxyacid oxidase 1, liver
14 Gstt3
15 Gyg
GYG1
GYG1
16 Hao1
ORM1-like 1 (Saccharomyces. cerevisiae)
Poly(A) binding protein, cytoplasmic 4-like
Protein phosphatase 1, regulatory subunit 1B
Pyridine nucleotide-disulfide oxidoreductase domain 1
Tumor protein p53 inducible nuclear protein 2
20 ORMDL1
21 Pabpc4l
22 Ppp1r1b
23 PYROXD1
24 TP53INP2
Motif refers to type of apical loop as defined in supplemental Table 3.
Nuclear receptor subfamily 4, group A, member 3
19 NR4A3
NM_021202
NM_024854
NM_144828
NM_001101479
NM_016467
NM_173199
NM_013587
NM_201531
NM_201531
NM_010403
NM_004130
NM_004130
NM_013755
NM_133994
NM_008184
NM_144779
NM_010118
NM_001364
IRE sequence
TAGGTCAGCG.T.G.CAGAG.CAGTGA.TGCTGG.AGGACACACC
GTCAGTTACA.G.C.TGATA.CAGAGA.TGTGGC.CTGTCTATGT
TTTCCTGGGT.G.C.GGGGA.CAGTGC.TCCTCC.TCCTCCTCCG
GTCTTTGGGT.C.C.ATTTG.CAGGGT.TAAAGTG.ATGCAGGAAG
TAAGTTAAAG.G.G.CATCA.CAGTGA.GGGTGT.AGTAGATAAA
CCGCTCACCG.C.C.TCCGG.GAGCCG.CTGGGC.TTGTACACCG
CTTCCCAGAA.C.C.CTCAG.CAGTGT.CTGAGG.CTCAGAGAAA
CAAAGGCTAA.G.C.TGGGC.CAGTGG.GCCTAA.CCCTTTATGG
ATTTTGTGAA.T.C.GTGAA.TAGTAC.TTTACA.TTCAAAATTT
TCATTTATAG.T.C.ACATT.CAGTGT.AAAGTA.CATATTTTGT
CAGATGAGAG.G.C.TTTTT.TAGGAT.AAGAGG.TGAGAACTGG
GTCAGCAGCA.C.C.AGACC.CAGGGT.GGCCTG.ACTGCTTCAA
ATCAGCAGCA.C.C.AGACC.CAGGGT.GGCCTG.ACTGTTTCAA
TGACTTGGTG.G.C.CATCA.CAGAGC.TGATGC.ATCCTGTCAG
TATGCAGGCT.C.C.ATCTC.CAGAGT.GGGAAG.GCCCAGTCTT
GTCGTCGGGA.C.C.CGGCC.GGGAGG.GGCCGC.GGCGGCCGCA
TTTACACTCT.C.C.AGATT.CAGAGC.GGTCTT.CTAAACTGCA
CCTCCTTGCG.T.C.AGTCC.CAGTGA.GGGATA.AGCGCCTGGC
TGGCAGTGGG.T.C.TGCAG.CAGTGA.CTGCCA.CCCCTTATAA
AAGAGATAGA.G.C.CATCA.GAGTGA.TGGGGC.TTCTTCACAG
GACTACGAAG.T.C.GACGG.CAGAGA.CTATCA.CTTTGTCATT
AGCAGGCTCG.T.C.TCCAT.CAGTGA.ATGGAA.CCTGAGCTCA
TGCGCGCGCG.G.C.CTGGA.CAGAGA.TTCAGC.GCGCGGCGCT
ATCTTGAGTT.T.C.CAACA.CCGTGC.TGCTTGA.TAGAATGACT
TGTGAGAGTA.G.C.TTTTT.GAGTGT.GTAAGC.CTACATTTGA
CTTGTATAAG.C.C.GCCTG.CAGAGA.CTGGTG.AAACTGTGCA
GCCGGCGGCG.G.C.TCCCG.CAGTGG.AGGGAC.CCCGACAAGT
GTGGGCAAAC.T.C.TTGCC.TAGTAT.GGATAGA.GCGACATCCC
GTTCCGCTTC.C.C. TTACC.CAGGGC.AGTGGG.GCCTCCCCAC
Position
3⬘UTR
CDS
5⬘UTR
CDS
3⬘UTR
5⬘UTR
3⬘UTR
3⬘UTR
3⬘UTR
3⬘UTR
3⬘UTR
CDS
CDS
CDS
3⬘UTR
5⬘UTR
CDS
5⬘UTR
CDS
3⬘UTR
CDS
3⬘UTR
CDS
3⬘UTR
N/A
CDS
CDS
3⬘UTR
CDS
18
2
1
8
18
14
1
1
9
1
10
8
8
2
2
16
2
1
1
17
2
1
2
4
17
2
1
9
8
Motif
Mismatch
N12-N21:A_G
N10-N23:G_G
N13-N20:A_G
N07-N25:C_C
N07-N25:G_A
N11-N22:A_A
N07-N25:G_G
N11-N22:A_C
N11-N22:A_C
N09-N24:A_A
N07-N25:C_C
N07-N25:C_T
N10-N23:G_A
N09-N24:T_C
N10-N23:A_G
N11-N22:C_A
N12-N21:T_T
N12-N21:T_T
N13-N20:G_A
N13-N20:C_A
N22b:A
N21b:C
N21b:A
Bulge
SANCHEZ et al
associated protein 1
Low-density lipoprotein receptor-related protein
Glutathione S-transferase, mu 6
18 Lrpap1
GTPase activating RANGAP domain-like 1
Garnl1
13 Gstm6
Potassium voltage-gated channel, subfamily F, member 1
GTPase activating RANGAP domain-like 1
12 Garnl1
Potassium voltage-gated channel, subfamily F, member 1
FXYD domain containing ion transport regulator 5
11 FXYD5
Kcnf1
NM_001003719
Early growth response 2
10 Egr2
17 Kcnf1
NM_019994
Discs, large homolog 2 (Drosophila)
NM_011807
NM_023158
NM_183170
DLG2
cDNA sequence BC051227
7 BC051227
NM_173505
Chemokine (C-X-C motif) ligand 16
Ankyrin repeat domain 29
6 ANKRD29
NM_001081436
AK134743
Discs, large homolog 2 (Drosophila)
Expressed sequence AI450353
5 AI450353
NM_178629
NM_173737
8 Cxcl16
RIKEN cDNA A430093A21 gene
4 A430093A21Rik
Reference ID
NM_001025573
9 Dlg2
RIKEN cDNA 4930579E17 gene
RIKEN cDNA 8430410A17 gene
2 4930579E17Rik
3 8430410A17Rik
RIKEN cDNA 2010107G12 gene
1 2010107G12Rik
Gene name
e174
Table 1. Bioinfomatic prediction of IREs by SIREs in novel IRP1 and IRP2 target mRNAs
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BLOOD, 24 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 22
SYSTEMS ANALYSIS OF THE MURINE IRE/IRP NETWORK
e175
Figure 4. Analysis of IRE motifs bound by IRP1, IRP2, or both IRPs. (A) Bioinformatic prediction of IRE motifs in IRP1 and IRP2 target mRNAs, IRP1-specific target mRNAs
and IRP2-specific target mRNAs. (B) Motif distribution of novel IREs (also see supplemental Table 3). Motif number and percentage (motif n. and motif %) are shown in the
3 IRP-target mRNAs lists. IREs without a mismatch or a 3⬘ bulge are depicted in blue, IREs with a mismatch are in red, and IREs with a 3⬘ bulge are shown in green.
(C) Enrichment of IRE motifs in IRP-target mRNAs. After normalization for n19 multiple IRE options in each IRE type, observed and percentage expected frequencies of IRE
motifs were calculated (␹2 test, 1 df) and statistically significantly overrepresented motifs are shown. Red font, cross sign “x” or bracket cross sign “(x)” indicate differences
compared with the canonical motif 1 IRE sequence. The cross sign or bracket cross sign indicate mismatches.
(motif 2; P ⫽ .0359). By contrast, group 2 (IRP1-specific binders) is
enriched in IRE motifs with a mismatch in the upper stem at a central
position n11-n22 (P ⫽ .0261). Group 3 (IRP2 binders) is enriched in
IREs with a canonical loop presenting or not a mismatch at position
n12-n21 of the upper stem, or CUGUGN19 loop with a mismatch
at either of the 3 upper base pairs of the stem (P ⬍ .0001 and
P ⫽ .0359, respectively; Figure 4C).
To complete the characterization of IRP binding to the transcriptome, we finally chose 16 of the 35 newly identified mRNAs that
bind both IRP1 and IRP2, and we generated in vitro RNA
transcripts for competitive IRP1 binding assays. These 16 mRNAs
include tissue-specific and multitissue IRP binders (Figures 1C and
2B), as well as transcripts within which IRE motifs could or could
not be identified (Figures 2B and 4A; Table 1). Six full-length IRP
target mRNAs (Ppp1r1b, Gyg, Gstm6, Cxcl16, Pfn2, and Pdcl3)
significantly compete for IRP1 binding at 40-fold molar excess
over an H-ferritin IRE probe (P ⬍ .001 or P ⬍ .01; Figure 5). Ten
of the novel IRP-associated mRNAs (0610007L01Rik,
2010107G12Rik, 8430410A17Rik, BC051227, Dhx32, Dirc2,
Gstt3, Lsm12, Ormdl1, and Pyrodx1) did not display significant
competition but neither did 2 positive controls with weak IREs
(Slc11a2 and Aco2; data not shown). Several reasons may explain
these observations: (1) like the Slc11a2 and Aco2 mRNAs, these
mRNAs also may interact weakly with IRP1; (2) the IRP1 binding
sites are part of the cellular mRNAs but excluded from the in vitro
transcript; or (3) IRP1 binding to the candidate mRNA and the
labeled ferritin IRE probe occurs to different sites of the protein and
is noncompetitive. In spite of all the specificity controls included in
the experiments, we also cannot formally exclude the possibility
that non–IRP-binding mRNAs specifically copurified by
(eg, hydrogen bounding) interactions with IRE-containing mRNAs.
IRE-like motifs were found in 4 of the 6 mRNAs that positively
compete in the EMSA experiments (Ppp1r1b, Gyg, Gstm6, and
Cxcl16; Table 1). No IRE-like motif was recognized by SIREs in
Pfn2 and Pdcl3 mRNAs. The mRNAs of Gstm6 and Cxcl16
contain one IRE-like motif in their 3⬘UTR, Ppp1r1b contains one
IRE in its 5⬘UTR, and Gyg contains one IRE in its coding region.
Exclusion of the region predicted to bear the IRE-like motif
prevents the observed competition in all but the Ppp1r1b mRNA
(Figure 5), supporting the notion that IRP binding occurs mainly
via the newly recognized motifs. Pfn2 and Pdcl3 clones lacking
3⬘-terminal sequences beyond the XcmI and EcoRV sites, respectively, fail to compete, suggesting that the RNA element responsible for IRP interaction is located within this region of the
transcripts (Figure 5).
Proteomics of cellular iron regulation
In addition to defining the target mRNAs of IRP1 and IRP2 in the
global transcriptome, we also wanted to explore the role of the
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e176
SANCHEZ et al
Figure 5. Validation of select novel IRP target mRNAs with in vitro transcripts.
In vitro transcribed full-length mRNAs were incubated at a 40⫻ molar excess against
an H-ferritin IRE-radiolabeled probe for the binding of recombinant IRP1. Wt
corresponds to an ⬃ 1-kb reporter mRNA bearing the 5⬘ IRE of H ferritin mRNA; mut
is the same reporter with a deltaC14 mutation of the IRE. Black bars represent
negative control mRNAs (Renilla) or no competitor signal. Yellow bars represent
positive controls (H-ferritin reporters and full-length Fth1 and Slc40a1 mRNAs). Red
and blue bars correspond to novel IRP target mRNAs with or without a bioinformatically predicted IRE-like motif, respectively. Tested full-length and indicated 5⬘
restriction enzyme truncation mRNAs are grouped together. Above each group a
schematic representation is shown indicating the restriction enzyme used to assess
truncated forms and the location of the predicted IRE motif (round hairpin) or the
putative IRP-binding RNA region (squared hairpin). P values are reported (***P ⬍ .001,
**P ⬍ .01, *P ⬍ .05, unpaired 2-tailed Student t test compared with the mutant
H-ferritin IRE construct, mut, or with each corresponding non-IRE construct). Data
are presented as mean ⫾ SEM.
IRE/IRP network in shaping the cellular proteome. To this end, we
used the recently described method of pSILAC,19 a method that
quantifies relative differences in de novo protein biosynthesis, on
IRP-deficient BMDM cells from macrophage-specific IRP1 and
IRP2 knockout mice. Furthermore, we investigated iron regulation
of the proteome of mouse hepatoma Hepa1-6 cells by the same
method; the IRE/IRP regulatory system is intact in these cells and
iron regulation integrates IRP-dependent and IRP-independent
proteomic changes.
In Hepa1-6 cells, we performed 2 independent pSILAC experiments to compare untreated cells with iron supplemented (100␮M
hemin) or iron-deficient cells (200␮M DFO, an iron chelator),
respectively. In total, we confidently detected 2364 proteins in the
iron-replete cells and 3594 proteins in their iron-starved counterparts. The results show that the biosynthetic labeling of 73 (3.1%)
proteins is increased and of 480 (20.3%) proteins is decreased by
hemin treatment; DFO treatment up-regulates the labeling of
67 (1.86%) proteins and down-regulates 920 (25.6%) proteins.
Opposite regulation by iron is seen for 22 proteins, with 12 proteins
more highly expressed after hemin treatment and repressed by
DFO, and 10 proteins with reciprocal regulation (supplemental
Table 4). Ferritin H and L are among the 12 proteins that are
increased by iron and decreased by iron deficiency, displaying a
14- and a 8.6-fold increase by hemin and a 1.5- and a 1.6-fold
decrease under iron-chelating conditions, respectively. The Tfrc
labeling is strongly decreased by hemin (⫺5.4 fold) and induced by
DFO (1.4-fold) treatment. Of these 22 proteins, only the mRNAs
encoding Ftl1, Fth1, and Tfrc bear IREs that are recognized by
SIREs. Moreover, none of the 19 remaining bidirectionally ironregulated proteins correspond to IRP-associated mRNAs, suggesting that iron regulation of these proteins occurs by IRPindependent or only indirectly IRP-dependent mechanisms.
In pSILAC experiments with IRP-deficient BMDM cells, the
absence of both IRPs causes the up-regulation of 63 (2.03%)
BLOOD, 24 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 22
proteins and the down-regulation of 188 (6.06%) proteins (supplemental Table 5). These changes may be a direct consequence of IRP
ablation or a secondary consequence of complete IRP deficiency,
which causes marked cellular and mitochondrial iron deficiency in
hepatocytes.16 Detectably IRP-regulated proteins include the positive control IRP-target mRNAs ferritin L and H (6.4- and 5.3-fold
up-regulated, respectively) and Tfrc (⫺2.5 fold down-regulated).
Of the 248 remaining IRP-regulated proteins, 3 proteins are
encoded by mRNAs that were found to be directly bound by IRP1
or IRP2 (Lpl, Psmd10, and Tomm40), and IREs were predicted in
54 mRNAs by SIREs. This result raises the possibility that cellular
responses to the IRE/IRP network reach beyond the currently
known targeted mRNAs.
Finally, we integrated all global datasets (IRP-binding transcriptome and iron/IRP-regulated proteome) in a Venn diagram analysis,
generating 2 4-set Venn diagrams (Figure 6A-B). Apparently, the
majority of iron-regulated proteins are not likely to be direct IRP
targets. Ferritin L and H proteins/mRNAs were detected in all
assays, showing an opposite iron regulation in Hepa 1-6 cells
(group a and b), an increase in their expression in BMDMs lacking
both IRPs (group c), and positively detected in IRP immunopurification experiments (group d; Figure 6A). Thirty-three additional
proteins/mRNAs meet 2 of these criteria (supplemental Table 6).
The Tfrc protein/mRNA was oppositely iron-regulated in Hepa 1-6
cells (groups e and f), down-regulated in BMDM cells lacking both
IRPs (group g), and positively IRP immunopurified (group d;
Figure 6B). As such, it is the only protein/mRNA that displays this
complete regulatory response. Forty-five additional proteins meet
2 of the 4 criteria (supplemental Table 6).
These data place the ferritins and Tfrc at the center of cellular
iron- and IRP-dependent regulation. More importantly, they uncover and specifically identify numerous proteins and mRNAs
through which iron regulation, IRP regulation, or both connect with
other aspects of cell biology and physiology.
Discussion
Posttranscriptional regulation of gene expression by the IRP/IRE
regulatory system plays a central role in the control of cellular iron
metabolism. In the course of the past 25 years, research on this system
has laid foundations for understanding of cellular iron homeostasis, even
if the list of IRP-target mRNAs is still limited to few that encode core
iron metabolism proteins. With this work, we begin to connect this core
with other cellular functions that need to respond to changes in iron
metabolism. To this end, we have deciphered the whole-genome
repertoire of mRNAs associated with IRP1 and IRP2 from 5 tissues, and
we defined the proteomic changes in IRP-deficient cells and in an
iron-perturbed hepatic cell line. In isolation and in combination, these
experiments have yielded a wealth of new information that will help
instruct future experiments.
We have identified new mRNAs that can interact either with
both IRPs or specifically only with one of them. Each of the
3 classes considerably enlarges the IRP regulatory repertoire,
whereas the latter 2 classes offer first examples of IRP-specific
cellular target mRNAs. These mRNAs encode proteins involved in
different cellular functions, including metal ion binding proteins,
transferases, ligases, helicases, and transcription or DNA binding
factors, according to functional annotation clustering analyses
using Database for annotation, visualization and integrated discovery (supplemental Table 7). Interestingly, 9 of the novel IRP1 ⫹ 2
target mRNAs (Lnx1, Mkrn1, Egr2, Nr4a3, Pex12, Garnl1, Cxcl16,
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BLOOD, 24 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 22
SYSTEMS ANALYSIS OF THE MURINE IRE/IRP NETWORK
e177
Figure 6. Integrative data analysis combining iron
proteomics and microarray data. (A) Combination of
categories a, b, c, and d. (B) Combination of categories
e, f, g, and d. Category definitions are as follows:
a, proteins up-regulated by hemin in Hepa1-6 cells in
pSILAC experiments; b, proteins down-regulated by
DFO in Hepa1-6 cells in pSILAC experiments; c, proteins
up-regulated in IRP-deficient BMDMs (pSILAC experiments); d, IRP target mRNAs detected by IPs and
microarrays; e, proteins down-regulated by hemin in
Hepa1-6 cells in pSILAC experiments; f, proteins upregulated by DFO in Hepa1–6 cells in pSILAC experiments; and g, proteins down-regulated in IRP-deficient
BMDMs (pSILAC experiments).
Kcnf1, and Dhx32) fall into the category “metal ion binding” that
also includes the already known IRP-regulated mRNAs Fth1, Ftl1,
Aco2, Slc11a2, and Slc40a1. For 7 of these genes (Lnx1, Mkrn1,
Egr2, Nr4a3, Pex12, Garnl1, and Cxcl16), the encoded proteins are
reported to interact selectively and noncovalently with zinc ions.
Iron and zinc are known to compete for the absorptive pathway
through binding to the IRE-containing Slc11a2 metal transporter.33
Our results raise the possibility of an extensive fine tuning
coordination of iron and zinc metabolism via the IRE/IRP system.
Concerning exclusive IRP1 or IRP2 targets, both lists are
enriched in functional term categories related to RNA splicing,
regulation of cell migration, and zinc finger (RING type) motifs.
IRP2-associated mRNAs are exclusively enriched in functional
terms related to ubiquitin-mediated proteolysis/ligase activity (Pml,
Ube2j2, Ube2q2, and Herc4) and the cytokine receptor/Jak-STAT
signaling pathway (Il6ra, Il6st, Csf2rb, Tnfrsf1B, and Cx3cr1;
supplemental Table 7). The discovery of common and specific
subsets of IRP target mRNAs implicated in a wide spectrum of
functions may, at least in part, explain phenotypic differences
between IRP1 and IRP2 knockout mice34-35 and connects the
regulation of intracellular iron homeostasis with these functions,
defining starting points for future explorations.
Previous in vitro studies reported IRP1- or IRP2-specific RNA
sequences generated by systematic evolution of ligands by exponential enrichment 26-28 (in this study, motifs 9, 10, 11, 12, and 17 for
IRP1 and motifs 4, 5, and 15 for IRP2). Our data confirm and
extend these results: mRNAs containing IREs with motif
4 CN(5)CCGUG(A/U/C) are specifically enriched in immunoprecipitation for IRP2, representing 9.3% of the total of predicted IREs
for this group (Figure 4B). Although many of the newly identified
IRP-associated mRNAs bear IRE-like motifs, some lack such
elements that we could recognize and entirely different IRPbinding sites or other not considered IRE structures29 may exist.
Indeed, this applies to 2 of the mRNAs for which we validated
IRP1 binding with in vitro transcripts (Pfn2 and Pdcl3).
None of the previously known IRE motifs were identified entirely
within the coding region. Notably, one of the specifically validated
mRNAs identified here (encoding glycogenin, the scaffold protein for
glycogen synthesis) contains a conserved IRE motif within its CDS.
Other functional cis-acting regulatory elements have been found previously within the CDS of mRNAs.36,37 Additional IRE-like motifs have
been reported in different species (eg, humans, primates, rats) that seem
not to be conserved in the mouse (CDC14A, APP, AHSP, and
CDC42BPA).17,38-40 None of these mRNAs were identified as IRPtarget mRNAs in this study using mouse total RNA as starting material.
Although we believe that this work identifies IRP-binding mRNAs in a
way that satisfies stringent biochemical criteria, it is important to point
out that further in vivo studies are needed to evaluate the functionality
and physiologic relevance of these candidate IREs and IRP-binding
mRNAs and to elucidate the underlying regulatory mechanisms.
We also complemented the exploration of transcriptomic IRPbinding targets by a proteomic approach by pSILAC, a method that
assesses overall changes in proteins synthesis (including changes in
de novo protein synthesis or profound changes in protein stability).
This work, for example, shows that the synthesis of the heme
b-binding protein succinate dehydrogenase (Sdh) subunit C is
strongly increased by hemin (⬎ 84 fold). Interestingly, a functional
IRE is present in the 5⬘UTR of the Drosophila melanogaster Sdh
subunit B mRNA,41 but no IRE-like motif is found in the murine or
human Sdh subunit C mRNA, and neither of these RNAs was
found to be associated with IRPs in our experiments. These
observations suggest that contrasting with flies, the induction of
these proteins in mammals is not driven by an IRP-dependent
mechanism, even if the regulatory outcomes are similar.
Although other membrane-associated transporters are identified
in our pSILAC experiments, Slc11a2 and Slc40a1 were not
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BLOOD, 24 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 22
SANCHEZ et al
detected. Possible explanations include low expression levels in the
tested cells, insufficient amount of de novo synthesis, or proteomic
methodologic limitations. The Aco2 protein was detected but not
found to be regulated by iron manipulation in Hepa1-6 cells or by
IRP deficiency in BMDMs. This observation is probably explained
by the well recognized low binding affinity of the 5⬘ IRE of Aco2
mRNA for IRP1, and this mRNA is known to be less stringently
regulated at the translational level compared with ferritin mRNAs.42
Analysis of the regulated proteins using the STRING algorithm43 reveals the down-regulation of the mitochondrial electron
transport chain, including 24 NADH dehydrogenases (complex I)
and all 4 proteins of complex II (Sdha, Sdhb, Sdhc, and Sdhd)
under conditions of IRP deficiency (supplemental Methods; data
not shown). These findings are very interesting in the context of
recent data showing that a general biologic function of IRPs lies in
securing mitochondrial iron sufficiency and function.16
The IRP-regulated proteins and mRNAs identified in this study
could physiologically respond to iron changes as well as to
iron-independent signals that alter the activity of the IRPs.
Collectively, our results begin to interconnect the well-characterized
core IRP regulon with key aspects of cell biology and physiology
and provide opportunities to further deepen the molecular and
cellular understanding of iron homeostasis; iron-related diseases;
and other IRP-dependent, iron-independent pathways.
Acknowledgments
The authors thank Dunja Ferring-Appel (EMBL) for excellent
technical assistance with mouse work. They are grateful to Monica
Campillos (EMBL) and Ildefonso Cases (IMPPC) for support in
the development of the bioinformatic web-server SIREs.
This work was supported by the Young Investigator Award of
the Medical Faculty, University of Heidelberg (Germany) and the
postdoctoral fellowship Beatriu de Pinós, Generalitat de Catalunya
(Spain) and Ramón y Cajal Program, Spanish Ministry of Science
and Innovation (RYC-2008-02352) to M.S. and by grants from the
Forschungsschwerpunktprogramm des Landes Baden-Württemberg
(RNA and disease) to M.W.H. and M.U.M.
Authorship
Contribution: M. Sanchez designed and performed experiments
and analyzed data; B.G. performed animal experiments, BMDM
work, pSILAC labeling of BMDMs, and assisted in manuscript
preparation; B.S. performed proteomics experiments and data
analysis; T.B.-I. performed microarray experiments; J.B. performed microarray data analysis; M. Selbach, V.B., M.U.M., and
M.W.H. oversaw the study and designed experiments; M.U.M.
assisted in manuscript preparation; and M. Sanchez and M.W.H.
wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Matthias W. Hentze, EMBL, Meyerhofstrasse
1, 69117 Heidelberg, Germany; e-mail: [email protected]; or Martina U. Muckenthaler, Department of Pediatric Oncology, Haematology and Immunology, University Hospital of Heidelberg, and
Molecular Medicine Partnership Unit, Im Neuenheimer Feld 156,
69120 Heidelberg, Germany; e-mail: martina.muckenthaler@med.
uni-heidelberg.de.
References
1. Hentze MW, Muckenthaler MU, Galy B,
Camaschella C. Two to tango: regulation of mammalian iron metabolism. Cell. 2010;142(1):24-38.
2. Muckenthaler MU, Galy B, Hentze MW. Systemic
iron homeostasis and the iron-responsive
element/iron-regulatory protein (IRE/IRP) regulatory network. Annu Rev Nutr. 2008;28:197-213.
3. Hentze MW, Caughman SW, Rouault TA, et al.
Identification of the iron-responsive element for
the translational regulation of human ferritin
mRNA. Science. 1987;238(4833):1570-1573.
4. Hentze MW, Caughman SW, Casey JL, et al. A
model for the structure and functions of ironresponsive elements. Gene. 1988;72(1-2):201-208.
5. Addess KJ, Basilion JP, Klausner RD, Rouault TA,
Pardi A. Structure and dynamics of the iron responsive element RNA: implications for binding of
the RNA by iron regulatory binding proteins. J Mol
Biol. 1997;274(1):72-83.
6. Muckenthaler M, Gray NK, Hentze MW. IRP-1
binding to ferritin mRNA prevents the recruitment
of the small ribosomal subunit by the cap-binding
complex eIF4F. Mol Cell. 1998;2(3):383-388.
7. Schlegl J, Gegout V, Schlager B, et al. Probing
the structure of the regulatory region of human
transferrin receptor messenger RNA and its interaction with iron regulatory protein-1. RNA. 1997;
3(10):1159-1172.
8. Binder R, Horowitz JA, Basilion JP, Koeller DM,
Klausner RD, Harford JB. Evidence that the pathway of transferrin receptor mRNA degradation
involves an endonucleolytic cleavage within the
3⬘ UTR and does not involve poly(A) tail shortening. EMBO J. 1994;13(8):1969-1980.
9. Walden WE, Selezneva AI, Dupuy J, et al. Structure of dual function iron regulatory protein 1
complexed with ferritin IRE-RNA. Science. 2006;
314(5807):1903-1908.
10. Gray NK, Quick S, Goossen B, et al. Recombinant iron-regulatory factor functions as an ironresponsive-element-binding protein, a translational repressor and an aconitase. A functional
assay for translational repression and direct demonstration of the iron switch. Eur J Biochem.
1993;218(2):657-667.
11. Haile DJ, Rouault TA, Tang CK, Chin J, Harford JB,
Klausner RD. Reciprocal control of RNA-binding
and aconitase activity in the regulation of the ironresponsive element binding protein: role of the
iron-sulfur cluster. Proc Natl Acad Sci U S A.
1992;89(16):7536-7540.
12. Salahudeen AA, Thompson JW, Ruiz JC, et al. An
E3 ligase possessing an iron-responsive
hemerythrin domain is a regulator of iron homeostasis. Science. 2009;326(5953):722-726.
13. Vashisht AA, Zumbrennen KB, Huang X, et al.
Control of iron homeostasis by an iron-regulated
ubiquitin ligase. Science. 2009;326(5953):718-721.
14. Smith SR, Ghosh MC, Ollivierre-Wilson H,
Hang Tong W, Rouault TA. Complete loss of iron
regulatory proteins 1 and 2 prevents viability of
murine zygotes beyond the blastocyst stage of
embryonic development. Blood Cells Mol Dis.
2006;36(2):283-287.
15. Galy B, Ferring-Appel D, Kaden S, Grone HJ,
Hentze MW. Iron regulatory proteins are essential
for intestinal function and control key iron absorption molecules in the duodenum. Cell Metab.
2008;7(1):79-85.
16. Galy B, Ferring-Appel D, Sauer SW, et al. Iron
regulatory proteins secure mitochondrial iron sufficiency and function. Cell Metab. 2010;12(2):
194-201.
17. Sanchez M, Galy B, Dandekar T, et al. Iron regulation and the cell cycle: identification of an iron-
responsive element in the 3⬘-untranslated region
of human cell division cycle 14A mRNA by a refined microarray-based screening strategy. J Biol
Chem. 2006;281(32):22865-22874.
18. Sanchez M, Galy B, Hentze MW, Muckenthaler MU.
Identification of target mRNAs of regulatory RNAbinding proteins using mRNP immunopurification
and microarrays. Nat Protoc. 2007;2(8):2033-2042.
19. Schwanhäusser B, Gossen M, Dittmar G,
Selbach M. Global analysis of cellular protein
translation by pulsed SILAC. Proteomics. 2009;
9(1):205-209.
20. Clausen BE, Burkhardt C, Reith W, Renkawitz R,
Forster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice.
Transgenic Res. 1999;8(4):265-277.
21. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise
comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids
Res. 2002;30(9):e36.
22. Thermann R, Hentze MW. Drosophila miR2 induces pseudo-polysomes and inhibits translation
initiation. Nature. 2007;447(7146):875-878.
23. Campillos M, Cases I, Hentze MW, Sanchez M.
SIREs: searching for iron-responsive elements.
Nucleic Acids Res. 2010;38[suppl 2]:W360W367.
24. Macke TJ, Ecker DJ, Gutell RR, Gautheret D,
Case DA, Sampath R. RNAMotif, an RNA secondary structure definition and search algorithm.
Nucleic Acids Res. 2001;29(22):4724-4735.
25. Bengert P, Dandekar T. A software tool-box for
analysis of regulatory RNA elements. Nucleic Acids Res. 2003;31(13):3441-3445.
26. Henderson BR, Menotti E, Kuhn LC. Iron regulatory proteins 1 and 2 bind distinct sets of RNA
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 24 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 22
target sequences. J Biol Chem. 1996;271(9):
4900-4908.
27. Butt J, Kim HY, Basilion JP, et al. Differences in
the RNA binding sites of iron regulatory proteins
and potential target diversity. Proc Natl Acad Sci
U S A. 1996;93(9):4345-4349.
28. Henderson BR, Menotti E, Bonnard C, Kuhn LC.
Optimal sequence and structure of iron-responsive
elements. Selection of RNA stem-loops with high
affinity for iron regulatory factor. J Biol Chem.
1994;269(26):17481-17489.
29. Meehan HA, Connell GJ. The hairpin loop but not
the bulged C of the iron responsive element is
essential for high affinity binding to iron regulatory
protein-1. J Biol Chem. 2001;276(18):1479114796.
30. Gunshin H, Mackenzie B, Berger UV, et al. Cloning and characterization of a mammalian protoncoupled metal-ion transporter. Nature. 1997;
388(6641):482-488.
31. Kohler SA, Menotti E, Kuhn LC. Molecular cloning of mouse glycolate oxidase. High evolutionary
conservation and presence of an iron-responsive
element-like sequence in the mRNA. J Biol
Chem. 1999;274(4):2401-2407.
32. Sanchez M, Galy B, Muckenthaler MU, Hentze MW.
33.
34.
35.
36.
37.
SYSTEMS ANALYSIS OF THE MURINE IRE/IRP NETWORK
Iron-regulatory proteins limit hypoxia-inducible
factor-2alpha expression in iron deficiency. Nat
Struct Mol Biol. 2007;14(5):420-426.
Troost FJ, Brummer RJ, Dainty JR, Hoogewerff JA,
Bull VJ, Saris WH. Iron supplements inhibit zinc
but not copper absorption in vivo in ileostomy
subjects. Am J Clin Nutr. 2003;78(5):1018-1023.
Galy B, Ferring D, Minana B, et al. Altered body
iron distribution and microcytosis in mice deficient
in iron regulatory protein 2 (IRP2). Blood. 2005;
106(7):2580-2589.
Cooperman SS, Meyron-Holtz EG, OlivierreWilson H, Ghosh MC, McConnell JP, Rouault TA.
Microcytic anemia, erythropoietic protoporphyria,
and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2. Blood. 2005;
106(3):1084-1091.
Lin X, Parsels LA, Voeller DM, et al. Characterization of a cis-acting regulatory element in the
protein coding region of thymidylate synthase
mRNA. Nucleic Acids Res. 2000;28(6):13811389.
Tai N, Schmitz JC, Chen TM, Chu E. Characterization of a cis-acting regulatory element in the
protein-coding region of human dihydrofolate reductase mRNA. Biochem J. 2004;378(Pt 3):9991006.
e179
38. Rogers JT, Randall JD, Cahill CM, et al. An ironresponsive element type II in the 5⬘-untranslated
region of the Alzheimer’s amyloid precursor protein transcript. J Biol Chem. 2002;277(47):4551845528.
39. dos Santos CO, Dore LC, Valentine E, et al. An
iron responsive element-like stem-loop regulates
alpha-hemoglobin-stabilizing protein mRNA.
J Biol Chem. 2008;283(40):26956-26964.
40. Cmejla R, Petrak J, Cmejlova J. A novel iron responsive element in the 3⬘UTR of human MRCKalpha. Biochem Biophys Res Commun. 2006;
341(1):158-166.
41. Gray NK, Pantopoulos K, Dandekar T, Ackrell BA,
Hentze MW. Translational regulation of mammalian and Drosophila citric acid cycle enzymes via
iron-responsive elements. Proc Natl Acad Sci
U S A. 1996;93(10):4925-4930.
42. Goforth JB, Anderson SA, Nizzi CP, Eisenstein RS.
Multiple determinants within iron-responsive elements dictate iron regulatory protein binding and
regulatory hierarchy. RNA. 2010;16(1):154-169.
43. Jensen LJ, Kuhn M, Stark M, et al. STRING 8–a
global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res.
2009;37:D412-416.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2011 118: e168-e179
doi:10.1182/blood-2011-04-343541 originally published
online September 22, 2011
Iron regulatory protein-1 and -2: transcriptome-wide definition of binding
mRNAs and shaping of the cellular proteome by iron regulatory proteins
Mayka Sanchez, Bruno Galy, Bjoern Schwanhaeusser, Jonathon Blake, Tomi Bähr-Ivacevic, Vladimir
Benes, Matthias Selbach, Martina U. Muckenthaler and Matthias W. Hentze
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