From the Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges s/Lausanne, Switzerland
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Iron regulatory proteins (IRPs) control the
synthesis of several proteins in iron metabolism by binding to
iron-responsive elements (IREs), a hairpin structure in the
untranslated region (UTR) of corresponding mRNAs. Binding of IRPs
to IREs in the 5' UTR inhibits translation of ferritin heavy and light
chain, erythroid aminolevulinic acid synthase, mitochondrial aconitase,
and Drosophila succinate dehydrogenase b, whereas IRP
binding to IREs in the 3' UTR of transferrin receptor mRNA prolongs
mRNA half-life. To identify new targets of IRPs, we devised a
method to enrich IRE-containing mRNAs by using recombinant IRP-1 as
an affinity matrix. A cDNA library established from enriched
mRNA was screened by an RNA-protein band shift assay. This revealed
a novel IRE-like sequence in the 3' UTR of a liver-specific mouse
mRNA. The newly identified cDNA codes for a protein with high
homology to plant glycolate oxidase (GOX). Recombinant protein
expressed in bacteria displayed enzymatic GOX activity. Therefore, this
cDNA represents the first vertebrate GOX homologue. The IRE-like
sequence in mouse GOX exhibited strong binding to IRPs at room
temperature. However, it differs from functional IREs by a mismatch in
the middle of its upper stem and did not confer
iron-dependent regulation in cells.
RNA-binding proteins play a central role in RNA processing,
nucleo-cytoplasmic transport, localization, translation, or stability. This makes it desirable to identify all the targets of a given RNA-binding protein. Because many RNA recognition elements cannot be
identified by hybridization or predicted with certainty by search
programs, we developed an experimental approach based on the
interaction between an RNA-binding protein and its target RNAs. As a
model system we are investigating the iron regulatory proteins 1 and 2 (IRP-1 and IRP-2),1 which
become active in iron-deprived cells and bind with high affinity to
structural RNA motifs, the iron-responsive elements (IREs) (1-5). IRPs
repress translation of mRNAs with a cap-proximal IRE, as found in
the mRNA of ferritin heavy (H) and light (L) chain (1, 6),
mitochondrial aconitase (7, 8), erythroid 5-aminolevulinic acid
synthase (9, 10), and Drosophila succinate dehydrogenase b
(SDHb) (8, 11, 12). As a consequence, IRP binding lowers iron storage
and utilization. Moreover, binding of IRPs to five IREs in the 3'
untranslated region (UTR) of transferrin receptor mRNA protects
this otherwise unstable mRNA from degradation (3, 13). This leads
to more receptor synthesis and enhanced iron uptake.
Functional IREs form stem loop structures with a conserved CAGUGN loop
sequence (where N is any nucleotide except G). An upper stem of five
perfectly paired bases is separated from a lower stem by a single
cytosine on the 5' side or by a cytosine preceded by two nucleotides
with one unpaired nucleotide on the 3' side. Adopting an in
vitro selection procedure, we and others have identified IREs with
alternative loop sequences that bind to IRPs and of which some show a
preferential interaction with IRP-1 or IRP-2 (14-16). Such IRE mutants
confer translational control in vivo when inserted into the
5' UTR of a reporter construct (17). However, none of these alternative
IREs was detected in naturally occurring mRNAs to date.
Here we devised a method to enrich IRE-containing mRNAs by
using recombinant human IRP-1 as an affinity matrix (Fig. 1). We then
constructed an enriched cDNA library from mouse liver mRNA and
screened it for IREs by RNA-protein band shift assays. This revealed a
clone with strong homology to plant glycolate oxidase (GOX, or
short-chain Cell Culture--
HL-60 and Ltk Purification of Recombinant Human IRP-1--
Bacteria expressing
glutathione S-transferase (GST)-tagged human IRP-1 (18) were
grown to A600 0.7 and induced with 0.1 mM isopropyl- Isolation of RNA--
Total RNA was extracted as described by
Chomczynski and Sacchi (19). Poly(A)+ RNA was prepared
using the Poly(A)Tract mRNA isolation kit (Promega).
Affinity Purification of RNA--
GST-tagged human IRP-1 (100 µg) was immobilized on 50 µl of glutathione-Sepharose (Amersham
Pharmacia Biotech). Beads were washed extensively with binding buffer
(10 mM HEPES, pH 7.6, 3 mM MgCl2,
40 mM KCl, 5% glycerol) containing 5 mg/ml heparin (Serva) and binding buffer alone. Total RNA (5 mg), renatured for 5 min at
70 °C and 5 min at 42 °C, was adsorbed to the IRP-coated beads in
4 ml of binding buffer with 0.2% 2-mercaptoethanol, 1 mM
dithiothreitol, and 1 unit/µl RNasin. After gentle agitation for 30 min at 25 °C, unbound RNA was removed, and the beads were washed
three times with binding buffer containing 5 mg/ml heparin and twice
with binding buffer alone. Bound RNA was recovered by phenol-chloroform extraction and precipitated with 2.5 volumes of ethanol.
cDNA Synthesis and Library Construction--
cDNA was
synthesized from enriched RNA using Superscript II RNaseH Library Screening--
Ferritin H and L chain cDNAs were
identified by hybridization (on GeneScreen Plus membranes; DuPont NEN)
with [
To screen for new IRE-containing mRNAs, groups of 16 colonies
(excluding the ferritin clones) were grown in 5 ml of L broth, and
plasmid DNA was prepared using the QIAprep spin miniprep kit (Qiagen).
The plasmid mixture was linearized with NotI and purified on
QIAquick columns (Qiagen). The eluted DNA was transcribed in vitro with T7-RNA polymerase in the presence of
[ RNA Band Shift Assays--
RNA-protein gel retardation assays
were carried out as described previously (3). For library screening,
RNA transcripts were incubated with 2 ng of purified recombinant human
IRP-1. Unprotected RNA was degraded with 0.5 units of RNase T1
(Calbiochem) for 10 min, and 5 mg/ml heparin (Serva) was added with
loading buffer before migration on a 6% nondenaturing gel. Heparin was omitted in competition band shift reactions. Competitor RNAs were tracer-labeled with [ Northern Blots--
RNAs were electrophoresed on 1.2% agarose
gels containing 6% formaldehyde, transferred to GeneScreen Plus nylon
membranes (DuPont NEN), and fixed by UV cross-linking in a
Stratalinker (Stratagene) and baking for 1 h at 80 °C under
vacuum. Hybridization was overnight at 65 °C in buffer containing
50% formamide with [ In Vitro Transcription and Translation--
Coupled in
vitro transcription and translation of the mouse GOX cDNA was
carried out with the TNT coupled reticulocyte lysate system (Promega)
according to the manufacturer's instructions using T7-RNA polymerase.
Proteins were labeled by the incorporation of
[35S]methionine (Amersham Pharmacia Biotech).
Expression of Recombinant GOX and Enzyme Assay--
GOX was
expressed as a GST fusion protein in E. coli DH5 Construction of Plasmids--
pBSII-GOXIRE for transcription of
band shift probes was constructed by cloning a
DraI-NotI fragment of the GOX cDNA
(nucleotides 1853-2028 plus the poly(A) tail) between sites
EcoRV and NotI of pBluescript II SK(+). For
protein expression, the coding region of the GOX cDNA was amplified
by polymerase chain reaction and cloned into the SalI and
NotI sites of pGEX-5X3 (Amersham Pharmacia Biotech) in frame
with the GST sequence. The GOX IRE was inserted into the vector pL5-GH
(6) as described (17). Plasmid pFer-GH was kindly provided by Dr.
Matthias Hentze (EMBL, Heidelberg, Germany).
Translation Assay in Cells--
Ltk GST-tagged Human IRP-1 Can Be Used as an Affinity Matrix for
IRE-containing mRNAs--
The approach for the identification of
targets of IRP-1 is depicted in Fig.
1A. It comprises the selection
IRE-containing mRNA on an IRP-1 affinity column, the construction
of an enriched cDNA library, and its screening by RNA-protein band
shift assays. As shown in Fig. 1B, an affinity column with
recombinant IRP-1 can selectively bind IRE-containing mRNAs. For
this, the human IRP-1 mutant C437S that binds IREs constitutively (23)
was expressed with a GST tag in bacteria, purified to remove bacterial
RNA, and adsorbed on glutathione-Sepharose. Total input RNA, unbound RNA, washes, and bound RNA were analyzed by Northern blotting (Fig.
1B). Quantification by the PhosphorImager revealed specific retention of 50% of the IRE-containing ferritin tracer RNA as well as
human transferrin receptor mRNA. In contrast, <0.5% of the
control
The column was also tested for the binding of human SDHb mRNA when
we noticed an IRE in Drosophila SDHb mRNA (11). The
Northern blot was rehybridized with a rapid amplification of cDNA
ends polymerase chain reaction product of the human SDHb cDNA 5'
end including part of the coding sequence (24). Human SDHb mRNA was
not retained on the IRP-coated beads (Fig. 1B). This agrees with conclusions drawn from the human SDHb gene sequence that revealed
no IRE-like motif upstream of the coding region (25). It suggests that
human SDHb expression is not regulated by IRP-1.
Analysis of Enriched mRNAs: Putative Glycolate Oxidase mRNA
Is Bound by IRP-1 in Vitro--
To isolate new mRNAs with an IRE,
we incubated the IRP-1 column at a preparative scale with total mouse
liver RNA. Bound mRNAs were eluted, reverse-transcribed, and cloned
directionally to obtain an oligo(dT)-primed cDNA library enriched
in IRE-containing inserts. 4608 recombinant clones were identified by
blue-white selection and transferred to 18 master plates with 256 clones each. Among 22 randomly picked recombinant cDNAs, we found
clones coding for mouse ferritin H and L chains. Additional ferritin cDNAs were expected at high frequency in the library and identified by hybridization of colony lifts. 56 ferritin H chain and 214 ferritin
L chain clones were found, representing a frequency of 1.2 and 4.6%, respectively.
Clones that did not code for ferritins were screened for the presence
of an IRE by an RNA-protein band shift assay. Plasmids were linearized
behind the cDNA insert, transcribed in vitro with T7-RNA
polymerase, and incubated with recombinant purified IRP-1, first in
groups of 16, then in subgroups of 4, and finally as single clones.
Because some transcripts were RNase T1-resistant and resembled
IRE·IRP band shift complexes in their migration, we had to exclude
false positives by omitting IRP-1 in the assay. 14 cDNA clones were
finally identified as IRE-positive and sequenced. Of these, 11 had
escaped detection by hybridization and coded for ferritin L or H chain.
The remaining three clones originated of a single mRNA species with
homology to plant glycolate oxydase and contained in the 3' UTR an
IRE-like sequence close to the polyadenylation site (Fig.
2A, bold). However, a mismatch
in the upper stem distinguished this putative IRE from other known IREs (Fig. 2B). We also noticed different 3' end sequences among
the isolated clones, one with the poly(A) tail beginning at 20 bases from the polyadenylation site and another one extending 40 bases further (Fig. 2A, underlined) to a second poly(A) tail start
site. Both classes of cDNAs were also found in an independent
cDNA library (data not shown). This difference was not further
investigated and probably represents alternative polyadenylation
sites.
Rehybridization of our cDNA library with the longest cDNA
insert identified three more clones, one of them with a full-length cDNA of 2.0 kb. This clone contained an open reading frame of 1110 nucleotides coding for a protein of 370 amino acids with a predicted
molecular mass of 41.0 kDa and a pI of 7.6 (GenBank accession number
AI104312). In vitro transcription and translation of the
plasmid yielded a protein with the predicted size (Fig. 2C).
A search in the data bases revealed that this clone had >55% amino
acid identity (74% similarity) to GOX from plants (Fig. 3), suggesting strongly that it might
encode a mouse GOX. A kidney-specific isozyme from rat long-chain
hydroxyacid oxidase (26) was likewise related but less homologous than
the plant enzyme (48% identical and 59% similar). We also identified
several expressed sequence tag clones of human origin, which could be
aligned to the mouse sequence and served to reconstruct part of the
human GOX sequence (Fig. 3). It exhibited 87.9% amino acid identity
(92.2% similarity) with the mouse protein. Moreover, a genomic
sequence from Caenorhabditis elegans (GenBank accession
number AF016448) previously predicted to encode GOX (27) was 48.9%
identical (60.2% similar) to that of mouse GOX (Fig. 3).
cDNA Codes for a Liver-specific Mouse Glycolate
Oxidase--
Spinach GOX has been crystalized, and amino acid residues
involved in the active site, the substrate binding, and tight
interaction with FMN are known (28). Here we found that all of these
residues are conserved in our cDNA (Fig. 3), supporting the idea
that it encodes a mouse GOX. To test this hypothesis, we investigated the enzymatic activity of recombinant protein. The cDNA was
subcloned into the plasmid pGEX-5X3 for expression with an
amino-terminal GST tag in bacteria. A spectrophotometric assay for GOX
activity (20) was set up for crude bacterial extracts. Control extracts carrying plasmid without insert showed no reduction of
2,6-dichloroindophenol in the presence of excess sodium glycolate (660 µM) as a substrate (Fig.
4A). In contrast, extracts
from GOX-transformed bacteria oxidized glycolate at a rate of 69 pmol/s
per µg of crude protein. Moreover, GOX activity co-purified with the
fusion protein on glutathione-Sepharose beads (Fig. 4B).
This confirmed that the newly isolated cDNA encodes mouse GOX.
In animals, two isozymes of Mouse GOX IRE Binds to IRPs with High Affinity in Vitro but Not in
Cells--
The presence of an IRE-like sequence in the 3' UTR
suggested that GOX mRNA might be regulated by IRPs. Therefore, rat
hepatoma cells (FTO2B) were either treated with iron or the iron
chelator desferrioxamine or with iron after desferrioxamine. GOX
mRNA levels were measured on Northern blots and normalized to
These results suggested that IRP might be involved in the control of
GOX expression. In support, we mapped the IRP-1 binding region of the
mRNA between nucleotides 1884 and 1974 in the 3' UTR encompassing
the IRE-like sequence (Fig. 2A, bold). An A:A mismatch in
the upper stem of this putative IRE (Fig. 2B) suggested, however, that it might affect protein binding. Therefore, the affinity
of the GOX IRE for IRPs was compared with that of ferritin H chain IRE
in competition band shift assays (Fig.
6). Cytoplasmic extract prepared from
iron-deprived FTO2B cells was incubated with labeled GOX or ferritin
IRE in the presence of increasing molar excess of nonlabeled competitor
RNA (GOX IRE, ferritin IRE, or tRNA). Surprisingly, at 25 °C, both
probes bound equally well to IRP-1 and IRP-2 (Fig. 6). Encouraged by
this result, we inserted the putative IRE into the 5' UTR of vector
pL5-GH containing a constitutive ferritin promoter and the human growth
hormone gene (hGH) (6). A wild-type ferritin IRE in this vector confers IRP-mediated repression of hGH translation in iron-deprived cells (Ref.
10 and Fig. 7A). However, we
were unable to detect translational regulation in response to iron
deprivation in stably transfected mouse L cells with the GOX IRE
construct (Fig. 7A). Yet, control band shift experiments
with extracts from the same cells revealed strong induction of IRP-1 in
response to iron deprivation (Fig. 7B). Thus, it seemed
plausible that the GOX IRE was not binding to IRPs in cells. Therefore,
we measured carefully the IRE-IRP interaction at higher temperatures
in vitro (Fig. 8). We found a
strong temperature dependence, the interaction being entirely lost
between 34 and 37 °C. The stem loop of the GOX IRE is probably thermodynamically unstable because of the A:A mismatch in the upper
stem.
Screening for Targets of mRNA-binding Proteins--
We
explored whether affinity chromatography with immobilized RNA-binding
IRP-1 can contribute to the identification of new targets for
post-transcriptional gene regulation by this protein. Such a method
would be generally useful in view of the rapidly growing list of
RNA-binding proteins. Only few methods to search for targets of
RNA-binding proteins have been described. They comprise either
systematic evolution of ligands by exponential enrichment with
naturally occurring mRNAs (32) or immunoprecipitation of
mRNA-protein complexes followed by reverse transcription-polymerase chain reaction (33). The present study indicates that mRNA affinity purification on recombinant protein and construction of an enriched cDNA library combined with screening by RNA-protein band shift assays is a viable approach. Our cDNA library was clearly enriched for IRE-containing clones. Although a normal human liver cDNA library comprises ~0.5% ferritin H and L chain clones (34), the
frequency was increased to 5.8% in the enriched library (1.2% ferritin H chain clones and 4.6% L chain clones). This was less than
expected from the mRNA enrichment step but can be explained by a
high frequency of void plasmids.
The data with human SDHb mRNA (Fig. 1B) illustrate that
by affinity purification it is also possible to determine whether a
candidate mRNA binds to a regulatory protein in vitro.
Knowledge of the entire mRNA or precise target sequence is not
required, because a Northern blot with column fractions can be
hybridized with a probe corresponding to any fragment of the candidate
RNA. From the results with this particular mRNA we conclude that
IRPs are probably not involved in regulation of human SDHb expression.
Our data demonstrate that screening by in vitro RNA-protein
band shift requires certain controls, notably for the specificity and
temperature of the interaction. In the mouse GOX mRNA, the IRE-related sequence binds IRPs at 25 °C but not at 37 °C. It fits with the lack of translational repression of the GH construct with
the 5' GOX IRE despite IRP activation (Fig. 7). These results indicate
that GOX mRNA is not regulated by IRPs and that the stimulation of
expression by high iron in a liver cell line must be attributable to a
different mechanism.
At 25 °C, quite unexpectedly, the GOX IRE bound as strongly to rat
IRP-1 and IRP-2 as the ferritin H chain IRE (Fig. 6), despite an A:A
mismatch in the center of the upper stem (Fig. 2B). We have
previously made a similar observation with a mutant ferritin H chain
IRE containing a G:G mismatch in the same position and that displayed
~25% of wild-type binding affinity (14). These results seem to
reflect a certain tolerance for noncanonical base pairing in the upper
stem. Its thermodynamic instability explains probably why this IRE does
not bind at higher temperature and, when placed in the 5' UTR, does not
render translation of a human growth hormone reporter mRNA
iron-dependent in cells.
Considering the relatively limited number of clones analyzed in the
present screen, we cannot exclude that further IRE-containing candidate
mRNAs may exist in the liver. However, if further clones are to be
found, they can be expected at a frequency at least as low as that of
the GOX mRNA, which was 50-100-fold less frequent than that of
ferritin mRNA.
Possible Role of Mammalian Glycolate Oxidase--
The second main
conclusion from our study concerns the first isolation of a mammalian
GOX cDNA. Its amino acid sequence was 55% identical (74% similar)
to GOX (also termed short-chain 2-hydroxyacid oxidase) from plants. The
plant enzyme contains an FMN cofactor and catalyzes oxidation of
glycolate to glyoxylate. The three-dimensional structure of spinach GOX
has been resolved by x-ray crystallography (28), which allowed
determination of active site residues and side chains involved in FMN
binding. All of these amino acids are perfectly conserved in the
isolated clone (Fig. 3), suggesting that it codes for mouse GOX. This
conclusion is supported by the enzymatic activity of the purified
recombinant protein (Fig. 4).
From these results and evidence detailed below we conclude that our
clone is full length. The sequence encompassing the start codon
(GCCACAAUGU) matches well the Kozak consensus
(GCC(A/G)CCAUGG) for translation start sites (35), and the
size of the predicted protein was in good agreement with the in
vitro transcription-translation product analyzed on SDS-PAGE (Fig.
2C). Second, several homologous clones were isolated from a
commercial mouse liver cDNA phage library. Only one of those clones
contained a slightly longer 5' sequence (not shown) but had no
additional upstream start codon. Third, a probe derived from the
isolated clone detected a single band of 2.2 kb on a Northern blot, the
size expected for the predicted, polyadenylated mRNA (Fig.
5A). Fourth, alignment to homologous sequences in the data
base revealed high conservation close to the amino terminus (Fig.
3).
Mammalian GOX activity had been discovered as early as 1940 (36), but
to our knowledge no vertebrate GOX cDNA or gene has been cloned to
date. In animals, two 2-hydroxy acid oxidases have been reported with
expression in either liver or kidney, one with specificity for short
chain 2-hydroxyacids and the other for long chain 2-hydroxyacids,
respectively (30, 37). Our cDNA corresponds to the liver enzyme
(Fig. 5B). The sequence of a rat kidney long chain
2-hydroxyacid oxidase (26) is similar and belongs to the same family of
proteins (Fig. 3). Mammalian liver GOX has been located in peroxisomes
(38-40). This localization might be mediated by the carboxyl-terminal
sequence SKI (Fig. 3), which resembles the consensus SKL sequence for
peroxisomal import (41, 42). However, studies with mutants of the
localization tripeptide behind luciferase suggested that SKI is a poor
import signal (43). Further work is required to analyze this issue for
mammalian GOX.
Glycolate oxidase contains an FMN cofactor (20, 44) that catalyzes the
oxidation of glycolate to glyoxylate and possibly to oxalate (45-47).
GOX could be implicated in the detoxification of glycolate via the
synthesis of oxalate. Alternatively, it might provide building blocks
for the synthesis of glycine and serine, because the carbon atoms of
14C-labeled glycolate are detected in these amino acids
(48).
In oil seed plants, GOX acts as part of the glyoxylate cycle. This
pathway is required in early stages of seedling growth for the
conversion of fatty acid stores into sugars (49). Because of its
importance in plant growth, GOX cDNAs have been cloned in several
plant species. The question arises of whether lipid mobilization and
gluconeogenesis involving glyoxylate cycle enzymes are a speciality of
plants only. Both isocitrate lyase and malate synthase activity have
been detected in liver peroxisomal fractions and in adipose tissue of
mammals (50, 51), where they are induced by fasting. This suggests that
under certain conditions, mammalian GOX might also feed glyoxylate into
this pathway. The present cDNA clone should help clarify the
physiological role of GOX.
INTRODUCTION
Top
Abstract
Introduction
References
-hydroxy acid oxidase, EC 1.1.3.15). In plants, this
enzyme participates in the glyoxylate cycle and catalyzes the oxidation
of glycolate to glyoxylate. An IRE-like sequence was found in the 3'
UTR of the mouse mRNA and analyzed with respect to its possible
involvement in iron-dependent, post-transcriptional regulation.
EXPERIMENTAL PROCEDURES
cells were
cultured in
-minimal essential medium; FTO2B rat hepatoma cells were
cultured in 45% Dulbecco's modified Eagle's medium and 45% F-12
(Life Technologies, Inc.); and B16.F1 mouse melanoma cells were
cultured in Dulbecco's modified Eagle's medium. All media were
supplemented with 10% fetal calf serum. Cells were deprived of iron in
medium with 100 µM desferrioxamine (Desferal, a gift from
Novartis, Basel, Switzerland) for 16-20 h or iron-loaded in medium
with ferric ammonium citrate (60 µg/ml) for 4-5 h.
-D-thiogalactopyranoside
(Boehringer Mannheim) for 5 h. Cells were harvested and lysed by
sonication in phosphate-buffered saline and 1% Triton X-100. The
lysate was cleared at 10,000 × g for 10 min and
adsorbed on glutathione-Sepharose beads (Amersham Pharmacia Biotech)
for 1 h at 4 °C. Beads were washed in a Bio-Rad Polyprep
column, and bound IRP-1 was eluted in 50 mM Tris-Cl, pH
8.0, and 5 mM glutathione. The yield of IRP-1 was ~150
µg/liter of bacterial culture. The eluate was diluted 2-fold in
buffer A (20 mM Tris-Cl, pH 8.0, 5% glycerol, 8 mM 2-mercaptoethanol) and applied to a MonoQ column
(Amersham Pharmacia Biotech). Bound protein was eluted with 0-300
mM KCl in buffer A, and IRP-1 recovered between 140 and 200 mM KCl.
reverse transcriptase (Life Technologies) and an oligo(dT) primer with
a NotI site. EcoRI adaptors were then ligated.
The cDNAs were digested with NotI for 16 h at
37 °C, ligated into an EcoRI-NotI-cleaved pBluescript II SK(+) vector (Stratagene), and transformed into Escherichia coli XL1-Blue. Bacteria were plated on
Luria-Bertani plates containing 100 µg/ml ampicillin (Sigma), 1.5 mM isopropyl-
-D-thiogalactopyranoside (Boehringer Mannheim), and 60 µg/ml
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (Boehringer Mannheim). White colonies were manually transferred onto 18 16 × 16 array master plates.
-32P]dATP-labeled (Amersham Pharmacia Biotech),
random-primed cDNA probes. Hybridization was carried out at
65 °C overnight in hybridization buffer (10% (w/v) dextran sulfate
and 1% SDS) with 150 µg/ml salmon sperm DNA.
-32P]CTP (Amersham Pharmacia Biotech). After ethanol
precipitation, the labeled RNA was used to perform band shift assays.
Positive groups were reanalyzed as subgroups of four colonies and
finally as single clones. Plasmid DNA of final positives was analyzed by automated DNA sequencing.
-32P]CTP (Amersham Pharmacia
Biotech), purified on 3% NuSieve agarose gels by migration onto NA45
DEAE paper (Schleicher and Schüll) and elution in 500 mM Tris-Cl, pH 7.2, and 2.5 M ammonium acetate at 65 °C for 10 min. After phenol extraction and ethanol
precipitation, RNAs were dissolved in water and diluted to equal molar
concentration. Cytoplasmic extracts were prepared from iron-deprived
FTO2B cells by lysis in binding buffer supplemented with 0.3% Nonidet
P-40 and 1 mM phenylmethylsulfonyl fluoride. Nuclei were
spun down at 13,000 × g for 5 min at 4 °C, and the
supernatant was stored in aliquots at
70 °C. Protein concentration
was determined by the Bio-Rad protein assay.
-32P]CTP-labeled (Amersham
Pharmacia Biotech) antisense riboprobes.
and
purified on glutathione-Sepharose as described for IRP-1. The yield was
0.5 mg/liter of culture. Enzyme activity was determined at 25 °C by
incubation with 165 µM 2,6-dichloroindophenol (sodium salt hydrate; Fluka), 660 µM sodium glycolate (ICN) in 66 mM potassium phosphate, pH 8.3, and 1 mM EDTA,
and the decrease in absorption was measured at 605 nm (20).
were stably
transfected by the calcium phosphate method (21). Incorporation of
[35S]methionine and immunoprecipitation of secreted human
growth hormone (hGH) were carried out as described previously (17, 22).
RESULTS
-actin and glyceraldehyde-3-phosphate dehydrogenase mRNA
without an IRE was retained on the column. Thus, immobilized IRP-1 can
be used to enrich IRE-containing mRNAs at least 100-fold.
View larger version (54K):
[in a new window]
Fig. 1.
Enrichment of IRE-containing mRNAs on an
IRP-1 column. A, approach for the isolation of target
mRNAs of IRP-1. B, glutathione-Sepharose beads coated
with purified recombinant human IRP-1 were incubated with
poly(A)+ RNA (4.2 µg) of HL-60 cells supplemented with
in vitro transcribed, 32P-labeled ferritin
tracer RNA. Nonspecifically bound RNA was removed by three washes with
binding buffer containing 5 mg/ml heparin and two washes with
binding buffer alone. Bound RNA was recovered by
phenol-chloroform extraction. Equal fractions of the total input RNA
(lane T), unbound RNA (lane U), washes
(lanes 1-5), and bound RNA (lane B) were
analyzed on a Northern blot and sequentially hybridized with probes for
transferrin receptor (TfR), -actin,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and SDHb.
The ferritin tracer mRNA was directly detected by
autoradiography.
View larger version (38K):
[in a new window]
Fig. 2.
Isolation of a putative glycolate oxidase
cDNA clone with an IRE-like motif. A, the nucleotide
sequence at the 3' end of the isolated cDNA contains an IRE-like
sequence (bold) with a classical IRE loop
(boxed), 29 bases upstream of the putative polyadenylation
signal (italic). A 40-nucleotide sequence is present in some
alternatively polyadenylated transcripts (underlined).
B, the hypothetical secondary structure of the putative IRE
(right) resembles other known functional IREs, as for
example the human transferrin receptor IRE C (left). It has
the CAGUGU loop sequence and a single C bulge. However, it differs from
other IREs by an A:A mismatch (boxed) in the upper stem.
TfR, transferrin receptor. C, in vitro
transcription and translation of the putative mouse GOX cDNA clone
in presence of [35S]methionine results in a polypeptide
of ~42 kDa on an SDS-polyacrylamide gel.
View larger version (136K):
[in a new window]
Fig. 3.
Evolutionary conservation of glycolate
oxidase. Using the program Pileup of the Genetics Computer Group
package (52), the amino acid sequence of rat kidney long chain
2-hydroxyacid oxidase (L-HAO; GenBank accession number
X67156) was aligned to the deduced amino acid sequence of the mouse GOX
cDNA as well as to other GOX sequences from human (partial sequence
reconstructed from several expressed sequence tags), C. elegans (GenBank accession number AF016448), ice plant (GenBank
accession number U80071), pumpkin (GenBank accession number D14044),
spinach (GenBank accession number J03492), Arabidopsis
thaliana (GenBank accession number AL021710), and rice (GenBank
accession number AF022740). Active site residues (A) and
amino acids involved in FMN (F) or substrate (S)
binding of the spinach enzyme (28) are indicated.
View larger version (14K):
[in a new window]
Fig. 4.
Enzymatic activity of recombinant mouse
glycolate oxidase. The activity of GOX was measured at 25 °C by
the reduction of 2,6-dichloroindophenol (DCIP) coupled to
the oxidation of glycolate. Enzyme activity was detected in crude
extract of bacteria expressing GST-tagged GOX protein (A;
) but not in extract from mock-transfected bacteria with the
parental plasmid (A;
). The enzyme was adsorbed on
glutathione-Sepharose (B;
), and only low residual
activity was found in the soluble supernatant (B;
). No
activity could be adsorbed from extracts of mock-transfected bacteria
(B;
).
-hydroxyacid oxidase were reported (29,
30), one expressed in the kidney with preference for long chain
-hydroxyacids and the liver-specific short chain
-hydroxyacid
oxidase, also known as GOX. This prompted us to investigate the tissue
specificity of the mouse GOX mRNA. Northern blot analysis of RNA
from various mouse tissues and fibroblast cells revealed GOX mRNA
expression exclusively in liver but not in spleen, skeletal muscle,
kidney, embryos, or fibroblasts (Fig. 5A).
View larger version (58K):
[in a new window]
Fig. 5.
Liver-specific expression of glycolate
oxidase mRNA and effect of iron levels. A, total RNA (20 µg) extracted from mouse Ltk cells or mouse tissues was
analyzed on Northern blots with an antisense riboprobe specific for
mouse GOX mRNA. Cross-hybridization to 28 S rRNA provides a loading
control. B, FTO2B rat hepatoma cells were cultured for
20 h in medium with either 60 µg/ml ferric ammonium citrate
(FAC), 100 µM desferrioxamine
(Des), or 100 µM desferrioxamine for 20 h
followed by 60 µg/ml ferric ammonium citrate (Des/FAC) in
fresh medium for an additional 5 h. Poly(A)+ RNA (2.5 µg) from each batch of cells, as well as untreated control cells, was
analyzed on a Northern blot next to 15 µg of mouse liver total RNA.
The blot was first hybridized to a GOX-specific antisense riboprobe and
then stripped and rehybridized to a mouse
-actin probe. The
intensity of radioactive signals was quantified on a
PhosphorImager.
-actin mRNA (Fig. 5B). No effect was observed when
iron-deprived cells were compared with controls. But when iron salt was
added, GOX mRNA was up-regulated 2.3- and 2.8-fold, respectively.
The RNA binding activity of IRPs in FTO2B cells was tested by band
shift experiments and responded as expected (31) to different
iron-loading conditions (not shown).
View larger version (98K):
[in a new window]
Fig. 6.
RNA-protein band shift assay analyzing mutual
competition of mouse GOX IRE and human ferritin H IRE. Equimolar
amounts of the human ferritin H IRE (Fer H; 31 pg, 2 × 104 cpm) or mouse GOX IRE (150 pg, 7.6 × 104 cpm) were mixed with the indicated molar excess of
unlabeled competitor RNA (ferritin H IRE, mouse GOX IRE, or yeast tRNA)
and analyzed by band shift assay as described under "Experimental
Procedures" with 2 µg of protein extract from iron-deprived FTO2B
cells.
View larger version (71K):
[in a new window]
Fig. 7.
Effect of the GOX IRE in a 5' position on
translation of human growth hormone. The putative GOX IRE was
subcloned into the 5' UTR of the vector pL5-GH (6), which codes for hGH
transcribed from a constitutive promoter. Mouse Ltk cells
were stably transfected and labeled for 4 h with
[35S]methionine, and secreted hGH was immunoprecipitated
(A). To detect IRP-mediated inhibition of translation, cells
were preincubated overnight with 100 µM iron chelator
desferrioxamine (Des) and compared with cells treated with
60 µg/ml ferric ammonium citrate (Fe). A 10-fold
inhibition of hGH translation was observed with the positive control
construct harboring the ferritin IRE (Fer-GH) (10), but no
difference was visible with the GOX IRE construct (Gox-GH).
B, cytoplasmic extracts of the same cells were analyzed for
RNA binding activity of IRPs with a 32P-labeled ferritin H
chain IRE. The IRE-IRP band shift assay shows strong activity of IRP-1
and IRP-2 in iron-deprived cells (Des;
2-me)
but very weak activity in iron-loaded cells (Fe;
2-me). The in vitro addition of 2%
2-mercaptoethanol (+2-me) to extracts activates fully all
cytoplasmic IRP-1. This serves as a control for equal loading.
View larger version (45K):
[in a new window]
Fig. 8.
Temperature dependence of IRP binding to the
GOX IRE. Cytoplasmic extracts made from B16.F1 cells were mixed at
different temperatures with a molar excess of
[ -32P]CTP-labeled GOX IRE and analyzed by the band
shift assay. RNase T1 was added after 10 min to digest excess IRE and
to prevent its binding to IRP during gel migration at 4 °C. IRP-1
and IRP-2 are not inactivated at 37 °C, as shown in the last
lane, where the sample was shifted back to 25 °C before adding
RNase T1.
DISCUSSION
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jovan Mirkovitch for the FTO2B cell line, Martin Irmler for carrying out the in vitro transcription-translation experiment, and Markus Nabholz for critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Institute of Pharmacology and Toxicology,
University of Lausanne, CH-1005 Lausanne, Switzerland.
§ To whom correspondence should be addressed: Swiss Institute for Experimental Cancer Research, Genetics Unit, 155 Ch. des Boveresses, CH-1066 Epalinges s/Lausanne, Switzerland. Tel.: 41-21-692-58-36; Fax: 41-21-652-69-33; E-mail: lukas.kuehn{at}isrec.unil.ch.
The abbreviations used are: IRP, iron regulatory protein; GOX, glycolate oxidase; GST, glutathione S-transferase; hGH, human growth hormone; IRE, iron-responsive element; UTR, untranslated region; SDHb, succinate dehydrogenase b; H, heavy; L, light.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|