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INTRODUCTION |
Insulin-like growth factor I
(IGF-I)1 is a ubiquitous
peptide that has a fundamental role in both prenatal and postnatal
development (1, 2). The effects of IGF-I are mediated through the IGF-I receptor (IGF-IR),which resembles the insulin receptor in primary and
tertiary structure (3). The IGF-IR plays a central role during the cell
cycle, as demonstrated by the fact that overexpression of IGF-IR
abrogates requirements for exogenous growth factors (4). In vascular
smooth muscle cells (VSMCs),the IGF-IR mediates the mitogenic effects
of various growth factors such as platelet-derived growth factor,
endothelial growth factor, fibroblast growth factor
, thrombin, and
angiotensin II, consistent with an important role in cardiovascular
growth responses (5-8). A variety of studies have shown that
relatively small changes in IGF-IR expression have important
proliferative or antiproliferative effects (9-11). However, studies
characterizing growth factor and tumor suppressor gene regulation of
the IGF-IR have focused on transcriptional mechanisms (12-16).
Some features of the IGF-IR gene suggest that it could be regulated at
the posttranscriptional level. Like housekeeping genes, which appear to
be transitionally regulated, the IGF-IR promoter gene is devoid of TATA
and CAAT elements and is also very GC-rich. Moreover, the human and rat
5' untranslated regions (UTRs) are unusually long, 1038 and 943 bp, respectively, and the rat 5' UTR has been reported to possess two
additional AUGs upstream in frame with the initiator AUG (17).
In eukaryotic cells, different modes of translation initiation are used
depending on the nature of the mRNA to be translated and on the
physiological state of the cell. The most frequently used are the
"scanning mechanism" and "internal initiation." In the scanning
mechanism, the initiation of translation requires the formation of a
"43 S complex," which binds to the 5'-m7G cap structure
of the mRNA and scans along the 5' UTR up to the initiator AUG.
Then, the 60 S subunit attaches to this complex and translation is
initiated (18). However, the presence of highly structured 5' UTR will
cause the repression of this cap-dependent mechanism, and
many mRNAs with structured 5' UTRs are poorly translated using this
mechanism (19).
A cap-independent mechanism called internal initiation was first
demonstrated in picornaviruses, which lack a 5'-m7G cap and
have long structured 5' UTRs in their RNA. The presence of an internal
ribosome entry site (IRES) has been shown in different picornaviruses,
such as encephalomyocarditis virus (EMCV), human rhinoviruses, and
hepatitis A virus (see Ref. 20 for review). This mechanism requires
secondary structures that allow ribosomes to bind directly next to the
initiator AUG and permit translation to start without previous
scanning. Regulation of IRESs sometimes also involves cellular factors
such as the polypyrimidine tract-binding protein (PTB), a member of the
heterogeneous nuclear ribonucleoprotein family involved in nuclear
splicing regulation (21).
Recently, IRES elements have been found in several cellular mRNAs.
These include growth factors such as vascular endothelial growth factor
(22), fibroblast growth factor (23), and IGF-II (24). IRESs are also
found in a variety of other mammalian genes: proto-oncogenes
(c-myc, (25) and c-sis (26)),
transcription factors (NF-
B repressing factor) (27), hematopoiesis
transcription factors (28), cardiac voltage-gated potassium channel
(Kv1.4 (29)), and the
subunit of mitochondrial H+-ATP
synthase (30).
In this study, we investigated whether IGF-IR mRNA translation
could be driven by an internal initiation mechanism. We provide evidence for the first time that the mRNA encoding a growth factor receptor (IGF-IR) is translated by an internal initiation mechanism. Moreover, we show that the 5' UTR of the IGF-IR mRNA can be
considered to possess an IRES that binds PTB. These findings have
important implications for understanding mechanisms of control of
IGF-IR expression and hence mechanisms of cellular growth control.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Vascular smooth muscle cells were isolated
from rat thoracic aorta as described previously (31). They were grown
in DMEM supplemented with 10% FCS, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Human MCF7 cells
were grown in DMEM without phenol red, supplemented with 2 mM glutamine, 10% fetal bovine serum, and antibiotics.
Rat2 (ATCC CRL-1764, embryo fetus fibroblasts) were grown in DMEM
supplemented with 5% fetal bovine serum, 2 mM glutamine,
and antibiotics. COS-1 monkey cells were grown in DMEM supplemented
with 10% fetal bovine serum, 2 mM glutamine, and antibiotics.
Plasmid Constructions--
The rat IGF-I receptor coding
sequence and partial 5' UTR was previously cloned in our laboratory
(10). The DNA fragment corresponding to the other part of the 5' UTR
(+1/+640) was kindly provided by Dr. H. Werner (plasmid -2350/+640).
The plasmid pIGFIR-Trunc was constructed by cloning the
BamHI/EcoRI fragment purified from pTrunc-smp8 (a
kinase-deficient rat IGF-IR cDNA construct, kindly provided by Dr.
J. Du, Emory University) into the corresponding sites of plasmid pBSK- (Stratagene).
The pBSK-430 plasmid was obtained by inserting the
SacI/PvuII Klenow-filled fragment of plasmid p611
(10) into the SmaI site of the pBSK- vector. A
BamHI fragment was then removed, and the resulting clone
contained 430 bp (+513/+943) of the sequence coding for the 5' UTR plus
130 bp of the adjacent coding sequence. Plasmid pBSK-943 was obtained
by inserting the Klenow-filled fragment AflIII of plasmid
-2350/+640 into the SmaI site of the 5' UTR (position
+513). This clone contains the sequence coding for the entire 5' UTR
(943 bp) and 130 bp of the coding sequence.
Construction of the bicistronic vector: The Renilla
luciferase (RLuc) reporter gene from plasmid pRL-TK
(Promega) was extracted by NheI/XbaI digestion
and inserted into the Klenow-filled XhoI site of plasmid pCI
(Promega) giving the pC-RL plasmid. Firefly Luciferase
reporter gene (FLuc) was then subcloned from the pGL3 plasmid (Promega) into the SmaI site of pC-RL. In the
resulting plasmid, called pBiC, the transcription of the first cistron
corresponding to RLuc is under the control of the
cytomegalovirus promoter.
The sequence coding for the 5' UTR of the IGF-IR mRNA was amplified
by PCR using plasmid pBSK-943 as template and oligonucleotides 5'-AAAGAATTCAGTGTGTGGCGGCGGCGG-3' and
5'-AAAGTCGACTCCTTTTATTTGGGACGA-3', which contained an
EcoRI site and a SalI site, respectively. The PCR
product was then inserted in the pGEM-T easy vector (Promega), giving
the pGEM-943 plasmid, and sequenced. The
EcoRI/SalI fragment containing the sequence
coding for the 5' UTR was then introduced in the EcoRI and
SalI sites of pBiC vector giving the pBiC-943 vector. A
stable hairpin (
G = -46.2
kcal·mol
1, calculated by using
Zucker's RNA folding software) was created by introduction of a
double-stranded autocomplementarity oligonucleotide 5'-GCTAGCTGAACTGGGAGTGGACACCTGCTAGC-3' and
5'-GCTAGCAGGTGTCCACTCCCAGTTCAGCTAGC-3' in the
NheI site of plasmid pBiC-943 upstream of the
Renilla luciferase sequence. The resulting plasmid was
called pHBiC-943. Plasmid pBiC-
, containing 413 bp of the sequence
coding for the 5' UTR, was constructed by extraction of the
SmaI/SalI fragment of pGEM-943 and ligation in
the Klenow-filled EcoRI/XbaI site of pBiC vector.
Dual Luciferase Assay--
COS-1 cells were plated in 24-well
plates and transfected with 1 µg of bicistronic plasmid per well with
LipofectAMINE reagent. After 24 h, cells were washed and lysed
according to the manufacturer's instructions. FLuc and
RLuc activities were measured by using the dual-luciferase
reporter assay system (Promega) in a EG&G Berthold luminometer (Bad
Wildbad, Germany). For rapamycin treatment, 80% confluent COS-1 cells
were serum-starved for 48 h and then stimulated with DMEM
containing 10% FCS in the presence or absence of various
concentrations of rapamycin.
UV Cross-linking Assay--
UV cross-linking experiments were
done according to Huez et al. (22). Cytoplasmic extracts
from MCF-7 and rat VSMCs were prepared as follows: subconfluent cells
were scraped in phosphate-buffered saline and pelleted by
centrifugation. The cell pellet was resuspended in 500 µl of lysis
buffer (10 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithiothreitol) and frozen-thawed three times. The extract was
centrifuged at 12,000 × g for 10 min, and the
supernatant (S10) was brought to 5% (v/v) glycerol, aliquoted, and
stored at
80 °C.
Probe A was synthesized and 32P-labeled by T3 polymerase
from plasmid pBSK-943 linearized at the EcoRI site. Probes
B, C, and D were synthesized and 32P-labeled by T3
polymerase from plasmid pBSK-430 linearized at the EcoRI,
BbsI, and AccIII sites, respectively. Probe EMCV
was transcribed and 32P-labeled by T7 polymerase from
plasmid pIRES (Promega) linearized at the XbaI site.
32P-labeled RNA probes (106 cpm) were incubated
with 6 µg of S10 extract, preincubated with 2.5 µg of yeast tRNA
(Ambion) for 15 min at 30 °C, in buffer containing 5 mM
Hepes (pH 5.2), 25 mM KCl, 2 mM
MgCl2, 3.8% glycerol, 0.2 mM dithiothreitol,
and 1.5 mM ATP in a final volume of 10 µl. Samples were
then transferred to ice and irradiated with UV Stratalinker
(Stratagene) at 10 cm from the bulbs and routinely with 400,000 µJ/cm2 at 254 nm for 4 min. The samples were then treated
with a mixture of 5 units of RNase One (Promega) and 3 µg of
RNase A at 37 °C for 30 min and, when indicated, with 1 mg/ml of
proteinase K (Ambion) at 37 °C for 20 min. Laemmli buffer was added,
and the samples were heated for 2 min at 95 °C and loaded on a
12.5% polyacrylamide denaturing gel. The gel was fixed in 40%
methanol-10% acetic acid for 30 min, dried, and submitted to autoradiography.
Immunoprepitation of UV Cross-linked Proteins--
After UV
cross-linking as described above, the samples (10 µl) were diluted to
450 µl with NETS buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, and 0.05% Nonidet P-40) and mixed with either 5 µl of monoclonal antibody directed against PTB (generously provided
by Dr. E. Wimmer, Stony Brook, NY) or preimmune serum. After a 2-h
incubation at 4 °C, the immunocomplexes were immobilized on protein
G-Sepharose (Amersham Pharmacia Biotech). The unbound materials
were washed five times with the same buffer. Bound proteins were
analyzed by SDS-polyacrylamide gel electrophoresis and identified by autoradiography.
Distribution of Specific mRNAs among Polyribosomal
Subfractions--
Polyribosomes were prepared from Rat2 cells. 80%
confluent cells were serum-starved for 48 h and then stimulated
with DMEM containing 10% FCS in the presence or absence of 50 ng/ml of
rapamycin. Sucrose gradient fractionation of ribosomes was carried out
on postmitochondrial supernatants as described previously (32). Cells
were washed in cold phosphate-buffered saline in the presence of
cycloheximide (100 µg/ml), scraped off the plates, and collected in
phosphate-buffered saline. After centrifugation, cells were resuspended
in cold buffer containing 250 mM sucrose, 50 mM
Tris-HCl, pH 7.4, 25 mM KCl, 5 mM
MgCl2, 100 µg/ml cycloheximide and lysed by addition of
Nonidet P-40 to a final concentration of 0.07%. Nuclei and
mitochondria were pelleted by two successive centrifugations at 750 and
12,000 × g. The postmitochondrial supernatant was
layered onto a 10-50% linear sucrose gradient made in the same buffer containing 2 mM dithioerythritol, and centrifuged in
a Beckman SW41Ti rotor at 40,000 rpm for 90 min at 4 °C. Twenty
fractions of identical volume were collected from the top of the
gradient. Each fraction was treated with 100 mg/ml proteinase K and 1%
SDS for 30 min before phenol-chloroform extraction and ethanol
precipitation of the RNA. RNA from each fraction was resuspended in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 unit of
RNAsin. The quantitative distribution of the various mRNAs among
the different fractions was then analyzed by slot-blot analysis. The
specific IGF-IR and
-actin probes were the 1768-bp
BamHI/NcoI fragment of plasmid pIGFIR-Trunc and the 1100-bp PstI fragment of plasmid pAL41-
(33),
respectively. The probes were gel-purified and labeled by random primer
DNA labeling. The blots were prehybridized for 1 h at 65 °C and
hybridized over night. After autoradiography each fraction was
quantified using a PhosphorImager system (Molecular Dynamics).
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RESULTS |
Characteristics of the IGF-IR mRNA 5' UTR--
The features of
the IGF-IR mRNA 5' UTR prompted us to examine whether this region
had the potential to adopt a highly structured secondary conformation.
Predictions of the rat and human IGF-IR 5' UTR secondary structures
were performed using Zucker's algorithm, and the structures were
folded with the ESSA software (Fig.
1) (34). Extended base pairing is
apparent all along this 5' UTR, which has a
G of -465.7
kcal·mol
1 and several hairpin structures.
Remarkably, a long polypyrimidine (poly-U) stem-loop located just
upstream of the initiator AUG (between position +850 and +950) was
identified. Sequence comparison of the rat and human 5' UTR showed the
same stem-loop at nearly the same location. Analysis of this stem-loop
sequence revealed a typical binding site for PTB. Moreover, the poly-U
sequence is embedded in a Y-shaped structure (+780/+960), which is an
important element of picornavirus IRES (35). These features led us to investigate whether the rat IGF-IR mRNA can be translated by
internal ribosome entry.

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Fig. 1.
Secondary structure of the IGF-IR 5'UTR
mRNA. Secondary structure of the rat IGF-IR 5'UTR mRNA
based on Zucker's algorithm and folded by the ESSA program (34). The
5' and 3' ends (nt 1 and 1143, respectively) are indicated. Position of
the poly-U tract and the initiator AUG are also indicated. The
right part of the figure shows the poly-U tract region of
the human 5' UTR, which is highly conserved between rat and human
IGF-IR mRNA.
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The 5' UTR of the IGF-IR mRNA Contains an IRES--
To
determine the presence of an IRES in the 5' UTR of the IGF-IR mRNA,
we used a bicistronic vector strategy in which the first cistron is
translated by a cap-dependent scanning mechanism and the
second requires translation by internal entry (36). The basic
bicistronic vector was constructed by cloning the RLuc gene
as the first cistron under the control of the cytomegalovirus promotor and the FLuc gene as the second cistron (Fig.
2A, pBiC). The sequence coding
for the entire or the partial 5' UTR of the IGF-IR mRNA (pBiC-943
or pBiC-
), as well as that coding for the encephalomyocarditis virus
(EMCV) IRES as a positive control (pBiCV) were introduced between the
RLuc and FLuc genes. These plasmids were
transfected into COS-1 cells and tested for the two luciferase enzyme
activities. The ratio between the two activities was then calculated,
and the results were normalized using the pBiC
FLuc/RLuc as the reference. The
FLuc/RLuc ratio was 8-fold higher in cells transfected with pBiCV in which the FLuc was under the
control of the EMCV IRES, than in cells transfected with the pBiC
control plasmid. In cells transfected with pBiC-943, the ratio was
18-fold higher than that of the control plasmid pBiC. We also tested
whether the 5' UTR of the rat IGF-IR mRNA can drive internal
initiation in human cells (MCF-7) and similar results were obtained
(data not shown). In cells transfected with pBiC-
, which contains
only a part of the sequence coding for the 5' UTR of the IGF-IR (region +513/+943), translation of the FLuc gene was reduced because
the ratio was only 3-fold higher than that obtained with the pBiC vector. We also constructed the pBiC-
inv, which contains the last
430 nt (+513/+943) of the 5' UTR, and 100 bp of the coding sequence
cloned in the opposite orientation. This construct was used to prove
that the IRES activity was specific for the 5' UTR sequence. RNA
synthesized from this plasmid was clearly incapable of allowing
translation of FLuc compared with the pBiC-
plasmid.

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Fig. 2.
Presence of an IRES in the 5' UTR of the
IGF-IR mRNA. A, representation of bicistronic
constructs containing in the intercistronic region between
RLuc and FLuc: the EMCV IRES, the IGF-IR 5' UTR
mRNA, a part of the IGF-IR 5' UTR mRNA (region +513/+943), and
an antisense sequence (region +1066/+513) containing 413 nt of the UTR
and 100 nt of the coding sequence. After transfection in COS-1 cells,
luciferase activities were measured as described under "Experimental
Procedures." The ratios between FLuc and RLuc
genes were calculated, and values represent the fold increase of the
ratio calculated for each construct relatively to that obtained for the
control plasmid pBiC. The bars represent the average ± S.E. of four independent transfection experiments. B,
comparison of luciferase activities in COS-1 cells transfected with
pBiC-943 and pHBiC-943, the latter containing a hairpin upstream
of the RLuc gene. The luciferase activities were measured
24 h postinfection, and results are summarized in the histogram on
the right.
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To prove that the IRES activity was not due to translation initiation
from the first cistron or by a mechanism of reinitiation, we introduced
a hairpin (
G = -46.2) just upstream of the RLuc gene (pHBiC-943). The relative activities measured for each enzyme are
presented in Fig. 2B. Activity of RLuc encoded by
the first cistron of plasmid pBiC-943 was higher than that of the
FLuc coded by the second cistron. In contrast, when a
hairpin was inserted upstream of the RLuc gene, activity of
the RLuc enzyme was strongly decreased, as expected, whereas
that of the second enzyme FLuc persisted and was even
increased. These results showed that translation of the second cistron
was totally independent of that of the first one. Thus, synthesis of
the FLuc enzyme was initiated from the IRES contained in the
rat 5' UTR IGF-IR.
Rapamycin Treatment Does Not Affect the Efficiency of Translation
of the IGF-IR IRES in Vitro--
To demonstrate further that the
IGF-IR 5' UTR contained an IRES, we investigated its ability to drive
translation in the presence of rapamycin which was known to
specifically reduce cap-dependent translation both in
vivo and in vitro (37). To this end bicistronic mRNAs containing either the EMCV IRES or the IGF-IR 5' UTR were expressed in COS-1 cells (Fig. 3). In the
pBiCV construct the translation of RLuc was
cap-dependent, whereas the translation of FLuc
under the control of the EMCV IRES was cap-independent (Fig.
3A). Rapamycin caused a dose-dependent
inhibition (with a maximum of 55% at 100 ng/ml) of the
cap-dependent translation of RLuc, whereas the
cap-independent translation was not significantly affected. Similar
results were obtained with the pBiC-943 construct, in which the
translation of FLuc was under the control of the IGF-IR 5'
UTR (Fig. 3B). A dose-dependent inhibition of
RLuc was observed with a maximum of 35% at 100 ng/ml of
rapamycin. Interestingly, the IGF-IR 5' UTR retained the ability to
drive translation in a cap-independent manner as the level of
FLuc activity did not significantly decrease. Thus, these
results proved that the IGF-IR 5' UTR could initiate translation
through a cap-independent mechanism.

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Fig. 3.
Effect of rapamycin on bicistronic mRNA
translation. Exponentially growing COS-1 cells were serum-starved
for 48 h and then transfected with bicistronic constructs. 3 h after transfection, rapamycin (0-100 ng/ml) and serum (10%) were
added. Cells were harvested 24 h after rapamycin treatment, and
luciferase activities were measured as described under "Experimental
Procedures." The level of luciferase activities in nontreated cells
was set as 100%. The experiments were carried out in duplicate,
and the results are presented as mean ± S.D. A, pBiCV
generates bicistronic mRNAs containing the IRES derived from the
untranslated region of poliovirus. B, pBiC-943 generates
bicistronic mRNAs containing the 5' UTR of rat IGF-IR.
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Distribution of the Endogenous IGF-R mRNA among Polyribosomes
Is Not Affected by Rapamycin Treatment--
The effect of rapamycin on
the efficiency of translation of the endogenous IGF-IR mRNA was
evaluated by analyzing the distribution of IGF-IR mRNA among
polyribosomes before and after rapamycin treatment. For these
experiments, a transformed fibroblast cell line (Rat2) was used because
it contains sufficient IGF-IR mRNA for detection by slot blot
hybridization.
-Actin mRNA was used as a control representing
mRNA initiated by cap-dependent translation. Exponentially growing cells were treated for 3 h with or without rapamycin, and the distribution of IGF-IR and
-actin mRNAs was determined among polyribosomal subfractions (Fig.
4). Distribution of IGF-IR mRNA from
control and rapamycin-treated cells among the different fractions was
very similar (Fig. 4, top panel). 83 and 87% of total
IGF-IR mRNA was associated with polyribosomes containing at least
two ribosomes (fractions 5-20) in control and rapamycin-treated cells.
Thus, rapamycin did not induced a significant change in the IGF-IR
mRNA distribution. In contrast, there was less
-actin mRNA
associated with polyribosomes (fractions 5-20) in cells treated with
rapamycin than in control cells (Fig. 4, bottom panel, 70%
versus 81%). An increase in the amount of
-actin
mRNA in fractions containing less than two ribosomes (fractions 1-4) was observed. Each
-actin mRNA molecule can theoretically bind 10 ribosomes; thus, the 11% shift in
-actin mRNA
distribution from fractions containing polysomes to fractions
corresponding to monosomes (fractions 1-4) corresponds to a
significant decrease of the efficiency of translation. In contrast,
rapamycin treatment did not modify the efficiency of translation of
the endogenous IGF-IR mRNA.

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Fig. 4.
Distribution of rat IGF-IR mRNA and
-actin mRNA among polyribosome fractions
extracted from cells treated with or without rapamycin. Rat2 cells
were treated with or without (control) 50 ng/ml of rapamycin for 3 h just before harvesting. The postmitochondrial supernatants were
fractionated by centrifugation on sucrose gradients. Twenty fractions
of identical volume were collected. Distribution of the indicated
mRNA was analyzed by slot-blot. Numbering of the 20 fractions is
from the top to the bottom of the gradient. The
positions of the 40, 60, and 80 S ribosomal particles and of
polyribosomes are indicated. The amount of radioactivity in the
hybridized slots were quantified using a PhosphorImager system
(Molecular Dynamics). Results are expressed as the percentage of the
sum of the radioactivity present in the 20 fractions.
Histograms represent the relative proportion of mRNA
present in untranslated form (fractions 1-4) versus
mRNA actively translated (fractions 5-20), corresponding to
polyribosomes.
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PTB Binds to the 5' UTR of the rat IGF-IR mRNA--
Several
RNA-binding proteins are known to interact with the IRES of the EMCV,
poliovirus, and mammalian mRNAs. To determine whether the IGF-IR 5'
UTR was able to bind particular proteins, we used rat VSMCs and
performed UV cross-linking experiments. Specifically, we tested whether
the part of the UTR containing the typical PTB binding site (see Fig.
1) was able to bind proteins present in VSMCs. The
32P-labeled B RNA probe (Fig.
5A) corresponding to the last
430 nt of the 5' UTR, containing the poly-U tract and the first 123 nt
of the adjacent coding sequence, was incubated with S10 VSMC extract
and then UV-irradiated. Analysis of proteins cross-linked to the
labeled RNA was carried out by SDS-polyacrylamide gel electrophoresis (Fig. 5B, lane 1). Two major cross-linked complexes of 60 and 35 kDa were detected, and both complexes disappeared after
proteinase K treatment (Fig. 5B, lane 2), confirming the
proteic nature of these signals. The specificity of the interaction was
demonstrated by competition experiments. In the presence of a 50- or
150-fold molar excess of unlabeled B probe (Fig. 5B, lanes 3 and 4), both signals were strongly and progressively
decreased.

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Fig. 5.
UV cross-linking of rat and human cellular
factors to the IGF-IR 5' UTR mRNA and EMCV IRES. A,
schematic representation of the different 32P-labeled rat
RNA probes, obtained from T3 in vitro transcription and
corresponding to the complete or partial IGF-IR 5' UTR mRNA.
B, S10 extracts from rat VSMCs were incubated with
106 cpm of probe B or EMCV probe (lane 5).
Competition experiments were carried out by addition of unlabeled probe
B at a molar excess of 50-150-fold (lanes 3 and
4), unlabeled EMCV probe (lanes 7 and
9), and unlabeled nonspecific RNA (lanes 9 and
10) at a molar excess of 150-300-fold. UV irradiation was
performed as described under "Experimental Procedures," and samples
were treated with a mixture of RNase One and RNase A before analysis by
SDS-polyacrylamide gel electrophoresis. Lane 2 corresponds
to sample treated with proteinase K. C, the different
32P-labeled probes indicated were cross-linked with
proteins extracted from S10 VSMCs and MCF-7 cells. Proteins are
revealed by autoradiography of the dried gels. Molecular mass
markers, in kDa, are shown in B and C.
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We then investigated the possibility that the 60-kDa protein could
correspond to PTB. We used a 32P-labeled RNA probe
corresponding to the EMCV IRES as a positive control for PTB binding.
The 60-kDa protein bound to the B probe comigrated with a factor bound
to the EMCV IRES (Fig. 5B, lanes 5 and 6).
Moreover, labeling of the 60-kDa protein was specifically and
progressively abolished when a 150- or 300-fold molar excess of
unlabeled EMCV IRES was used as competitor (Fig. 5B, lanes 7 and 8). By contrast, an unknown protein with an apparent
molecular mass of 35 kDa was not linearly competed by this probe.
Further, no competition was observed when an unlabeled nonspecific RNA probe was used (Fig. 5B, lanes 9 and 10). To
localize, within the 5' UTR, the site of interaction with the 60-kDa
protein, we carried out complementary experiments using the different
RNA probes described in Fig. 4A. Probe C contained the
poly-U tract but not the coding sequence. In probe D, the poly-U tract
was removed. The 60-kDa protein was able to bind to both probes B and C
(Fig. 5C, lanes 1 and 2). However, this protein
did not interact at all with the probe D (lane 3). This
indicated that the presence of the poly-U tract is absolutely necessary
for binding of the 60-kDa protein. These results are consistent with
the hypothesis that the 60-kDa protein could be PTB and with the
predicted secondary structure of the region of the RNA recognized by
this protein (Fig. 1). As the poly-U tract is also present in the 5'
UTR of the human IGF-IR mRNA, UV cross-linking assays were
performed with S10 extract of MCF-7 human cells. Again, two proteins of 60 and 35 kDa were revealed with probes A, which corresponds to the
entire rat IGF-IR 5' UTR. These two complexes were formed with probes B
and C (Fig. 5C, lanes 4-6) whereas only that at 35 kDa was
found for probe D (Fig. 5C, lane 7). These results revealed
that the same proteins might be present in rat and human extracts and
that they were able to bind to the rat IGF-IR 5' UTR.
To demonstrate the identity of the 60-kDa protein, UV cross-linked
complexes obtained with MCF-7 and VSMC extracts and probe B were
immunoprecipitated using a monoclonal anti-PTB antibody (Fig.
6A). The 60-kDa complex was
immunoprecipitated with both the EMCV probe and the probe containing
the last half part of the IGF-IR 5' UTR mRNA (probe B) (Fig.
6A, lanes 1-4). The 35-kDa protein was also detected after
immunoprecipitation of UV cross-linked complexes from human MCF-7 S10
extracts incubated with probe B but was barely detectable in
immunoprecipitates from VSMCs. The detection of this protein after
immunoprecipitation might result either from interaction of this
protein with PTB or from residual background. Immunoprecipitation
performed with protein G alone and protein G plus preimmune serum (Fig.
6A, lanes 5 and 6) did not reveal these proteins.
Finally, to verify that the interaction between probe B and PTB was
direct, the immunoprecipitated complex (Fig. 6B, top panel)
was then analyzed by Western blot, using the same anti-PTB antibody.
This analysis (Fig. 6B, bottom panel) revealed a unique
immunoreactive band in both cell types, which migrated at around 60 kDa. This result demonstrated that the PTB was indeed present in the UV
cross-linked complex.

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Fig. 6.
Immunoprecipitation and Western blot analysis
of the UV cross-linked proteins. A, a monoclonal
anti-PTB antibody (lanes 1-4) was used to immunoprecipitate
human (lanes 1 and 2) and rat (lanes 3 and 4) proteins cross-linked to the EMCV IRES (lanes
1 and 3) or the rat IGF-IR 5' UTR fragment +513/+1066
(see probe B in Fig. 4A) (lanes 2 and
4). Lane 5 and 6 represent UV
cross-linked proteins incubated with protein G and protein G plus
preimmune serum, respectively. B, the proteins cross-linked
to the IGF-IR 5' UTR (probe B) were immunoprecipitated using an
anti-PTB antibody. After SDS-polyacrylamide gel electrophoresis, the
proteins were transferred to polyvinylidene difluoride membrane and
revealed either by autoradiography of the membrane (top
panel) or by Western blot using the same antibody (bottom
panel).
|
|
 |
DISCUSSION |
IGF-I receptor gene expression is controlled by a number of
physiological conditions and cell metabolic states. In this study, we
demonstrate that initiation of translation of this growth factor receptor mRNA can occur by internal ribosome entry, providing a
potential additional level of control of its expression. To our
knowledge, this is the first IRES to be characterized in the mRNA
of a growth factor receptor.
Because the rat IGF-IR 5' UTR is long and displayed a high G-C content,
the question about the ability of ribosomes to linearly scan along the
IGF-IR 5' UTR mRNA according to the conventional scanning model was
raised. Internal ribosome entry was first described for picornavirus
(38),and different elements involved in the internal ribosome entry
process are now well known. The rat IGR-IR 5' UTR mRNA does not
present the conventional motifs of picornavirus IRES, such as a GNRA or
CAAA loop (39). Nonetheless, our prediction of the secondary structure
revealed that this 5' UTR mRNA presents a conserved
polypyrimidine-rich stem-loop structure composed of two different
loops. The first contains two repeated UUUC motifs described to be the
consensus binding site for PTB (40) and a stretch of 15 uridines.
Interestingly the second loop contains a third UUUC sequence localized
21 nt upstream of the AUG, as is the case of a number of picornavirus
IRESs (41). Moreover, this stem-loop structure is embedded in a
Y-shaped conformation described for other cellular mRNAs, such as
vascular endothelial growth factor and c-myc (22, 42). The
predicted secondary structure of the 5' UTR of the human IGF-IR
mRNA revealed some differences between rat and human 5' UTRs.
First, the human 5' UTR is longer than the rat and contains a GNRA loop
localized 625 nt upstream of the initiator AUG. Moreover, the two
upstream in-frame AUGs described for the rat IGF-IR mRNA are not
found in the human IGF-IR mRNA.
We initially investigated the ability of the rat IGF-IR 5' UTR to
direct internal translation initiation. For this purpose, we used the
bicistronic strategy (36). The translation efficiency of the second
cistron was clearly increased when the IGF-IR 5' UTR mRNA was
cloned in the intercistronic region, and the efficiency of the IGF-IR
IRES was higher than that of the control EMCV IRES. Furthermore, we
tested whether this IRES was also functional after inhibition of
cellular cap-dependent translation by rapamycin treatment.
These experiments were first conducted in vitro and demonstrated that the IGF-IR 5' UTR conserved the ability to initiate translation when the cap-dependent translation machinery is
blocked. This result was also confirmed in vivo as the
endogenous IGF-IR mRNA was still engaged into polyribosomes under
rapamycin treatment.
The limits of the IGF-IR IRES remain to be defined, although an
important element is contained in the first 400 nt of the 5' UTR, and
it will be of primary interest to determine whether this IRES is
composed of short modules as described recently for the Gtx homeodomain
protein mRNA (43).
UV cross-linking experiments performed with rat vascular smooth muscle
cell and human MCF-7 cells revealed that at least two proteins can bind
to the rat IGF-IR 5' UTR mRNA and that the patterns using rat and
human cell extracts were very similar. Moreover, these proteins were
detected both with the EMCV IRES and the rat IGF-IR 5' UTR mRNA.
The apparent 35-kDa protein could correspond to the previously
described p36 protein known to interact with EMCV IRES (44). We
demonstrated by immunoprecipitation experiments that the 60-kDa protein
corresponds to the PTB splicing factor. Based on the predicted
secondary structure, the potential binding site should be localized
within the stem-loop structure described above. This hypothesis was
confirmed by the fact that the removal of this structure completely
abolished the binding of the PTB in human and rat cellular extracts.
PTB, also known as hnRNP 1, is a predominantly nuclear protein, present
in lower abundance in the cytoplasm of many cells, which binds to
pre-mRNA elements (45). The role of this factor both in the
splicing mechanism and in IRES-directed translation initiation remains
unclear. Nonetheless, it has been shown recently that this protein
enhances the translational activity of different viral IRESs (46).
The biological significance of IRESs has only recently been
appreciated. There is evidence that posttranscriptional regulation involving IRES-dependent translation plays a critical role
in expression of several proto-oncogenes and homeodomain protein genes.
The proto-oncogene c-myc is a critical regulator of cell proliferation, differentiation, and apoptosis, and recently it was
demonstrated that the c-myc IRES was active during apoptosis (47). Moreover, IRESs were found in mRNA that direct the
translation of important mammalian regulating proteins, such as
fibroblast growth factor-2, IGF-II, and platelet-derived growth factor.
A translational mechanism mediated by an IRES confers clear advantages to the mRNA bearing such an element. The vascular endothelial growth factor IRES is active during hypoxia when protein synthesis is
inhibited (48), likely playing an important role in angiogenesis. The
platelet-derived growth factor IRES is more active during cell
differentiation, in which protein synthesis rates are also reduced
(26). Thus, IRES-dependent translational regulation allows
translation under conditions that are not favorable for cap-dependent translation, such as apoptosis, mitosis,
development, or viral infection.
Accumulating evidence has shown that the IGF-IR is at a convergence
point in the control of cell growth and that a functional IGFI/IGF-IR
autocrine loop is required for the mitogenic effects of a variety of
growth factors, including platelet-derived growth factor (5),
endothelial growth factor (6), angiotensin II, and thrombin (8, 49).
Deletion of the IGF-IR is lethal, and overexpression of the receptor
has transforming potential. Thus, IGF-IR gene expression must be
tightly regulated to guarantee correct protein levels at the
appropriate time and location. In quiescent cells,
cap-dependent translation is strongly inhibited, and
proteins of the cap-dependent machinery are poorly
expressed. We can postulate that the internal ribosome entry process
allow the translation of key proteins such as growth factor receptors to permit the cell to engage in mitosis. In this respect, it is interesting to note that a recent study reported that in human kidney
cells, the level of IGF-IR remained unchanged after peroxide injury
(50). This is consistent with the mechanism of cap-independent translation initiation of the IGF-IR mRNA, as has been hypothesized recently for the insulin receptor based on microarray analysis (51).
Additionally, we have recently shown that native low density lipoprotein up-regulates IGF-IR on VSMCs without affecting
IGF-IR mRNA levels, suggesting that translational control
mechanisms could be important (52).
In summary, we have shown that the 5' UTR of the IGF-IR mRNA
contains an IRES that binds PTB and allows cap-independent translation. Because of the critical role played by the IGF-IR in cell cycle control
and development, our findings have important implications for
understanding mechanisms of normal and abnormal cell proliferation and
mechanisms for developmental control of gene expression.