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Molecular and Cellular Biology, November 1998, p. 6178-6190, Vol. 18, No. 11
INSERM U397, Endocrinologie et Communication
Cellulaire, Institut Fédératif de Recherche Louis
Bugnard, CHU Rangueil, 31403 Toulouse cedex 04, France
Received 3 April 1998/Returned for modification 2 June
1998/Accepted 4 August 1998
The mRNA of vascular endothelial growth factor (VEGF), the major
angiogenic growth factor, contains an unusually long (1,038 nucleotides) and structured 5' untranslated region (UTR). According to
the classical translation initiation model of ribosome scanning, such a
5' UTR is expected to be a strong translation inhibitor. In
vitro and bicistronic strategies were used to show that the VEGF mRNA
translation was cap independent and occurred by an internal ribosome
entry process. For the first time, we demonstrate that two
independent internal ribosome entry sites (IRESs) are present in this
5' UTR. IRES A is located within the 300 nucleotides upstream from the
AUG start codon. RNA secondary structure prediction and site-directed mutagenesis allowed the identification of a
49-nucleotide structural domain (D4) essential to IRES A activity. UV
cross-linking experiments revealed that IRES A activity was correlated
with binding of a 100-kDa protein to the D4 domain. IRES B is located in the first half of the 5' UTR. An element between nucleotides 379 and
483 is required for its activity. Immunoprecipitation experiments
demonstrated that a main IRES B-bound protein was the polypyrimidine
tract binding protein (PTB), a well-known regulator of picornavirus
IRESs. However, we showed that binding of the PTB on IRES B does not
seem to be correlated with its activity. Evidence is provided of an
original cumulative effect of two IRESs, probably controlled by
different factors, to promote an efficient initiation of translation at
the same AUG codon.
The vascular endothelial growth
factor (VEGF) is a potent endothelial cell mitogen that plays a crucial
role in the regulation of both physiologic and pathologic angiogenesis
(10, 44). VEGF is involved not only in embryogenic
development and differentiation of the vascular system, in wound
healing, and in reproductive function but also in pathologic angiogenic
processes such as proliferative retinopathies, tumor growth, arthritis,
and psoriasis (10).
Numerous studies have been devoted to understanding the expression
regulation of this factor, especially at the transcriptional level. A
wide range of cytokines or oncogenic proteins, including interleukins
1 Surprisingly, very little is known about the possible translational
control of VEGF messenger except for a stimulatory effect on VEGF
translation in CHO cells overexpressing the cap binding protein
eukaryotic initiation factor 4E (eIF4E) (25). However, the
VEGF mRNA presents unusual features also found in other RNAs of
viral origin or transcribed from cellular proliferation regulator genes, such as the fibroblast growth factor 2 (FGF-2) gene, the platelet-derived growth factor (PDGF) gene, or the c-myc
proto-oncogene. The 5' untranslated region (UTR) of the mRNA
is unusually long, as the transcription starting point is located 1,038 nucleotides (nt) upstream from the AUG initiation codon and heavily
structured due to a high percentage of G and C residues. The 5' UTR
region also contains three noncanonical upstream CUG codons in frame with the initiator AUG codon. All of these elements make the use of a
conventional ribosome scanning model for translation initiation very
difficult (26).
A cap-independent mechanism involving an internal ribosome entry site
(IRES) has been identified in picornavirus messengers, which are
uncapped and present a long 5' UTR (20, 41). The presence of
an IRES has also been reported for many viral (cardiovirus, rhinovirus, and aphthovirus) (21) and some cellular
human (Bip, FGF-2, IGF-II, eIF4G, PDGF, and c-Myc) (2, 13, 33, 37, 50, 51, 53) messengers and in Drosophila antennapedia
and ultrabithorax mRNAs (39, 59). The IRESs discovered
so far differ in their primary sequences but show similarities in their secondary structures which appear to be crucial to IRES function (21, 28). In several picornaviruses, the internal entry
process has been shown to require cellular factors including the
polypyrimidine tract binding protein (PTB) (1), which is
also involved in the nuclear splicing regulatory pathway (40,
56).
We show here that the mRNA of the major angiogenic factor is
translated by an internal ribosome entry process. Furthermore, we
demonstrate that the VEGF 5' mRNA leader contains two independent IRESs which are able to promote efficient translation at the AUG start
codon. The patterns of cellular proteins binding to the two IRESs are
clearly different. These data suggest that different factors could
control the activities of these IRESs.
Plasmid constructions.
The VEGF cDNAs and the DNA fragment
corresponding to 5' UTR of the messenger were kindly provided by J. Abraham (52) and cloned into a pKS-derived plasmid. PCR was
performed with sense oligonucleotide
5'AAATCTAGAATTCGCGGAGGCTTGGGGCA3' and antisense oligonucleotide 5'GGTATCGATTGGATGGCAGTAG3' to construct a
translational fusion between VEGF and chloramphenicol acetyltransferase
(CAT). The amplified fragment extending from positions 1 to 1205 (corresponding to the 5' UTR and the 167 nt downstream from the AUG
codon) was cloned in a previously described pSCT-derived vector
(45) upstream from the CAT coding sequence, under the
control of cytomegalovirus (CMV) and T7 promoters. This chimeric
construct, called pVC, was expected to encode one VEGF-CAT protein of
32 kDa.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Two Independent Internal Ribosome Entry Sites Are Involved in
Translation Initiation of Vascular Endothelial Growth Factor
mRNA
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(31) and 6 (8), insulin-like growth factor 1 (IGF-1) (14), tumor growth factor (TGF-)
(5), c-Src (36), v-Raf (15), and Ras
(46), and oxygen tension have been shown to regulate VEGF
gene transcription (48). VEGF may also be
posttranscriptionally regulated. The VEGF pre-mRNA undergoes alternative splicing which generates four polypeptide isoforms of 121, 165, 189, and 206 amino acids (11), the functions of which
have not yet been fully defined. VEGF mRNA stability is also
influenced by hypoxic conditions or by IGF-1 expression (49, 57). Finally, posttranslational modifications of VEGF isoforms, including plasmin and urokinase proteolysis or glycosylation, have been
described (11, 43).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
G = 
40 kcal/mol) (described in
reference 53) was introduced into the pVC construct
between the promoters and the 5' end of VEGF cDNA, leading to the pHVC
construct. Bicistronic vectors were constructed from the previously
described pSCTCAT plasmid, which contains the CAT gene downstream from
the CMV promoter (45). The IVS2 intron was removed from
this plasmid, and a synthetic intron, obtained from plasmid pRLCMV
(Promega), was added just downstream from the CMV promoter. A second
CAT gene was then introduced downstream from the intron. This
intermediate vector was called pCC. All or part of the 5' UTR leader of
VEGF plus 167 nt of the coding sequence was then cloned in the
intercistronic region between the two CAT genes. The bicistronic vector
containing the entire 5' UTR (nt 1 to 1205), designated pCVC, was
expected to encode two CAT proteins of 24 and 32 kDa. The
above-described hairpin (G = 40 kcal/mol) was
introduced into pCVC between the CMV promoter and the first cistron.
This construct was designated pHCVC. Different deletions of the VEGF 5'
UTR were performed. Removal of the first 475 nt in the 5' UTR (up to
the BamHI site) resulted in plasmid pCVC1, removal of the
first 654 nt (up to the XmnI site) resulted in pCVC2,
removal of the first 745 nt (to Nhe site) yielded pCVC3,
removal of the first 846 nt (to the BanII site) resulted in
pCVC4, and removal of the first 917 nt (up to the Sma site)
gave pCVC5. We then constructed another bicistronic plasmid to
determine the 3' border of the IRES. This plasmid contained the entire
VEGF 5' UTR, in which the AUG codon of VEGF was directly fused to a
chimeric CAT gene (fCAT) resulting from translational fusion of part of
the nucleolin gene with the CAT gene (pSVNC82) (9). This
chimeric fCAT gene was used to discriminate between the products of the
two CAT cistrons encoded by this bicistronic vector during Western
immunoblotting. This 5' UTR-fCAT fusion was obtained by PCR
amplification using oligonucleotides T7 (matching sequence upstream
from the 5' UTR), and 5'
AAACCTAGGGCCCAAGTTCATGGTTTCGGAG 3' (matching nt 1029 to 1046) on plasmid pVC. This PCR fragment was digested at the
ApaI site (underlined sequence) and fused to fCAT in the pCC
vector deleted from the second CAT cistron. This CAT-VEGF 5' UTR (nt 1 to 1046)-fCAT-containing plasmid was called pCVC0. Several deletions
were then made from this plasmid in the VEGF 5' UTR. The plasmid was
called pCVC30 when the first 745 nt were deleted, pCVC60 when nt 654 (XmnI site) to 917 (Sma site) were removed,
pCVC70 when nt 554 (NaeI site) to 1013 (NaeI site) were deleted, pCVC90 when nt 1 to 91 (AgeI site) as
well as nt 554 to 1013 were removed, and pCVC80 when nt 332 (SacI site) to 1013 (NaeI site) were deleted.
D4. Another deletion plasmid (pRVL3) constructed from pRVL2
consisted of a deletion of nt 917 (Sma site) to 1013 (NaeI site). pRVL4 contained a 5' UTR fragment limited by nt
801 to 1046 in the intercistronic space. This fragment was obtained by
PCR from plasmid pCVCO, using oligonucleotide 5'
TTTGGATCCGAAGGAAGAGGAGAGGGGGC 3' (matching residues
798 to 817) and a reverse one located in the CAT gene (5'
GCAACTGACTGAAATGCC 3'). This amplified fragment was then
restricted at the ApaI (see description of pCVCO above) and
BamHI (underlined) sites to obtain only the corresponding VEGF sequence. pRVL6 was obtained after cloning of the 5' UTR fragment
(nt 91 to 554 and 1013 to 1039) contained in plasmid pCVC90 between the
two LUC genes. pRVL7, pRVL8, pRVL9, and pRVL10 were constructed from
pRVL6 after deletion of nucleotides 483 (PvuI site) to 1013 (NaeI site), 379 (BssHII site) to 1013, 332 (SacI site) to 1013, and 189 (DraI site) to 1013, respectively. pRVL11 contained nt 134 to 483 and 1013 to 1039. This
fragment was obtained from plasmid pRVL6 by PCR using
oligonucleotide 5' AAAGAATTCAGATCTTTGATATTCATTGATCCGGG 3'
(matching residues 134 to 155) and a reverse one located in the
LUCf gene. This amplified fragment was then restricted by the
EcoRI (underlined) and the PvuI sites and
recloned in the intercistronic space. pRVL12 was constructed in the
same way, using oligonucleotide 5'
AAAGAATTCAGATCTTGAATCGGGCCGACGGCT 3' (matching
residues 241 to 260), and thus contained nt 241 to 483 and 1013 to 1039 between the two LUC genes. pRVL13 consisted of a deletion of
nucleotides 379 (BssHII site) to 1013 (NaeI site) from pRVL12.
Monocistronic vectors derived from plasmid pSCTCAT in which VEGF 5' UTR
fragments had been introduced upstream from the CAT gene were
constructed for cross-linking experiments. pVC30 contained nt 745 to
1046 upstream from the CAT gene. We also cloned the mutated 5' UTR
fragment contained in pRVL2D4 (nt 745 to 858 and 907 to 1046)
upstream from the CAT gene. This plasmid was called pVC30D4. pVC1
was a pVC-derived plasmid in which nt 1 to 475 were deleted. Probes C
and D (see Fig. 8A) are derived from plasmids pSKV3 and pKSV4,
pKS-derived vectors in which were inserted the VEGF 5' UTR regions
contained in plasmids pRVL11 (nt 134 to 483 and 1013 to 1046) and
pRVL12 (nt 241 to 483 to 1013 to 1046), respectively.
Two new plasmids, pVC80 and pVC90, were constructed for in vitro
translation by inserting the fragments of the VEGF 5' UTR contained in
plasmids pCVC80 and pCVC90 (nt 1 to 332 and 1013 to 1046 and nt 91 to
554 and 1013 to 1046) into the pSCTCAT vector upstream from the CAT
gene. We also constructed vector pVC5, a derivative from pVC in which
nt 1 to 917 were deleted.
Probes transcribed from two pKS-derived vectors, called pKSV1 and
pKSV2, were used for the RNase protection assays. We inserted nt 1 to
745 (bordered by EcoRI and NheI sites) into
plasmid pKS digested at the XbaI and EcoRI sites
in pKSV1. In pKSV2, we inserted nt 1 to 1046 (bordered by the
XbaI and ApaI sites) into plasmid pKS digested by
XbaI and ApaI.
In vitro translation.
Plasmids pVC, pVC90, pVC80, pVC30, and
pVC30D4 were linearized downstream from the 3' end of the CAT coding
sequence at the BglII site. pSCT11A (53) was
linearized downstream from the 3' end of the FGF-2 coding sequence at
the HindIII site. Capped or uncapped RNAs were generated
in vitro, using the T7 mMessage mMachine kit (Ambion) according to the
manufacturer's instructions, with or without adding
m7GpppG (0.5 mM) to the transcription reaction. RNA
transcripts were quantified by absorbance at 260 nm and ethidium
bromide staining on an agarose gel, which also allowed verification of
their integrity. In vitro translation in rabbit reticulocyte lysate
(RRL; Promega) was performed as previously described (45),
in the presence of [35S]methionine (Amersham).
Translation products were analyzed by polyacrylamide gel
electrophoresis (PAGE) in a sodium dodecyl sulfate (SDS)-12.5%
polyacrylamide gel (45); the dried gels were scanned with a
PhosphorImager apparatus (Molecular Dynamics), and quantification of
the bands was performed with Imagequant software.
DNA transfection and Western immunoblotting. COS-7 monkey cells were transfected with Fugene-6 transfection reagent (Boehringer) according to manufacturer's instructions or by the DEAE-dextran method as described previously (45). Forty-eight hours after transfection, either (i) the cells were scraped in phosphate-buffered saline (PBS) and the pellets were resuspended in 1% SDS solution and sonicated or (ii) the cells were lysed in 1 ml of Tri-Reagent (Euromedex). Following the latter method and after total RNA extraction (see below), the proteins were precipitated with 1 volume of isopropanol. The protein pellet was then washed five times in 2 ml of a 0.3 M guanidine hydrochloride-95% ethanol buffer and once in 2 ml of ethanol. The protein pellet was then heated to 65°C for 20 min and resuspended in 1% SDS solution. Total proteins were quantified by the bicinchoninic acid assay (Pierce) (absorbance at 562 nm), and 10 µg of proteins from each cell lysate was used for Western immunoblotting. In summary, lysates were heated for 2 min at 95°C in SDS- and dithiothreitol (DTT)-containing sample buffer, separated in a 12.5% polyacrylamide gel, and transferred onto a nitrocellulose membrane. CAT proteins were immunodetected with rabbit polyclonal anti-CAT antibodies prepared in the laboratory (1/10,000 dilution). Antibodies were detected with an enhanced chemiluminescence kit (Amersham).
Cellular RNA purification. Total cellular RNAs were prepared by the Tri-Reagent method (Euromedex), derived from the guanidinium thiocyanate procedure (7). A total of 5 × 106 transfected cells were scraped, centrifuged, and lysed in 1 ml of Tri-Reagent. RNA was extracted after addition of 0.2 ml of chloroform and precipitated with isopropanol. After an ethanol wash and precipitation, the RNA was quantified by measuring the absorbance at 260 nm and checked for integrity by electrophoresis on an agarose gel and ethidium bromide staining.
RNase mapping.
A complementary-strand RNA probe was
generated in vitro by T7 or T3 RNA polymerase (Promega) according to
manufacturer's instructions, using a linearized DNA template and in
the presence of 50 µCi of [
-32P]UTP. Probe A was
transcribed by using T3 polymerase from plasmid pKSV2 linearized at the
XbaI site. Probe C was transcribed by using T3 polymerase
from plasmid pKSV2 linearized at the BamHI site. Probe B was
transcribed by using T7 polymerase from plasmid pKSV1 linearized at the
EcoRI site. 32P-labeled RNA was purified by
using the RNaid kit (Bio 101). The RNase protection experiments were
performed with an RPAII kit (Ambion) according to the manufacturer's
instructions. A 10-µg aliquot of total cellular RNA and a large
excess (2 × 106 cpm) of probe were precipitated with
ethanol; 10 µg of yeast RNA can be added to the total RNA before
precipitation to increase the size of the RNA pellet. All experiments
described were tested with and without addition of RNA. As a control
experiment, total RNA samples were incubated 15 min at 37°C in the
presence of 10 U of RNase-free DNase 1 prior to the precipitation in
order to avoid DNA contamination. The pellet was resuspended in 20 µl
of hybridization buffer, heated at 90°C for 4 min, and incubated at
42°C for 14 h. Then 200 µl of RNase digestion buffer
containing 20 U of RNase T1 and 1 µg of RNase A were
added, and the reaction mixture was incubated for 30 min at 37°C; 300 µl of inhibitor RNase buffer and 10 µg of carrier yeast RNA were
then added, and the reaction mixture was precipitated at 20°C for
20 min. The pellet was resuspended in 8 µl of gel loading buffer,
denatured, and fractionated on a 5% acrylamide-8 M urea gel at 200 V
for 1.5 h. The gel was then fixed, dried, and autoradiographed.
Control experiments showed that all probes described, used alone or
only with yeast RNA, were totally digested after RNase mix treatment.
UV cross-linking assay and immunoprecipitation.
Cytoplasmic
extracts from COS-7 cells were prepared as previously described
(53). Confluent cells were scraped in PBS and centrifuged.
The cell pellet was resuspended in lysis buffer (10 mM NaCl, 10 mM
Tris-HCl [pH 7.4], 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT),
and frozen-thawed three times. The extract was centrifuged at
12,000 × g for 10 min, and the supernatant (S10) was
brought to 5% (vol/vol) glycerol and frozen in aliquots at 80°C.
D4 linearized with
Bsp120.1. Probes A (nt 91 to 554 and 1013 to 1046) and B (nt
91 to 189) (Fig. 8A) were transcribed from plasmid pVC90 linearized with Bsp120.1 and DraI, respectively. Probes C
(nt 134 to 483) and D (nt 241 to 483) (Fig. 8A) were transcribed from
plasmids pKSV3 and pKSV4 linearized with NaeI.
32P-labeled RNA probes (1 × 106 to
1.5 × 106 cpm) were incubated with 6 µg of S10
extract, preincubated or not with 2.5 µg of calf liver tRNA
(Boehringer Mannheim) for 15 min at 30°C, in buffer containing 5 mM
HEPES (pH 7.5), 25 mM KCl, 2 mM MgCl2, 3.8% glycerol, 0.2 mM DTT, and 1.5 mM ATP in a final volume of 10 µl at 30°C for 15 min (34). Samples were transferred to ice and irradiated with a UV Stratalinker (Stratagene) by being fixed 10 cm from the bulbs
and routinely irradiated with 400,000 µJ/cm2 at 254 nm;
2.5 µg of calf liver tRNA was then added to calibrate the RNase
digestion when no prior incubation of the S10 extract with tRNA had
been performed. The samples were then treated with a mix of RNase ONE
(5 U; Promega) and 2.5 µg of RNase A at 37°C for 30 min and, when
indicated, with proteinase K (Sigma) at 37°C for 20 min at a final
concentration of 1 mg/ml. PAGE sample buffer was added, and the samples
were heated for 2 min at 95°C and loaded on an SDS-10 or 12.5%
polyacrylamide gel. The gel was fixed in 30% methanol-10% acetic
acid for 30 min, dried, and autoradiographed.
The cross-linked proteins were immunoprecipitated with Pansorbin as
follows. Ten microliters of the cross-linked 32P-labeled
sample (see below) was diluted to 150 µl in PBS-Nonidet P-40 (NP-40)
buffer (1× PBS, 50 mM NaF, 2 mM EDTA, 2 mM EGTA, 0.05% NP-40) and
precleared by incubation with 50 µl of Pansorbin for 10 min at room
temperature (RT). The supernatant was incubated for 30 min at RT with 5 µl of anti-PTB antibody (kindly provided by J. G. Patton)
(40) and then for 30 min at RT with 50 µl of Pansorbin.
After five washes in HEPES-NP-40 buffer (150 mM NaCl, 15 mM HEPES [pH
7.4], 1 mM EDTA [pH 7.4], 0.5% NP-40), the samples were analyzed by
PAGE (10 or 12.5% gel) as described above.
Dual luciferase assay. LUCf and LUCr activities were measured by using the Dual-Luciferase reporter assay system (Promega). Transfected COS-7 cell plates (60 by 15 mm) or 24-well dishes were rinsed twice with PBS, scraped, and homogenized in 400 µl of lysis reagent provided with the kit 24 to 48 h after transfection. The lysate was cleared by a 2-min centrifugation at 4°C. Chemiluminescent signals were measured in a luminometer (Berthold) equipped with automatic injectors. A 20-µl volume of extract was incubated with 100 µl of Luciferase Assay Reagent II (Promega) for 2 s, and LUCf activity was measured for a period of 15 s; 100 µl of Stop and Glo buffer (Promega), stopping the firefly enzymatic reaction and containing the substrate for LUCr enzyme, was then injected. Luminescence corresponding to LUCr activity was measured for 15 s starting 3 s after injection.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Identification of an IRES in the 5' UTR of the VEGF mRNA. Two alternative strategies involving hairpin-containing monocistronic vectors or bicistronic vectors were used to study whether VEGF expression was translationally regulated by an internal ribosome entry process. The first approach had been successful for identification of Moloney murine leukemia virus IRES (55), and the second had been used to identify IRESs in several viral and cellular mRNAs (22, 33, 41).
DNA plasmids were designed to contain the 1,038 nt of the VEGF 5' UTR and the first 167 nt of its coding sequence fused in frame with the CAT coding sequence (Fig. 1A). The predicted size of the chimeric VEGF-CAT protein encoded by this vector was 32 kDa. In the monocistronic plasmid (Fig. 1A, construct A [pVC]), this 5' UTR-VEGF-CAT sequence was controlled by the CMV and T7 promoters. In a second construct, a stable (G = 40 kcal/mol) hairpin was added downstream from the promoters to impair the cap-dependent ribosome scanning (construct B [pHVC]). This latter construct was expected to encode a 32-kDa VEGF-CAT protein only if
there was an IRES in the VEGF mRNA leader.
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Localization of IRES A in the VEGF mRNA 5' UTR. A number of additional bicistronic constructs containing shortened intercistronic UTRs, with or without a 5' hairpin (Fig. 2A), were designed to more precisely localize the IRES structure in the VEGF mRNA 5' UTR. Identical amounts of each DNA construct were used to transfect COS-7 cells, and the level of protein synthesis was determined as described above. The same quantity of COS-7 cell protein extracts was loaded in all lanes.
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293 and 192 upstream from
the AUG codon) is required for the formation of a functional IRES.
Hereafter this fragment will be referred to as the IRES A 5'
region. Although the 293 nt upstream from the AUG codon seem sufficient
for IRES function, Fig. 2B shows that the translation efficiency of the
VEGF-CAT mRNA decreased with shortening of the 5' UTR
(compare lanes A to H). This finding suggests that sequences located at
different positions in the 5' UTR are required for optimal IRES
efficiency.
Two additional constructs were made to determine the involvement of the
VEGF coding sequence located downstream from the AUG codon in the
internal entry process. They consisted of fusion of the full VEGF 5'
UTR or its 3' 293 bp up to and including the AUG codon with the
chimeric fCAT gene and composed of 560 neutral nucleotides of the
nucleolin coding sequence in frame with the CAT gene. This chimeric
fCAT reporter gene was used to discriminate the products of the two CAT
cistrons in Western immunoblotting. The results showed an efficient
expression of the fCAT protein from both constructs (Fig. 2C),
revealing that the first 167 coding nucleotides of the VEGF coding
region, present in the plasmids used previously (Fig. 2A), were not
required for efficient internal entry of the ribosomes on the
messenger.
Altogether, these data clearly localize an IRES within the 293 nt
upstream from the AUG start codon of VEGF mRNA. This IRES will
hereafter be called IRES A.
Verification of the bicistronic mRNA integrity in transfected COS-7 cells. Bicistronic RNA integrity was assessed by RNase protection assays (see Materials and Methods) to rule out the possibility that the observed VEGF-CAT protein could be expressed from unexpected monocistronic mRNA generated by a cleavage or a cryptic promoter located in the VEGF 5' UTR. The protection experiments were performed with total RNA from COS-7 cells transfected with bicistronic or monocistronic constructs containing either the complete leader sequence, a deletion of the first 475 nt, or a deletion of the last 293 nt (Fig. 3A). Three probes, A, B, and C, with expected sizes of 1054, 748, and 572 nt were used for hybridization with transfected COS-7 total RNA before RNase digestion; they were expected to produce protected fragments of 1,017, 696, and 535 nt, respectively, after RNase digestion. The data presented in Fig. 3B show that the protected fragments are unique and of the expected sizes, thus demonstrating that the intercistronic region is full size in the bicistronic mRNA.
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Characterization of the structural features of IRES A. Figure 4 shows the secondary structures, predicted by the Zuker procedure (6), of the entire or 293-bp fragment of the 5' UTR (nt 745 to 1038) demonstrated to be sufficient for IRES function (Fig. 2B). Extended base pairing is apparent all along this UTR fragment, bringing the AUG codon in the vicinity of the IRES A 5' region (nt 745 to 846). A stem-loop structure, called D5, bearing an unpaired GNRA (where N is C, G, A, or U and R is G or A; in this case GUGA) sequence is located in the first half of the leader between nt 416 and 434. This motif was shown to be common to picornavirus IRES and implicated in aphthovirus IRES function (32). Involvement of this predicted structure in the VEGF IRES activity is discussed later.
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cis elements involved in IRES A activity.
The predicted structural data shown in Fig. 4 were used to design
new deletions for further characterization of the VEGF IRES A. A new
bicistronic vector was produced in which the LUCr gene was subcloned as
the first cistron and the (LUCf) gene was subcloned as the second
cistron (Fig. 5) to improve sensitivity
and quantification of the assay. The VEGF complete or deleted 5' UTR
(constructs pRVL1, pRVL2, and pRVL5), as well as new deleted fragments
corresponding to removal of predicted domains D4 and D3 and the first
56 nt of IRES A (constructs pRVL2D4, pRVL3, and pRVL4,
respectively), were introduced into the intercistronic region. Either a
hairpin (G = 40 kcal/mol) or the
encephalomyocarditis virus (EMCV) IRES (35) was introduced
between the LUCr and LUCf genes as controls (construct pRHL or pREL,
respectively). These plasmids were transfected in COS-7 cells as
described above. The ratio between the activities of the two luciferase
enzymes observed in the cell extracts was calculated and compared with
the data obtained with the negative control construct pRHL (Fig. 5).
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D4), while deletion of the D3 domain (pRVL3)
resulted in only a 50% decrease in IRES activity compared to that
obtained with the pRVL2 construct. In comparison to pRVL2 construct
efficiency, deletion of the 5' 56 nt of IRES A resulted in a 60%
decrease of LUCf expression (pRVL4), indicating a very low IRES
activity, albeit higher than that obtained with the D4 deletion. This
result confirmed the observation made in Fig. 2. These results
indicated that the IRES A 5' end (nt 745 to 801) and the D4 domain (nt
858 to 907) are necessary for the IRES function. Both elements probably
form the core of the VEGF IRES, whereas the D3 domain (nt 917 to 1013)
seems also to play a role in IRES activity.
Identification of cellular factors bound to VEGF 5' UTR and to IRES A. UV cross-linking experiments were performed with COS-7 cell extracts and different 32P-labeled RNA probes corresponding to either the complete 5' UTR or deleted fragments of the 5' UTR (Fig. 6A) to identify cellular factors interacting with the VEGF 5' UTR and particularly with IRES A. As shown in Fig. 6B, the protein pattern differed according to the RNA probe used. The complete leader was able to bind at least nine proteins (Fig. 6B, lane A). Most of these proteins were able to bind to probe B, corresponding to the upstream part of the leader (lane B). In contrast, probes C, D, and E bound only a small number of proteins. Three major proteins migrating at 120, 100, and 85 kDa were detected with probes C and D, corresponding to the downstream part of the leader and both containing the IRES (lanes C and D, bands a, b, and c). Finally probe E, in which the D4 domain was deleted, was able to bind the p85 and the p120 proteins but not p100 (lane E). These results showed a correlation between the cross-linking of p100 to RNA and IRES activity and suggested a potential role of the 100-kDa protein in IRES function.
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The VEGF mRNA 5' UTR contains two distinct IRESs. It is clear from data presented in Fig. 2B and 5 that the complete 5' UTR behaved as a more efficient IRES than the IRES A fragment containing nt 745 to 1046. Furthermore, deletion of the D4 domain in the full-length 5' UTR led to a 30% reduced internal entry activity compared to that observed with the corresponding complete leader (data not shown), whereas the same deletion in the IRES A context almost abolished IRES activity. We also showed above that the binding of PTB occurred in the 5' part of the VEGF mRNA 5' UTR. These data led us to investigate the presence of a second IRES in the 5' part of the UTR.
To test this hypothesis, four new bicistronic plasmids containing different portions of the upstream half of the VEGF 5' UTR were designed (Fig. 7A, left) and used to transfect COS-7 cells, and the cell extracts were analyzed by Western immunoblotting with an anti-CAT antibody. It was apparent from these experiments that three VEGF mRNA leader fragments corresponding to nt 1 to 654, 1 to 554, and 91 to 554, respectively, were able to promote translation of the second cistron fCAT (Fig. 7A, right, lanes B, C, and E). fCAT expression was, however, less efficient with these three constructs (lacking the IRES A) than with the complete leader (lane A). In contrast, the fragment extending from positions 1 to 332 exhibited very low IRES activity (Fig. 7A, lane D).
|
PTB binding to IRES B is independent of IRES efficiency. As we had shown that PTB was the main protein bound to the upstream half of the VEGF RNA leader sequence (Fig. 6), we were prompted to see whether PTB interacted with IRES B. As for Fig. 6, this was investigated by UV cross-linking and immunoprecipitation experiments using several fragments of the 5' part of the leader with or without IRES activity as probes; the EMCV IRES was used as a control (Fig. 8). It was clearly apparent from these experiments that the RNA segments containing nt 91 to 189 and nt 134 to 483 were able to bind PTB with the same efficiency as the segment extending from nt 91 to 554 (Fig. 8, probes and lanes A, B, and C). In contrast, the fragment extending from nt 241 to nt 483 was no longer able to bind this protein (Fig. 8, probe and lane D). PTB binding to probe C was confirmed by immunoprecipitation with anti-PTB antibody (Fig. 8B, lane C).
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Activities of the two IRESs in RRL. The ability of both IRESs to promote cap-independent translation in vitro was analyzed by using monocistronic RNA containing different fragments of the VEGF 5' UTR fused to the CAT gene. A cap-dependent control which corresponded to the leader deleted FGF-2 mRNA (53) was included. Equal amounts of uncapped and capped monocistronic mRNAs were transcribed in vitro for each construct and assayed for translation in RRL (Fig. 9B). Cap independence was evaluated by calculating the ratio of CAT expression obtained from uncapped versus capped mRNA (NC/C ratio) (Fig. 9B and C).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this study, we demonstrate that the synthesis of VEGF, the major angiogenic factor, occurs through an internal ribosome entry site process. This observation, together with the previous discovery of an IRES in the mRNA of another important angiogenic factor, FGF-2 (53), suggests an involvement of IRES-dependent translation in the control of angiogenesis. It is also clearly apparent from our results that two IRESs are present in the 5' UTR of the VEGF mRNA and that they bind some different factors. This novel and interesting feature of VEGF mRNA suggests that VEGF expression is controlled at the translational level, probably in a specific way, by each of the two IRESs.
IRES A is located in a 293-nt-long fragment just upstream from the AUG codon (nt 745 to 1038 from the mRNA 5' end [Fig. 2]). Analysis of the cis elements involved in the activity of this IRES revealed a need for at least two elements: the 5' part of the IRES-containing fragment (IRES A 5' region, between nt 745 and 846) and the D4 domain (nt 858 to 907 [Fig. 5]). The D4 loop, containing nucleotides AGACA, differs from the sequences of the loop consensus motifs ACCC (21) and GNRA or CAAA (18, 32) conserved in picornavirus IRESs. The data from Fig. 5 also show a detectable but more marginal influence of the D3 domain (nt 917 to 1013) in IRES function. According to its AUG proximal location, this IRES seems to allow ribosome binding and translation initiation without scanning. This is reminiscent of the so-called type II IRESs described in the literature and including the cardiovirus IRESs (21).
IRES B is located in a 392-nt segment between nt 91 and 483 from the mRNA 5' end and exhibits optimal activity in the presence of nt 91 to 134 (Fig. 7). A 104-bp sequence limited by nt 379 and 483 is strictly necessary for IRES B activity. This sequence contains a predicted stem-loop structure bearing a GNRA motif, defined as an element shared by all picornavirus IRES (21, 32). The observation of such a predicted structure also suggests the presence of an IRES in this region. Although involvement of the motif would need to be confirmed, it seems that VEGF mRNA could be an example of cellular IRES the activity of which would require this unpaired GNRA sequence. Strikingly the 3' border of this IRES was located more than 500 nt from the AUG codon (position 1039). This is reminiscent of the picornavirus type I IRESs, in which the 3' border is located quite far upstream from the start codon (1). However, in IRES B, the spacer region, which does contain a second IRES, is longer than that of the picornavirus type I IRES, which is usually about 150 nt in length. Insertion of AUG codons in the poliovirus type I IRES, upstream from the authentic start codon, has demonstrated that ribosome scanning occurs from the IRES 3' border to reach the AUG codon (17). It cannot be excluded that this could be the case for VEGF IRES B, although the length of the spacer region and its predicted stable structure (Fig. 4) would argue against such a hypothesis. Alternatively, two other hypotheses can be proposed. One possibility is that the spacer region allows a jump between the IRES B and the AUG. Such a mechanism has been described for adenovirus (60) and cauliflower mosaic virus (12). In the case of adenovirus, the 5' part of the mRNA leader allows cap-independent translation to occur, while the 3' part is required for the jump. This hypothesis cannot be ruled out here. Another possibility is that the 5' part of the leader, although it contains an independent IRES, can also behave as an enhancer of IRES A. In this case, the two IRESs would together form a single super-IRES structure. Further investigations are necessary before we can choose between these hypotheses.
However, the existence of two independent IRESs is supported by their very different UV cross-linked protein patterns (Fig. 6). Interestingly, very few proteins are UV cross-linked to IRES A (Fig. 6). One, p100, seems to bind to the D4 domain, suggesting a potential role of this protein in IRES function.
Among the proteins which are bound to the 5' half of the VEGF mRNA leader (Fig. 6), most, including the PTB, are bound on the 5' part of IRES B (Fig. 8A). These experiments did not permit detection of protein whose binding was correlated to IRES B activity. This one-dimensional analysis was not, however, sufficient for conclusions to be drawn in an absence of IRES-specific protein, as different proteins may have the same electrophoretic mobility. Several proteins were bound to a polypyrimidine-rich sequence located between nt 189 and 241 (Fig. 8A; compare lanes B, C, and D). PTB is the major protein bound to the 5' part of the RNA leader, and its binding site was mapped to between nt 134 and 189, which suggests that it recognizes the short UUUC sequence present around position 172 of the leader, which corresponds to the PTB consensus binding site described for viral IRESs (19, 42). According to the predicted structural data, this consensus site is located in a paired sequence. However, whereas PTB plays a crucial role in the function of EMCV and foot-and-mouth disease IRESs (4, 23, 38), its binding to IRES B does not seem to be correlated to IRES activity. These data are consistent with several reports showing that PTB is not the universal internal entry factor and that its binding to RNA does not necessarily imply its requirement for IRES function (3, 24).
The occurrence of two IRESs in the VEGF mRNA, probably controlled by different factors, provides interesting possibilities for the regulation of VEGF expression at the translational level. As VEGF plays a central role in angiogenesis, its expression has to be finely regulated. Two independent IRES domains, sensitive to different environmental conditions or cellular contexts, could permit an additional degree of flexibility in the expression of this growth factor. It should also be mentioned that the VEGF 5' leader presents potential initiation codons located between the two IRESs, in the same open reading frame as the AUG 1039 initiation codon. It could thus be hypothesized that the upstream IRES controls the expression of longer VEGF isoforms, as has been observed for FGF-2 mRNA (53). However, the existence of such alternative VEGF isoforms remains to be demonstrated. It would also be interesting to know whether translation of the various splicing variants of VEGF mRNA is regulated differently by the two IRESs. Indeed, although we have shown that the second IRES does not extend downstream from the AUG codon, we cannot rule out that long-range interactions of IRES A or B with the downstream coding sequence of the messenger could affect or stabilize the IRES structure and influence its ribosome binding efficiency.
The discovery of IRESs in the mRNAs of two major angiogenic growth factors, VEGF and FGF-2, as well as in the mRNA of PDGF, a factor involved in vascular physiology, raises the question as to the possible involvement of this translation initiation mechanism in the control of angiogenesis. We have already shown that FGF-2 expression is translationally activated in response to stress or vascular lesion (8a, 54). Thus, the advantage of IRES-dependent regulation might be to allow an immediate response of the cell to exogenous stimuli. The control of such a process could have important repercussions in cardiovascular disease therapy.
The FGF-2 IRES seems to be constitutively activated in various transformed cell types (54), whereas c-myc is overexpressed in an IRES-dependent manner in Bloom's syndrome cells (58). The observation of an IRES-dependent translational activation of growth factor or proto-oncogene expression in tumors, added to previous descriptions of eIF4E-dependent translation enhancement (25, 27), favors the hypothesis that translational deregulation of gene expression may play a key role in cell transformation. Furthermore, it has been reported that overexpression of VEGF induces cell transformation in cooperation with FGF-2 (16). Thus, the existence of IRESs in both FGF-2 and VEGF mRNAs suggests that the processes leading to cell transformation and/or tumor neovascularization could involve an IRES-dependent activation of the expression of these angiogenic factors.
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ACKNOWLEDGMENTS |
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We thank B. Michot for predicted secondary structures, F. Bayard and J. Plouet for helpful discussions, and D. Warwick for English proofreading. We thank J. Abraham for the gift of the VEGF cDNA and promoter region and J. G. Patton for sending the anti-PTB antibody.
This work was supported by grants from the Association pour la Recherche contre le Cancer, the Ligue Nationale contre le Cancer, the Conseil Regional de Midi-Pyrenées, the European Community Biotechnology program (subprogram Cell Factory, Actions de Recherches Concertées, contract 94/99-181). I. Huez received fellowships from the Ministère de l'Education Nationale et de la Recherche Scientifique. L. Créancier received a fellowship from the European Community Biotechnology program.
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FOOTNOTES |
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* Corresponding author. Mailing address: INSERM U397, Endocrinologie et Communication Cellulaire, Institut Fédératif de Recherche Louis Bugnard, CHU Rangueil, Ave. Jean Poulhès, 31403 Toulouse cedex 04, France. Phone: 33 (5) 61 32 21 44. Fax: 33 (5) 61 32 21 41. E-mail: pratsh{at}rangueil.inserm.fr.
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