A The sequence of the VEGF 5¢-UTR V1 RNA probe is shown and consensus CSD and PTB protein binding site sequences are indicated.. Results The VEGF 5¢-UTR binds cytoplasmic and recombinant
Trang 1A multi-protein complex containing cold shock domain (Y-box)
Potential role in mRNA stabilization
Leeanne S Coles1,*, M Antonetta Bartley1,*, Andrew Bert1, Julie Hunter1, Steven Polyak2, Peter Diamond1, Mathew A Vadas1,3and Gregory J Goodall1,3
1
Division of Human Immunology, The Hanson Institute, Institute of Medical and Veterinary Science;2Division of Biochemistry, Department of Molecular Biosciences, The University of Adelaide;3Department of Medicine, The University of Adelaide,
North Terrace, Adelaide, South Australia, Australia
Vascular endothelial growth factor (VEGF) is a key
regu-lator of angiogenesis and post-transcriptional regulation
plays a major role in VEGF expression Both the 5¢- and
3¢-UTR are required for VEGF post-transcriptional
regu-lation but factors binding to functional sequences within
the 5¢-UTR have not been fully characterized We report
here the identification of complexes, binding to the VEGF
mRNA 5¢- and 3¢-UTR, that contain cold shock domain
(CSD) and polypyrimidine tract binding (PTB) RNA
binding proteins Analysis of the CSD/PTB binding sites
revealed a potential role in VEGF mRNA stability, in both
noninduced and induced conditions, demonstrating a
gen-eral stabilizing function Such a stabilizing mechanism had
not been reported previously for the VEGF gene We further
found that the CSD/PTB-containing complexes are large
multiprotein complexes that are most likely preformed in
solution and we demonstrate that PTB is associated with the
VEGFmRNA in vivo Complexformation between CSD proteins and PTB has not been reported previously Analysis
of the CSD/PTB RNA binding sites revealed a novel CSD protein RNA recognition site and also demonstrated that CSD proteins may direct the binding of CSD/PTB com-plexes We found the same complexes binding to an RNA-stabilizing element of another growth factor gene, suggesting
a broader functional role for the CSD/PTB complexes Finally, as the VEGF gene is also regulated at the tran-scriptional level by CSD proteins, we propose a combined transcriptional/post-transcriptional role for these proteins in VEGF and other growth factor gene regulation
Keywords: cold shock domain proteins; Y-boxprotein; polypyrimidine tract binding protein; mRNA stabilization; vascular endothelial growth factor
VEGF is an essential regulator of angiogenesis that acts on
vascular endothelial cells to induce proliferation and
promote cell migration [1–3] Disregulated VEGF
expres-sion is implicated in a number of diseases that are
characterized by abnormal angiogenesis [1–6] In the case
of solid tumors, the overexpression of VEGF, produced in
response to activated oncogenes, growth factors or low
oxygen conditions (hypoxia), plays a major role in
promo-ting tumor angiogenesis and progression [1–3,7] Both the
cancer cells themselves and nontumor support cells, such as
fibroblasts, are sources of VEGF [8] In contrast, in the case
of coronary artery disease, inadequate VEGF expression rather than VEGF overexpression, plays a role in disease progression A number of cell types, including cardiac myocytes, fibroblasts and endothelial cells produce VEGF
in response to hypoxia, but this natural response is not sufficient to prevent the further progression of heart disease [9–11] It is therefore important to understand the mecha-nisms of VEGF regulation to develop means to control VEGF expression
Post-transcriptional regulation plays a major role in VEGF expression, with regulation occurring at the level of splicing, mRNA stability and translation [2,7] The VEGF mRNA is normally unstable and its stability is increased in response to cytokines and stress conditions such as hypoxia [7,11–14] Regions in both the 5¢- and 3¢-UTR have been shown to be involved in VEGF mRNA stabilization [7,12,13,15–18] The presence of an internal ribosome entry site (IRES) in the VEGF 5¢-UTR ensures continual translation of the VEGF mRNA in stress conditions that normally decrease cap-dependent translation [19–21] Little
is known about the factors involved in VEGF post-transcriptional regulation Factors such as HuR and hnRNPL have been implicated in hypoxic stability via their
Correspondence to L S Coles, Division of Human Immunology,
The Hanson Institute, Institute of Medical and Veterinary Science,
Frome Road., Adelaide, South Australia, 5000, Australia.
Fax: + 61 88 2324092, Tel.: + 61 88 2223432,
E-mail: leeanne.coles@imvs.sa.gov.au
Abbreviations: CSD, cold shock domain; IRES, internal ribosome
entry site; VEGF, vascular endothelial growth factor.
*These authors contributed equally to this work.
(Received 16 October 2003, revised 14 December 2003,
accepted 16 December 2003)
Trang 2actions on the VEGF 3¢-UTR [17,18] but factors involved in
stability or translational regulation have not been identified
on the 5¢-UTR
The single-strand RNA and DNA binding, cold shock
domain (CSD) (also known as Y-box) proteins, play
diverse roles in both transcriptional and
post-transcrip-tional regulation of growth factor and stress response
genes [22–29] CSD proteins have several family members
which are defined by the presence of a central highly
conserved 70 amino acid region called the cold shock
domain [24,25,29] The central domain is required for
sequence-specific RNA binding, while the adjacent
C-terminal domain has a more nonspecific role in
stabilizing binding [24–27] There are two types of
nongerm cell CSD proteins and these are called dbpB
(also known as YB-1, MSY-1, chkYB-1b, EF1A, p50 and
FRGY1) and dbpA (MSY4, chkYB-2 and YB2/RYBa)
DbpB and dbpA CSD proteins are ubiquitously expressed
and are highly conserved across species Highly conserved
germ cell-specific CSD proteins also exist such as MSY-2
and FRGY2 [22–25,29] In addition there are CSD-related
proteins such as UNR (upstream of N-ras) which contains
multiple conserved CSD domains [30] CSD proteins
stabilize growth factor/stress response mRNAs in response
to stress signals [31–33] and also act as general mRNA
stabilizers [34–37] In addition, CSD and CSD-related
proteins have been shown to play a role in cap-dependent
and [26,27,38–42] IRES-dependent [43–46] translation and
in RNA splicing [47,48] In the case of the GM-CSF
(granulocyte-macrophage colony stimulating factor)
growth factor gene, CSD proteins have been shown to
play a combined role at both the transcriptional and
post-transcriptional levels [22,23,49,50] As we have recently
shown a role for CSD proteins in regulation of the VEGF
gene at the transcriptional level [51], and given the diverse
functions of CSD proteins, relevant to VEGF expression,
we investigated a role for CSD proteins in VEGF
post-transcriptional regulation
We now show here that CSD proteins can bind to both
the 5¢- and 3¢-UTR of the VEGF mRNA We find that
CSD proteins form a cytoplasmic complexon VEGF
mRNA that also contains the multifunctional
single-strand RNA/DNA binding protein, PTB [43–46,52–57],
and that the binding of this complexmay be involved in
general VEGF mRNA stabilization The
CSD/PTB-con-taining cytoplasmic complexalso forms on a stability
element in the interleukin-2 (IL-2) 5¢-UTR suggesting a
similar mechanism of regulation of stability of growth
factor mRNAs
Materials and methods
Plasmid constructs
The pGEM44, pGEM46 and pGEM47 constructs were
generated by cloning segments of the mouse VEGF
5¢-UTR, that were amplified by PCR from the pfVEGF
construct [15], into pGEM4Z (Promega) The pGEM44,
46 and 47 constructs contain, respectively, mouse VEGF
5¢-UTR sequences +1 to +325, +461 to +727 and +735 to
+1014 (relative to the transcription start site at +1) [58]
(Fig 1) The pGEMV1 construct, containing the VEGF
5¢-UTR CSD site 1 sequences (+150 to +185) was constructed by cloning double strand oligonucleotides (with EcoRI 5¢- and HindIII 3¢-ends) into pGEM4Z pGEMV37,
39, 15, 17 and 19 were similarly constructed, except that they contained mutant versions of the CSD site 1 sequences
Fig 1 The VEGF 5¢-UTR binds cytoplasmic and recombinant CSD proteins (A) Schematic of the mouse VEGF 5¢-UTR The sequences and coordinates (relative to the mRNA start site +1) [58] of consensus CSD binding sites (CSD site 1,2) are indicated The coordinates of RNA probe sequences are also indicated RNA probes were derived from pGEM44, pGEM46 and pGEM47, respectively (B) Balb/c 3T3 fibroblast cytoplasmic extracts were incubated without competitor (–)
or with unlabeled single-strand DNA competitor oligonucleotides containing wild-type (CSDwt) and mutant (CSDmut) CSD binding sites [49–51] 32 P-Labeled RNA probes (44 and 46) were then imme-diately added and RNase T1 digested complexes analyzed by gel shift assay Cytoplasmic complexes CC44a, CC44b and CC46 and unbound RNA probe are indicated (C) Cytoplasmic extracts were preincubated with anti-CSD polyclonal Ig (CSD), with preimmune serum (PI) or left untreated (–), followed by addition of the labeled VEGF 44 RNA probe in a gel shift assay Increasing amounts of anti-CSD Ig were added Pre-immune sera was used at the maximal concentration used for the anti-CSD Ig Cytoplasmic complexes CC44a and CC44b are indicated (D) Recombinant GST-dbpB/YB-1 was incubated with labeled 44, 46 and 47 RNA probes Complexes were competed with wild-type (CSDwt) or mutant (CSDmut) CSD binding site single-strand DNA competitors or left untreated (–).
Trang 3(Figs 2 and 3) pGEMV25 and pGEMV27 were
construc-ted by cloning wild-type and mutant double strand
oligo-nucleotides containing the IL-2 5¢-UTR +1 to +35
sequences [31] (Fig 4) pGEMVC1 was constructed by
cloning double strand oligonucleotides containing the
VEGF 3¢-UTR CSD site 3 (+1712 to +1747, relative to
the stop codon at +1, of the mouse VEGF 3¢-UTR) [59]
into pGEM4Z pGEMVC2 and VC3 contained mutations
in the CSD site 3 sequence (Fig 6)
The pfVEGF construct contained a reconstructed, tagged VEGF cDNA sequence [15], composed of the entire VEGF mouse 5¢-UTR (+1 to +1014), the coding region for the 164 amino acid form of VEGF and the VEGF 3¢-UTR (+4 to +2195) The major polyadenylation site is at +1861 [59] A
Fig 2 The VEGF 5¢-UTR CSD-containing cytoplasmic complexes also
contain PTB (A) Schematic of the VEGF 5¢-UTR CSD site 1 The
coordinates for the VEGF RNA probes 44 and V1 are indicated
rel-ative to the start of the VEGF mRNA (+1) [58] (B) Balb/c 3T3
fibroblast cytoplasmic extract was incubated with labeled VEGF 44 or
V1 RNA probes, followed by RNase T1 digestion in a gel shift assay.
The 44 and V1 RNA probes are derived from pGEM44 and
pGEMV1, respectively Cytoplasmic complexes CC44a and CCV1
and unbound RNA probe are indicated (C) Cytoplasmic complexes
CC44 and CCV1, in gel shift assay gels, were exposed to UV light to
cross-link proteins in each complexto RNA Cross-linked proteins
were then analyzed by SDS/PAGE and the sizes of cross-linked
pro-teins were calculated by subtraction of the molecular weight of bound
RNA probe The sizes of cross-linked proteins are indicated
Cross-link analysis is not quantitative as different proteins will crossCross-link to
different extents (D) Cytoplasmic extracts were incubated with
unlabe-led wild-type or mutant CSD (CSDwt, mut) or PTB (PTB wt, mut) [52]
binding site single-strand DNA oligonucleotides, or left untreated (–).
Labeled V1 RNA probe was then immediately added and complexes
analyzed in a gel shift assay The CCV1 complexis indicated (E)
Cytoplasmic extracts were preincubated with an anti-PTB monoclonal
antibody (PTB), with a control anti-GM-CSF monoclonal antibody
(GM), or without antibody (–) Labeled V1 RNA probe was then
added and complexes analyzed in a gel shift assay.
Fig 3 Sequence requirement for VEGF 5¢-UTR CCV1 CSD/PTB complex formation-PTB complexes form on the VEGF mRNA in vivo (A) The sequence of the VEGF 5¢-UTR V1 RNA probe is shown and consensus CSD and PTB protein binding site sequences are indicated The 3¢ consensus PTB site is also found, in this report, to bind recombinant CSD protein (labeled CSD*) The sequences of mutant RNA probes are given under the V1 sequence Only those bases that are changed in the mutant probes are indicated The RNA probes were generated from pGEMV1, V37, V39, V15, V17 and V19 constructs, respectively (B) Balb/c 3T3 fibroblast cytoplasmic extracts were incubated with labeled wild-type (V1) and mutant VEGF 5¢-UTR CSD site 1 RNA probes in a gel shift assay The CCV1 cytoplasmic complex
is indicated (C) Recombinant GST-dbpB/YB-1 and GST-PTB were incubated with labeled wild-type (V1) and mutant (V37, V39) VEGF 5¢-UTR RNA probes in a gel shift assay The recombinant protein complexes are indicated (D) PTB binding to VEGF mRNA in vivo was investigated using an RNA immunoprecipitation assay Cytoplasmic RNA/protein complexes (prepared in the presence of RNase inhibi-tors) were immunoprecipitated with anti-PTB monoclonal Ig (PTB), with an IgG2 isotype control (control), or without antibody (–) VEGF mRNA in RNA extracted from immunoprecipitated complexes was detected by RT-PCR The VEGF PCR product is indicated.
Trang 4polylinker is positioned between the coding region and the
3¢-UTR sequences to distinguish pfVEGF mRNA from
endogenous VEGF mRNA pfVEGFdel contains deletions
of the site 1, 2 and 3 CSD sites (Fig 7) The sequences +156
to +179 (CSD site 1) and +650 to +666 (CSD site 2) of the
5¢-UTR were deleted and the sequences +1727 to +1740
(CSD site 3) of the 3¢-UTR were deleted
Construction of the expression vector producing
recom-binant GST-dbpB/YB-1 (pGEXBT) has been described
previously [49,51] pGEXPTB, for production of
recombin-ant GST-PTB, was constructed by cloning of a 1.6kb EcoRI
fragment from pcDNA3PTB (gift from T Cooper, Baylor
College of Medicine, Houston, TX, USA), coding for
human PTB, into pGEX4T-2
Oligonucleotides Oligonucleotides for cloning into pGEM4Z and for use as competitors in gel shift assays were synthesized by Gene-works (Adelaide, Australia) and purified from nondenatur-ing polyacrylamide gels Snondenatur-ingle-strand oligonucleotides for competition of CSD protein-containing complexes were from the human granulocyte-macrophage-colony stimula-ting factor (GM-CSF) gene The wild-type (CSDwt) and mutant sequences (CSD site mutant; CSDmut) have been described previously (GM- and GMm23-, respectively) [49–51] The CSD wild-type sequence binds both dbpA and dbpB CSD proteins The wild-type (PTBwt) and mutant PTB (PTBmut) competitor single-strand DNA oligonucleo-tides are from the transferrin gene (DR1 sense and DR1 sense mut1, respectively) [52]
RNA probe preparation
32P-labeled RNA probes for gel shift analysis or RNase protection assays were generated by in vitro transcription from linearized plasmid templates (pGEM4Z constructs) using SP6 (for pGEM44,46,47) or T7 (for pGEMV1, V25 and VC1) RNA polymerase (Promega) and [32P]UTP[aP] Probes for RNase protection assays were processed as previously described [15] Probes for gel shift assays were purified from nondenaturing polyacrylamide gels and eluted into RNase-free water at 56C
Preparation of recombinant and cytoplasmic proteins The Escherichia coli strain MC1061 transformed with pGEXBT or pGEXPTB was induced with isopropyl thio-b-D-galactoside to produce recombinant GST-dbpB/YB-1 and GST-PTB [49,51] The fusion proteins for gel shift analysis were purified on glutathione-Sepharose beads (Promega) Cytoplasmic extracts were produced according
to the method of Schrieber et al [60]
FPLC gel filtration of cytoplasmic extracts Cytoplasmic extract from Balb/c 3T3 fibroblasts was applied at a flow rate of 0.35 mLÆmin)1to a Superdex200 column (10 mm diameter, 20 mL bed volume) pre-equili-brated with buffer containing 150 mM KCl, 20 mM Tris/ HCl pH 7.6, 20% glycerol, 1.5 mMMgCl2, 2 mM dithio-threitol, 0.4 mMphenylmethanesulfonyl fluoride and 1 mM
Na3VO4 The CCV1 complexwas eluted with the same buffer and 0.5 mL fractions collected The molecular mass
of the complexwas estimated from the column by comparison with the elution volumes of c-globulin, bovine serum albumin, ovalbumin, myoglobin and vitamin B12 Antibodies
The anti-CSD antibody is a rabbit polyclonal Ig raised against a peptide conserved in dbpA and dbpB/YB-1 CSD proteins across species [49,51] The anti-PTB Ig is a mouse monoclonal antibody (BB7; gift from D Black, UCLA, Los Angeles, CA, USA) A mouse monoclonal anti-(GM-CSF) Ig (gift from A Lopez, Hanson Institute, IMVS, Adelaide, Australia) and an IgG2 monoclonal
Fig 4 CCV1 complex formation on the IL-2 5¢-UTR stability element
in fibroblasts and Jurkat T cells (A) Sequence of the IL-2 5¢-UTR
wild-type probe V25 with consensus CSD and PTB sites indicated The
region +1 to +22 is involved in IL-2 mRNA stabilization in T cells
[31] The V27 mutant sequence is shown with only those bases differing
from the wild-type sequence shown (B) Balb/c 3T3 fibroblast
cyto-plasmic extracts were incubated with labeled VEGF (V1) and IL-2
(V25,V27) 5¢-UTR RNA probes, and analyzed by gel shift The CCV1
cytoplasmic complexis indicated (C) The Balb/c 3T3 CCV1
com-plexes binding to the VEGF (V1) and IL-2 (V25) RNA probes (after
RNase T1 digestion) were exposed to UV light, in gel shift gels, to
cross-link proteins to RNA Cross-linked proteins were sized by SDS/
PAGE The sizes of cross-linked proteins were calculated by
subtrac-tion of molecular masses of bound RNA probes (D) Balb/c 3T3
fibroblast and Jurkat T cell cytoplasmic extracts were incubated with
labeled VEGF (V1) and IL-2 (V25) RNA probes, digested with RNase
T1 and analyzed in a gel shift assay The CCV1 cytoplasmic complexis
indicated (E) The Jurkat T cell CCV1 cytoplasmic complexbinding to
the IL-2 V25 RNA probe was analyzed by UV cross-link analysis as
described above The sizes of cross-linked proteins are indicated.
Trang 5antibody isotype control (Silensus, Boronia, Victoria,
Aus-tralia) were used as controls for the anti-PTB antibody in gel
shift assays and RNA immunoprecipitations, respectively
RNA gel shift analysis, competitions and antibody
analysis
RNA gel shifts were performed using 32P-labeled RNA
probes in a 10 lL reaction mixof 0.5· TM buffer [49–51]
containing 200 mMKCl, 1 lg poly(dI.dC), 100 ng tRNA,
1 lg bovine serum albumin and either 1 lg cytoplasmic
extract or 25 ng recombinant protein (dbpB or
GST-PTB) Reactions were incubated at 4C for 20 min,
followed by treatment with or without RNase T1
(Worth-ington Biochemical Corp., NJ, USA) and analyzed on
6% nondenaturing polyacrylamide gels Competition with
single-strand DNA oligonucleotides was performed by
addition of protein and 50 ng of unlabeled probe, followed
by immediate addition of the 32P-labeled RNA probe
Antibody blocking experiments were performed by
incuba-ting protein and antibody for 5 min before adding the
32P-labeled probe Antibodies did not degrade RNA probes
under the gel shift conditions used
UV cross-linking
Cytoplasmic extracts were bound to 32P-labeled RNA
probes in a 25 lL gel shift reaction and fractionated on a
6% polyacrylamide gel as described above The gel was
exposed to UV light (340 nm) for 15 min and retarded
complexes were excised after exposure overnight to X-ray
film Protein in excised bands was analyzed by 12% SDS/
PAGE as described previously [49–51]
RNA immunoprecipitation assay
Balb/c 3T3 fibroblast extracts were prepared, as described
above, in the presence of RNase inhibitors (Promega) and
incubated with or without anti-PTB monoclonal Ig or with
an IgG2 isotype control for 60 min RNase inhibitors were
required to prevent the loss of RNA from extracts Protein
A sepharose CL-4B (Pharmacia, Biosciences, Uppsala,
Sweden) was added and further incubated for 60 min
Sepharose was extracted for bound RNA (TRIzol
reagent, Invitrogen) RNA was reverse transcribed using
Superscript II (Promega) and a PCR assay for VEGF
cDNA was performed using oligonucleotides from the
mouse VEGF cDNA of 5¢-CACAGACTCGCGTTGCA-3¢
and 5¢-TGGGTGGGTGTGTCTAC-3¢ PCR products
were analyzed by agarose gel electrophoresis The VEGF
PCR product is approximately 400 bp
Cell culture, stable transfection and cell stimulation
Mouse Balb/c 3T3 fibroblasts and rat C6 glioma cells were
grown in Dulbecco’s modified Eagle’s medium with 10%
fetal bovine serum Jurkat T cells were cultured in RPMI
media with 10% fetal bovine serum For cytoplasmic
extracts, cells were grown in normoxic conditions (normal
oxygen; 20% O2) For the production of stably transfected
cell lines, C6 glioma cells were transfected with linearized
pfVEGF or pfVEGfdel plasmids using lipofectamineTM
2000 (Gibco BRL Life Technologies, Melbourne, Australia) according to the manufacturer’s directions Cells were grown for 24–48 h and selected in 400 lgÆmL)1G418 [15] Serum stimulation of stably transfected cell lines was as described previously [15] Hypoxic conditions (1% O2) were generated in a hypoxic chamber (Edwards Instrument Company, Sydney, Australia)
Analysis of mRNA stabilityin vivo Stable transfectants (pfVEGF or pfVEGFdel) were serum stimulated (time 0) and concurrently incubated under normoxic or hypoxic conditions for 1, 1.5, 2, 3 or 4 h Serum stimulation provides a brief pulse of transcription from the c-fos promoter in pfVEGF/pfVEGFdel con-structs, allowing subsequent degradation of the mRNA to
be monitored as previously described by us in analysis of the pfVEGF construct [15] This system allows determination
of mRNA stability directly, rather than using indirect means such as nonspecific inhibitors of transcription RNA was isolated from treated cells using TRIzolrreagent (Invitro-gen) according to the manufacturers instructions, and pfVEGF/pfVEGFdelmRNA was detected by RNase pro-tection analysis using a32P-labeled transcript covering the polylinker sequence in the pfVEGF/pfVEGFdel constructs
as previously described [15] Neomycin phosphotransferase (neo) mRNA expressed from pfVEGF/pfVEGFdel con-structs was detected as described [15] Protected RNAs were separated on denaturing polyacrylamide gels and the amounts of specific32P-labeled protected pfVEGF/pfVEGF-del or neo mRNAs were quantitated by PhosphoImager analysis (Molecular Dynamics, Sunnyvale, CA, USA) Levels of pfVEGF/pfVEGFdel mRNA (with time 0 levels subtracted) were normalized with respect to the levels of neo mRNA at each time point
Results
The VEGF 5¢-UTR binds cytoplasmic and recombinant CSD proteins
Sequence specific RNA binding sites for CSD proteins have been determined in a few genes but a consensus sequence has not been established Analysis of the prota-mine 1 (Prm1) 3¢-UTR has revealed a preferred binding site
of 5¢-U/C/A–C/A–C–A–U/C–C–A/C/U-3¢ for mouse CSD proteins [38–40] This sequence is consistent with a prefer-red sequence for Xenopus CSD proteins (FRGY1/2) of 5¢-AACAUCU-3¢ [61] and with a 5¢-ACCACC-3¢ sequence from the Rous Sarcoma virus LTR that binds chicken CSD proteins [41]
Given a potential role for CSD proteins in VEGF post-transcriptional regulation, the VEGF 5¢-UTR was exam-ined for CSD protein binding sites Two potential sites at +157 and +650 were observed These were named CSD site
1 and CSD site 2 and have sequences of 5¢-AACCU CU-3¢ and 5¢-AACUUCU-3¢, respectively (Fig 1A) No other potential CSD protein binding sequences were observed
To determine if the VEGF 5¢-UTR could bind cytoplas-mic CSD complexes,32P-labeled RNA probes 44 (+1 to +325) and 46 (+461 to +727) covering the potential CSD sites (Fig 1A) were bound to cytoplasmic extracts from
Trang 6Balb/c 3T3 fibroblasts and analyzed by gel shift assay
(Fig 1B) The 44 and 46 probes formed strong complexes
with cytoplasmic proteins (CC44a, b and CC46,
respect-ively) and these complexes were readily competed by a
single-strand DNA oligonucleotide (CSDwt) from the
GM-CSFgene that is known to bind CSD proteins [49,50] These
complexes were less readily competed by a mutant version
of the GM-CSF CSD oligonucleotide (CSDmut),
suggest-ing the presence of CSD proteins (Fig 1B) In support of
this, formation of complexes on the 44 RNA probe were
blocked by preincubation of extracts with increasing
amounts of an anti-CSD polyclonal Ig (CSD), whereas
preimmune serum (PI) had no effect (Fig 1C) Cytoplasmic
complexes containing CSD proteins can therefore bind the
VEGF 5¢-UTR
To further support a role for CSD proteins binding the
VEGF 5¢-UTR, it was observed that a recombinant CSD
protein GST-dbpB/YB-1 could bind to both the 44 and 46
RNA probes but not to a probe which does not contain
a potential CSD site (probe 47; +735 to +1014) The 47
probe contains sequences required for mouse VEGF IRES
activity [19,20] As for cytoplasmic complexes, GST-dbpB/
YB-1 binding was specifically competed by the CSD wt
oligonucleotide (Fig 1D) Both cytoplasmic and
recombin-ant CSD protein complexes are therefore forming on the
VEGF 5¢-UTR
The VEGF 5¢-UTR CSD-containing cytoplasmic complexes
also contain PTB
To localize the binding site for the major complexon the 44
RNA probe (CC44a), Balb/c 3T3 fibroblast cytoplasmic
extract was bound, in a gel shift assay, to a shorter
32P-labeled RNA probe, containing the proposed CSD
binding site 1 (V1; +150 to +185, Fig 2A) A single major
complex(CCV1) formed on the V1 probe and migrated in a
similar position to the CC44a complexon the 44 RNA
probe (Fig 2B) To verify that the CC44a and CCV1
complexes were the same, complexes were analyzed by UV
cross-linking (Fig 2C) The CC44a and CCV1 complexes,
that had been separated in a gel shift gel, were exposed
to UV light to cross-link proteins in complexes to their
respective RNA probes Cross-linked proteins were then
separated by SDS/PAGE and the sizes of cross-linked
proteins were calculated by subtraction of molecular masses
of bound RNA fragments The number and size of proteins
cross-linked to RNA was identical for the 44 and V1 RNA
probes We similarly found that the 46 RNA probe binding
complex(CC46), that contains the CSD site 2, gave an
identical cross-link pattern (data not shown)
As expected the CSD-containing cytoplasmic complexes
binding to the CSD site 1 (and CSD site 2) contained a
protein of 50 kDa, consistent with the size of the CSD
protein, dbpB (also known as YB-1/p50) [42] Additional
proteins in the complexhad sizes of 60, 27 and 12 kDa The
single CSD-containing cytoplasmic RNA/protein complex
therefore contains at least four different proteins It has been
reported that PTB, another single-strand RNA/DNA
binding protein [43–46,52–57] can bind to a 50 base region
spanning the CSD site 1 sequence in the human VEGF
5¢-UTR [21] Given that we have detected a 60 kDa protein
of the approximate size for PTB (57 kDa), we further
investigated the CSD site 1, CCV1 complex The presence of PTB protein in the CCV1 complexwas confirmed in a gel shift assay, by competition of the CCV1 complexwith an unlabeled single-strand DNA oligonucleotide probe from the transferrin gene (PTB wt), that binds PTB [52] The CCV1 complexwas not readily competed by a transferrin gene oligonucleotide with a mutant PTB site (PTB mut) (Fig 2D) We confirmed using recombinant proteins that the PTB wt oligonucleotide could not bind CSD proteins and hence was specific for PTB (data not shown) The presence of PTB was further confirmed by preincubation of cytoplasmic extract with an anti-PTB monoclonal Ig (PTB) before binding to V1 RNA probe in a gel shift assay The formation of the CCV1 complexwas blocked by the anti-PTB Ig (anti-PTB) but not by an irrelevant monoclonal antibody (anti-GMCSF; GM) (Fig 2E)
Taken together, this data demonstrates that the CSD-containing cytoplasmic complexforming on the VEGF 5¢-UTR contains PTB in addition to further unknown proteins of 27 and 12 kDa The ability of anti-CSD and PTB antibodies and DNA competitors to effectively compete complexformation demonstrates the dependence
on the presence of both CSD and PTB proteins to form the CCV1 complex
VEGF 5¢-UTR CCV1 complex formation requires the consensus 5¢-ACCUCUU-3¢ sequence and a downstream 5¢-UUUUCUU-3¢ sequence
To determine the sequences required for CCV1 complex formation on the VEGF 5¢-UTR CSD site 1 RNA probe (V1), Balb/c 3T3 fibroblast cytoplasmic extract was bound
to mutant versions of the V1 probe (Fig 3A) and analyzed
in a gel shift assay (Fig 3B) Mutations were made in the predicted CSD protein binding site, 5¢-ACCUCUU-3¢, and also in an adjacent sequence containing a potential PTB site 5¢-UUUUCUU-3¢ 5¢-UCUU-3¢ sequences flanked by pyrimidine residues are commonly found in PTB binding sites [52–55,57] Mutation of either sequence, by a block mutation (V37, V39) or by mutation of the central UC residues to AA (V15, V17) reduced CCV1 binding, suggesting a role for both sequences in complexformation Consistent with this, a double mutation (V19) abolished CCV1 complexformation (Fig 3B)
To investigate the individual roles CSD and PTB proteins may play in directing CCV1 complexbinding to the 5¢-ACCUCUU-3¢ and 5¢-UUUUCUU-3¢ sequences, recombinant CSD dbpB/YB-1) and PTB (GST-PTB) binding to wild-type (V1) and mutant (V37, V39) RNA probes was examined (Fig 3C) Consistent with CCV1, GST-dbpB/YB-1 binding was reduced by mutation
of both sites (V37, V39) whereas PTB binding was only slightly reduced by mutation of the 3¢-pyrimidine-rich sequence (V39) The presence of the adjacent poly U stretch may provide an alternative contact site for the recombinant PTB protein CSD proteins may play a larger role in directing CCV1 complexformation than PTB via their ability to bind both the predicted CSD site, 5¢-ACCUC UU-3¢, and the downstream 5¢-UUUUCUU-3¢ sequence required for CCV1 complexformation
To confirm that PTB-containing complexes can form on the VEGF mRNA in vivo an RNA immunoprecipitation
Trang 7assay was performed Cytoplasmic RNA/protein complexes
from Balb/c 3T3 fibroblasts were immunoprecipitated with
an anti-PTB monoclonal antibody and RNA extracted
from immunoprecipitated complexes was assayed by
RT-PCR for mouse VEGF mRNA sequences (Fig 3D)
Cytoplasmic extracts, for immunoprecipitation, were made
in the presence of RNase inhibitors to prevent RNA loss
VEGFmRNA was readily detected by RT-PCR in samples
immunoprecipitated with anti-PTB monoclonal Ig (PTB)
VEGFmRNA was not, however, detected in
immunopreci-pitations performed with an IgG2 monoclonal antibody
isotype control or without the addition of antibody (–)
An IL-2 5¢-UTR stability element binds the same
cytoplasmic complex as the VEGF 5¢-UTR
The IL-2 5¢-UTR contains sequences, at +1 to +22, that are
required for mRNA stabilization in T cells Both
dbpB/YB-1 and another RNA binding protein, nucleolin, bind to this
region and are involved in stabilization [31] Inspection of
the IL-2 5¢-UTR stability element revealed a sequence,
5¢-ACUCUCUU-3¢, at +4 to +11, that was very similar to
the VEGF CSD site 1 CSD consensus sequence (Fig 4A)
The ability of the IL-2 sequence to bind the CCV1 complex
was tested in a gel shift assay using Balb/c 3T3 fibroblast
cytoplasmic extract (Fig 4B) An RNA probe containing
the +1 to +35 IL-2 5¢-UTR sequences (V25) bound a
similarly migrating complexto that observed on the VEGF
V1 probe (CCV1), and this complexwas abolished by
mutation of the 5¢-ACUCUCUU-3¢ sequence (V27) The
V27 block mutation is reported to reduce IL-2 mRNA
stability in T cells [31] UV cross-link analysis of the IL-2
complexrevealed that it was identical to the VEGF 5¢-UTR
CCV1 complex(Fig 4C) An identical complexfrom
fibroblast extracts can therefore form on both the VEGF
and IL-2 genes
For the CCV1 complexto be of relevance to the
regulation of expression of the IL-2 gene, it was important
to determine if the complexcould be formed using T cell
extracts Binding of the IL-2 V25 probe to Jurkat T cell
cytoplasmic extracts revealed the formation of a complex
comigrating with the fibroblast CCV1 complex(Fig 4D)
UV cross-linking demonstrated that the fibroblast and
T-cell complexes were identical (Fig 4E) The CCV1
complextherefore forms on a functional element in the
IL-2 5¢-UTR in T cells
The VEGF 5¢-UTR CCV1 complex may be preformed
To determine if cytoplasmic CSD/PTB-containing VEGF
5¢-UTR complexes can form in the absence of RNA, Balb/c
3T3 fibroblast extract was fractionated by FPLC gel
filtration and the fractions incubated with wild-type (V1)
or mutant (V19; Fig 3) VEGF CSD site 1 RNA probes in a
gel shift assay (Fig 5) CCV1 complexformation, binding
to the V1 probe, was observed in fractions with an
approximate molecular mass range of 400–490 kDa
(frac-tions 6, 7) No other complexes were observed across the
range of fractions analyzed (from 10 to 1000 kDa) (data not
shown) CCV1 complexformation, in fractions 6 and 7, was
abolished by the V19 mutation, verifying the nature of these
complexes (Fig 5) These data indicate that the CCV1
complexis preformed in solution The presence of a higher order preformed complexsuggests the possibility that CSD and PTB may interact Consistent with this, CSD proteins have been shown to interact with a number of partner proteins in solution [22,23]
Involvement of the VEGF 3¢-UTR in binding CSD and PTB proteins
As sequences in the 3¢-UTR are also involved in post-transcriptional regulation of VEGF expression, the mouse VEGF 3¢-UTR [59] was examined for potential CSD protein binding sites A single site at +1727, relative to the stop codon at +1, was observed with a sequence of 5¢-AACAUCA-3¢ This sequence is an exact match to the preferred binding site for mouse CSD proteins [38,40] and,
as for the VEGF 5¢-UTR sites, has a potential PTB site, 5¢-UCUU-3¢, immediately downstream at +1736 (Fig 6A)
We used gel shift assays to examine whether a CSD/PTB complexbinds to this region in the 3¢-UTR An RNA probe (VC1) containing sequences +1712 to +1747 of the mouse VEGF 3¢-UTR was bound to Balb/c 3T3 fibroblast cytoplasmic extracts and two major complexes were observed The faster migrating complex(CCVC1) was competed more readily with wild-type (wt) than mutant (mut) PTB and CSD protein binding oligonucleotides, suggesting that this complexcontains PTB and CSD proteins (Fig 6B) As expected, mutation of either the potential CSD site (VC2) or the PTB site (VC3) reduced the formation of the CCVC1 complex(Fig 6C) Consistent with this, recombinant GST-PTB and GST-dbpB/YB-1 bound to the VC1 probe and PTB and CSD protein binding were reduced by mutations in the PTB (VC3) and CSD protein (VC2) binding sites, respectively As was observed for the 5¢-UTR, CSD binding was also reduced by mutation
of the potential PTB site (VC3) (Fig 6D)
Fig 5 Gel filtration fractionation of the VEGF 5¢-UTR CCV1 com-plex Balb/c 3T3 fibroblast cytoplasmic extract was fractionated by FPLC gel filtration and fractions assayed by incubation with labeled VEGF 5¢-UTR wild-type (V1) and mutant (V19) RNA probes in a gel shift assay Consecutive 0.5 mL fractions containing CCV1 binding activity are shown The approximate sizes of protein fractions, deter-mined by comparison to elution profiles of protein standards, is given
in kDa The CCV1 complexis indicated.
Trang 8Hence as for the 5¢-UTR, both the potential CSD and
PTB 3¢-UTR sites are required for cytoplasmic complex
(CCVC1) formation, with recombinant PTB primarily
contacting the downstream PTB site and CSD protein
contacting both sites
TheVEGF mRNA CSD/PTB binding sites play a role
inVEGF mRNA stability
The binding of common cytoplasmic complexes to the IL-2
5¢-UTR stability element and the VEGF CSD/PTB sites,
suggested a possible role for these sites in VEGF mRNA
stability CSD proteins have been shown to be involved in
both inducible mRNA stabilization, as is observed for the
IL-2 mRNA [31–33], and general mRNA stabilization
[34–37] PTB proteins may also play a role in general
mRNA stabilization [62]
To investigate a role for the VEGF mRNA CSD/PTB
sites, we analyzed the in vivo stability of mRNAs produced
from constructs containing tagged VEGF mRNA coding
Fig 6 CSD and PTB proteins bind to the VEGF 3¢-UTR (A)
Sequence of the VEGF 3¢-UTR CSD site 3 RNA probe (VC1) The
sequences represent +1712 to +1747 of the mouse VEGF 3¢-UTR,
relative to the stop codon at +1 [59] Consensus CSD and PTB binding
sequences are indicated CSD* indicates that recombinant CSD
pro-tein can also contact the PTB site Mutant RNA probe sequences
(VC2, VC3) are shown with only those bases that differ from the wild
type indicated RNA probes were generated from pGEMVC1, VC2
and VC3 constructs (B) Balb/c 3T3 fibroblast cytoplasmic extract was
incubated with wild-type and mutant CSD (CSDwt,mut) or PTB
(PTBwt,mut) binding site single-strand DNA competitors or left
untreated (–) Labeled VC1 probe was then immediately added and
complexes analyzed in a gel shift assay The CCVC1 complex and
unbound RNA probe are indicated (C) Cytoplasmic extracts were
incubated with VEGF 3¢-UTR wild-type (VC1) and mutant (VC2,
VC3) RNA probes and analyzed in a gel shift assay (D) Recombinant
GST-dbpB/YB-1 and GST-PTB were incubated with VEGF 3¢-UTR
wild-type (VC1) and mutant (VC2,VC3) RNA probes Recombinant
complexes are indicated.
Fig 7 Deletion of the VEGF CSD/PTB sites affects VEGF mRNA stability in normoxic and hypoxic conditions (A) Diagrammatic rep-resentation of the pfVEGF [15] and pfVEGFdel constructs The c-fos promoter and sequences encoding the mouse VEGF mRNA are indicated A poly linker is located between the protein coding and 3¢-UTR sequences as a tag for detection of pfVEGF and pfVEGFdel mRNA The sequences deleted from the CSD sites 1 (+156 to +179),
2 (+650 to +666) and 3 (+1727 to +1740) in the pfVEGFdel con-struct are indicated with dashes (B) Stable transfectants, containing pfVEGF or pfVEGFdel were serum stimulated at time 0 to induce a brief pulse of RNA expression from the c-fos promoter from which mRNA degradation can be followed [15] Cells were simultaneously treated (at time 0) under normoxic or hypoxic conditions and the levels
of pfVEGF/pfVEGfdel mRNA expressed from constructs determined
by RNase protection assay mRNA levels were normalized with respect to neo mRNA Serum stimulation increased transfected mRNA levels approximately 10-fold in both the pfVEGF and pfVEGFdel stable transfectants and was maximal at the 1 h time point The levels of pfVEGF and pfVEGFdel mRNAs were approxi-mately 1.7-fold the levels of endogenous VEGF mRNA in respective cell lines at this time (data not shown) The percentage mRNA remaining, relative to the mRNA levels at the 1 h time point (given as 100%), is shown as a linear plot for experiments performed under normoxic and hypoxic conditions Data is the average of five experi-ments RNase protection gel data is shown below the linear plot for one representative experiment Three repeats were performed for each time point The pfVEGF, pfVEGFdel and neo transcripts are indi-cated (C) Presentation of data in (B) as a log plot.
Trang 9sequences with either wild-type or deleted CSD/PTB sites
(pfVEGF and pfVEGFdel, respectively) (Fig 7A) We have
reported previously the use of pfVEGF in stabilization
experiments [15] pfVEGF or pfVEGFdel stable
transfect-ants were serum stimulated and simultaneously exposed to
hypoxic or normoxic conditions Transfected wild-type or
mutant VEGF mRNA levels were then assayed by RNase
protection assay at time intervals following serum
stimula-tion to determine the stability of respective mRNAs
(Fig 7B,C)
It can be seen in Fig 7(B,C) that degradation of the
wild-type VEGF mRNA (pfVEGF) occurs less rapidly than for
the mutant mRNA (pfVEGF del) under both normoxic and
hypoxic conditions, indicating that the CSD/PTB sites play
a role in stabilizing the VEGF mRNA in both noninduced
and hypoxia-induced conditions The wild-type pfVEGF
mRNA is 1.3-fold more stable than the CSD/PTB site
deleted mRNA Mutation of the CSD/PTB sites, however,
had no effect on the ability of hypoxia to increase mRNA
stability relative to that seen under normoxic conditions
Both wild-type and mutant VEGF mRNAs were stabilized
approximately 1.4-fold by hypoxia The VEGF CSD/PTB
sites therefore are not involved in induced stabilization in
response to hypoxia but appear to be involved in general
stabilization of the VEGF mRNA Interestingly the
pres-ence of the CSD/PTB sites confers a similar degree of
increased stability to the VEGF mRNA to that produced
under hypoxic conditions
Discussion
Inappropriate or inadequate expression of VEGF plays a
key role in the progression of a number of diseases [1–6] It is
therefore important to determine the processes involved in
regulation of VEGF expression We had previously shown
that cold shock domain (CSD) (or Y-box) proteins
regu-lated VEGF expression at the transcriptional level in the
nucleus [51] We show here that CSD proteins may also play
a role in post-transcriptional regulation of VEGF
expres-sion in the cytoplasm, in conjunction with another
single-strand RNA/DNA binding protein, PTB
Conserved CSD/PTB binding sites in theVEGF mRNA
5¢- and 3¢-UTR
The 5¢- and 3¢-UTR of the VEGF mRNA are involved in
post-transcriptional regulation [7,11–15,17,17–21] and we
have identified CSD/PTB protein binding sites in both
these regions Two sites were found in the 5¢-UTR (CSD
sites 1 and 2) and one site was found in the 3¢-UTR (CSD
site 3) (Fig 8A) All three sites contain a sequence that is
similar to a preferred RNA binding sequence determined
for the mouse CSD proteins MSY1, 2 and 4 [38–40] This
preferred sequence is consistent with the RNA binding
sites identified for chicken, frog and human CSD proteins
[31,41,47,61] (Fig 8B) The VEGF sequences all show a
substitution of the fourth position of the preferred mouse
sequence from an A to a C or U residue Potential PTB
binding sequences, 5¢-UCUU-3¢ (flanked by U/C residues)
[52–55,57], were located immediately downstream of the
consensus CSD sequences in both the 5¢- and 3¢-UTR
CSD/PTB sites An additional potential PTB site
over-lapped the consensus CSD sequence in the VEGF 5¢-UTR site 1 (Fig 8A)
Consistent with the presence of potential PTB binding sites, cytoplasmic complexes binding to the VEGF 5¢- and 3¢-UTR sites contained both CSD and PTB proteins and it was observed that both the consensus CSD sequence and the downstream PTB sequence, within these sites, were required for full complexformation The ability of both antibody and oligonucleotide competitors to reduce or abolish complexformation demonstrated that CSD and PTB proteins were simultaneously bound to VEGF RNA The ability of CSD and PTB proteins to bind to the 5¢- and 3¢-UTR sequences was further demonstrated by the binding
of recombinant PTB and dbpB/YB-1 CSD protein to these sites Importantly, we found that PTB-containing com-plexes could be detected on the VEGF mRNA in vivo PTB binding was primarily affected by the mutation of the downstream PTB consensus sequences, while surprisingly, recombinant CSD binding was affected by mutation of either the consensus CSD site or the downstream PTB sequence CSD proteins therefore recognize not only the expected consensus RNA sequence but also the VEGF PTB binding sequences The binding of CSD proteins to PTB
Fig 8 Comparison of CSD protein RNA binding sites (A) The sequences of the VEGF 5¢- and 3¢-UTR CSD site 1, 2 and 3 sequences are shown and consensus CSD (5¢) and PTB (3¢) protein binding sites are underlined Both sequences are required for cytoplasmic CSD/PTB complexformation and recombinant CSD protein binding (B) A comparison of CSD protein RNA binding sites is shown relative to a preferred sequence derived for the mouse MSY1/2/4 CSD proteins [38–40] Bases in binding sites that vary from this sequence are indi-cated in lower case The sequences are for chicken chkYB-1b/2 pro-teins [41], for Xenopus FRGY1/2 propro-teins derived using the selex procedure [61], for dbpB/YB-1 binding to CD44 pre-mRNA [47], dbpB/YB-1 binding to the IL-2 5¢-UTR [31] and the VEGF 5¢-UTR CSD site 1 MSY1, ChkYB-1b and FRGY1 are dbpB/YB-1 proteins MSY-4 and chkYB-2 are dbpA proteins, and MSY2 and FRGY2 are germ cell-specific CSD proteins.
Trang 10sequences has not previously been reported The ability of
CSD proteins to recognize both types of sequence suggests
that CSD proteins may direct formation of the CSD/PTB
cytoplasmic complexes on the VEGF 5¢-and 3¢-UTR
Functional role of VEGF CSD/PTB binding sites
The stability of VEGF mRNA is increased by stress
conditions such as hypoxia [7,11–14,17,18] in response to
a number of signaling pathways [12,14,16] Investigation of
stabilization mechanisms in noninduced or normoxic
con-ditions has not previously been reported Our data
presen-ted here suggests that the VEGF CSD/PTB sites may be
involved in such mechanisms We observed, that deletion of
the VEGF 5¢- and 3¢-UTR CSD/PTB site sequences, results
in reduced VEGF mRNA stability in both normoxic and
hypoxic conditions while the degree of stabilization of the
VEGFmRNA under hypoxic conditions was not affected
It appears therefore that CSD/PTB complexes may be
playing a general protective role for the VEGF mRNA in
normoxic growing cells, but that they are not involved in
increased stabilization in response to hypoxia Consistent
with this finding, CSD proteins have been shown to play a
role in both induced [31–33] and general [34–37] mRNA
stabilization Recent data suggests that PTB proteins may
also play a role in this latter type of mRNA stabilization
[62] Factors such as HuR and hnRNPL proteins, have been
implicated in VEGF mRNA stabilization through binding
to the 3¢-UTR, but this is the first report of identification of
potential post-transcriptional regulatory factors binding to
the VEGF 5¢-UTR [17,18] CSD and PTB proteins may
function to stabilize structures required to enhance mRNA
stability, as proposed for other post-transcriptional roles
mediated by CSD and PTB proteins [42,45] As the second
5¢-UTR CSD/PTB site is downstream of a reported
alternative transcription start site, the presence of two
CSD/PTB sites in the VEGF 5¢-UTR may be to ensure that
at least one of these sites will be present in alternative forms
of the VEGF mRNA [63]
Given that CSD and CSD-related proteins can play a role
in both cap-dependent [26,27,42] and IRES-driven
transla-tion [43–46] it is possible that the CSD/PTB sites play a role
in translation as well as stabilization of the VEGF mRNA
A combined role in mRNA stability and translation has
been observed for the YB-1 and MSY-2 CSD proteins
[35–37] Sequence-specific CSD binding sites involved in
translational regulation have been found in both 5¢- and
3¢-UTR sequences [38–41] PTB proteins are also involved
in translational regulation and it has been demonstrated
that PTB in combination with a CSD-related protein,
UNR, is involved in IRES function [43–46] The CSD/PTB
sites are, however, outside the regions defined for IRES
activity in the mouse and human VEGF 5¢-UTR sequences
[19–21], hence a cap-dependent translational role would be
more likely for the VEGF CSD/PTB binding regions
Higher order CSD/PTB complex formation
We have shown that the VEGF 5¢-UTR
CSD/PTB-containing complexes are multiprotein complexes
contain-ing proteins of the appropriate size for PTB (60 kDa), the
dbpB/YB-1 CSD protein (50 kDa) and two additional
smaller unidentified factors (27 and 12 kDa), giving a combined molecular mass 180 kDa DbpB/YB-1 (also called MSY-1, chkYb-1b, p50 and FRGY1) is one of two ubiquitously expressed CSD proteins The other being represented by dbpA (also called MSY-4, chkYB-2 and YB2/RYBa) [22–25,29] Both CSD and PTB proteins have been shown to functionally interact with a number of partner proteins e.g [23,43] but the dbpA or dbpB/YB-1 CSD proteins have not previously been reported to complex, or interact, with PTB Although not investigated here, a role for dbpA in the CSD/PTB complexes can not
be ruled out, as dbpA and dbpB/YB-1 have very similar functions [38–41] The large CSD-related UNR protein, with a size of 97 kDa is, however, unlikely to be part of the complex
Our data also demonstrates that the VEGF 5¢-UTR cytoplasmic CSD/PTB complexis preformed in solution This indicates that the preformed CSD/PTB complex, with
an approximate size of 400–490 kDa, may contain addi-tional proteins to those detected by UV cross-link analysis (the combined size of cross-linked components is only
180 kDa) Alternatively the 400–490 kDa complexmay contain two molecules of each protein identified by UV cross-linking This is likely as CSD and PTB proteins have
in fact been found to be able to bind to RNA and DNA as dimers as well as monomers [28,29,42,51,56]
Comparison of the sequence requirements for CSD/PTB complexformation with the binding of recombinant dbpB/ YB-1 and PTB, suggests that CSD proteins may direct the binding of the multiprotein complexto the VEGF RNA, as discussed above Consistent with this, using recombinant proteins, we have found that dbpB/YB-1 enhances the binding of PTB to VEGF RNA (M A Bartley & L S Cole, unpublished observation) Similarly the CSD-related protein, UNR, has been shown to direct the binding of PTB to IRES sequences [45] CSD proteins have also been shown to affect the ability of transcription factors to bind to DNA [23]
Broader role for CSD/PTB complexes in growth factor gene regulation
We found that the CSD/PTB complexes binding to the VEGF 5¢-UTR bound to similar sequences in the IL-2 5¢-UTR (Fig 8B) The IL-2 sequence that binds the CSD/ PTB complexhas previously been shown to be part of a stability element that responds to the JNK signaling pathway
in T cells [31] We confirmed that the CSD/PTB complexes could in fact form in T cells Both the dbpB/YB-1 CSD protein and another RNA binding protein, nucleolin, were found to be required for the induced IL-2 mRNA stabiliza-tion [31] It therefore appears likely that dbpB/YB-1 may be able to partner with multiple proteins on the IL-2 5¢-UTR stability element and that these complexes may respond to different signaling events or operate under different condi-tions The CSD/PTB complexes, for example, could be involved in general protection of the IL-2 mRNA until appropriate signaling pathways are activated, whereby CSD proteins could partner with nucleolin to bring about enhanced stabilization A similar mechanism could be occurring on the VEGF mRNA, where CSD/PTB complexes protecting VEGF mRNA under normal growing conditions