Báo cáo khóa học: Phức hợp Multi-protein chứa Cold Shock Domain (Y-box) và Polypyrimidine Tract Binding Proteins hình thành trên mRNA Vascular Endothelial Growth Factor: Vai trò tiềm năng trong ổn định mRNA

A multi-protein complex containing cold shock domain (Y-box)

and polypyrimidine tract binding proteins forms on the

vascular

endothelial growth factor

mRNA

Potential role in mRNA stabilization

Leeanne S. Coles

*, M. Antonetta Bartley

*, Andrew Bert

, Julie Hunter

, Steven Polyak

, Peter Diamond

Mathew A. Vadas

1,3

and Gregory J. Goodall

1,3

Division of Human Immunology, The Hanson Institute, Institute of Medical and Veterinary Science;

Division of Biochemistry,

Department of Molecular Biosciences, The University of Adelaide;

Department 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¢-and3¢-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

VEGF mRNA in vivo. Complex formation 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-box protein;

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¢-and3¢-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)

Eur. J. Biochem. 271, 648–660 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2003.03968.x

actions 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¢-and3¢-UTR of the VEGF mRNA. We find that

CSD proteins form a cytoplasmic complex on VEGF

mRNA that also contains the multifunctional single-

strand RNA/DNA binding protein, PTB [43–46,52–57],

and that the binding of this complex may be involved in

general VEGF mRNA stabilization. The CSD/PTB-con-

taining cytoplasmic complex also 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¢-andHindIII 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].

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 (–).

FEBS 2004 CSD and PTB protein complexes on the VEGF mRNA (Eur. J. Biochem. 271) 649

(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

aminoacidformofVEGFandtheVEGF3¢-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-

ativetothestartoftheVEGF 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 complex to 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 crosslink 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 complex is 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.

650 L. S. Coles et al. (Eur. J. Biochem. 271)FEBS 2004

polylinker 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. Single-strand oligonucleotides for

competition of CSD protein-containing complexes were

from the human granulocyte-macrophage-colony stimula-

ting factor (GM-CSF)gene.Thewild-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

P-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 [

P]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 56 C.

Preparation of recombinant and cytoplasmic proteins

The Escherichia coli strain MC1061 transformed with

pGEXBT or pGEXPTB was induced with isopropyl thio-

-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

to a Superdex 200

column (10 mm diameter, 20 mL bed volume) pre-equili-

brated with buffer containing 150 m

KCl, 20 m

Tris/

HCl pH 7.6, 20% glycerol, 1.5 m

MgCl

,2m

dithio-

threitol, 0.4 m

phenylmethanesulfonyl fluoride and 1 m

. The CCV1 complex was eluted with the same

buffer and 0.5 mL fractions collected. The molecular mass

of the complex was 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 complex is 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 complex is

indicated. (E) The Jurkat T cell CCV1 cytoplasmic complex binding 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.

FEBS 2004 CSD and PTB protein complexes on the VEGF mRNA (Eur. J. Biochem. 271) 651

antibody 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

P-labeled RNA

probes in a 10 lL reaction mix of 0.5·TM buffer [49–51]

containing 200 m

KCl, 1 lg poly(dI.dC), 100 ng tRNA,

1lg bovine serum albumin and either 1 lg cytoplasmic

extract or 25 ng recombinant protein (GST-dbpB or GST-

PTB). Reactions were incubated at 4 C 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

P-labeled RNA probe.

Antibody blocking experiments were performed by incuba-

ting protein and antibody for 5 min before adding the

P-labeled probe. Antibodies did not degrade RNA probes

under the gel shift conditions used.

UV cross-linking

Cytoplasmic extracts were bound to

P-labeled RNA

probes in a 25 lLgelshiftreactionandfractionatedona

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% O

). 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

G418 [15].

Serum stimulation of stably transfected cell lines was as

described previously [15]. Hypoxic conditions (1% O

)were

generated in a hypoxic chamber (Edwards Instrument

Company, Sydney, Australia).

Analysis of mRNA stability

in 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 TRIzol

reagent (Invitro-

gen) according to the manufacturers instructions, and

pfVEGF/pfVEGFdel mRNA was detected by RNase pro-

tection analysis using a

P-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 specific

P-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,

P-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

652 L. S. Coles et al. (Eur. J. Biochem. 271)FEBS 2004

Báo cáo khóa học: A multi-protein complex containing cold shock domain (Y-box) and polypyrimidine tract binding proteins forms on the vascular endothelial growth factor mRNA Potential role in mRNA stabilization

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