Eur J Biochem 271, 648–660 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2003.03968.x 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 Coles1,*, M Antonetta Bartley1,*, Andrew Bert1, Julie Hunter1, Steven Polyak2, Peter Diamond1, Mathew A Vadas1,3 and Gregory J Goodall1,3 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 regulator 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 regulation 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 general 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 complexes We found the same complexes binding to an RNAstabilizing 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 transcriptional level by CSD proteins, we propose a combined transcriptional/post-transcriptional role for these proteins in VEGF and other growth factor gene regulation VEGF is an essential regulator of angiogenesis that acts on vascular endothelial cells to induce proliferation and promote cell migration [1–3] Disregulated VEGF expression 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 promoting 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 mechanisms 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 posttranscriptional 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) Keywords: cold shock domain proteins; Y-box protein; polypyrimidine tract binding protein; mRNA stabilization; vascular endothelial growth factor Ó FEBS 2004 CSD and PTB protein complexes on the VEGF mRNA (Eur J Biochem 271) 649 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-transcriptional 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 posttranscriptional 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 posttranscriptional 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 complex on VEGF mRNA that also contains the multifunctional singlestrand 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-containing 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 Fig 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] 32P-Labeled RNA probes (44 and 46) were then immediately 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 singlestrand DNA competitors or left untreated (–) 5¢-UTR CSD site 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 sequences 650 L S Coles et al (Eur J Biochem 271) Fig The VEGF 5¢-UTR CSD-containing cytoplasmic complexes also contain PTB (A) Schematic of the VEGF 5¢-UTR CSD site The coordinates for the VEGF RNA probes 44 and V1 are indicated relative to the start of the VEGF mRNA (+ [58] (B) Balb/c 3T3 1) 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 proteins were calculated by subtraction of the molecular weight of bound RNA probe The sizes of cross-linked proteins are indicated Crosslink analysis is not quantitative as different proteins will crosslink to different extents (D) Cytoplasmic extracts were incubated with unlabeled 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 (Figs and 3) pGEMV25 and pGEMV27 were constructed by cloning wild-type and mutant double strand oligonucleotides 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 (+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 sequence (Fig 6) Ó FEBS 2004 Fig 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 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 inhibitors) 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 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 Ó FEBS 2004 CSD and PTB protein complexes on the VEGF mRNA (Eur J Biochem 271) 651 Oligonucleotides Oligonucleotides for cloning into pGEM4Z and for use as competitors in gel shift assays were synthesized by Geneworks (Adelaide, Australia) and purified from nondenaturing polyacrylamide gels Single-strand oligonucleotides for competition of CSD protein-containing complexes were from the human granulocyte-macrophage-colony stimulating 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 oligonucleotides are from the transferrin gene (DR1 sense and DR1 sense mut1, respectively) [52] RNA probe preparation 32 Fig 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 wildtype probe V25 with consensus CSD and PTB sites indicated The region + 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 cytoplasmic 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 complexes 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 subtraction 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 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, and 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 recombinant GST-dbpB/YB-1 (pGEXBT) has been described previously [49,51] pGEXPTB, for production of recombinant 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 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 [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 56 °C Preparation of recombinant and cytoplasmic proteins The Escherichia coli strain MC1061 transformed with pGEXBT or pGEXPTB was induced with isopropyl thiob-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 mLỈmin)1 to a Superdex 200 column (10 mm diameter, 20 mL bed volume) pre-equilibrated with buffer containing 150 mM KCl, 20 mM Tris/ HCl pH 7.6, 20% glycerol, 1.5 mM MgCl2, mM dithiothreitol, 0.4 mM phenylmethanesulfonyl fluoride and mM Na3VO4 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 Ó FEBS 2004 652 L S Coles et al (Eur J Biochem 271) antibody isotype control (Silensus, Boronia, Victoria, Australia) 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 mix of 0.5· TM buffer [49–51] containing 200 mM KCl, lg poly(dI.dC), 100 ng tRNA, lg bovine serum albumin and either lg cytoplasmic extract or 25 ng recombinant protein (GST-dbpB or GSTPTB) Reactions were incubated at °C for 20 min, followed by treatment with or without RNase T1 (Worthington 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 incubating protein and antibody for before adding the 32 P-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 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 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 Sepharose was extracted for bound RNA (TRIzolÒ 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 lgỈmL)1 G418 [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 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, or h Serum stimulation provides a brief pulse of transcription from the c-fos promoter in pfVEGF/pfVEGFdel constructs, 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 TRIzolr reagent (Invitrogen) according to the manufacturers instructions, and pfVEGF/pfVEGFdel mRNA was detected by RNase protection analysis using a 32P-labeled transcript covering the polylinker sequence in the pfVEGF/pfVEGFdel constructs as previously described [15] Neomycin phosphotransferase (neo) mRNA expressed from pfVEGF/pfVEGFdel constructs was detected as described [15] Protected RNAs were separated on denaturing polyacrylamide gels and the amounts of specific 32P-labeled protected pfVEGF/pfVEGFdel or neo mRNAs were quantitated by PhosphoImager analysis (Molecular Dynamics, Sunnyvale, CA, USA) Levels of pfVEGF/pfVEGFdel mRNA (with time 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 protamine (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 preferred 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 posttranscriptional regulation, the VEGF 5¢-UTR was examined for CSD protein binding sites Two potential sites at +157 and +650 were observed These were named CSD site and CSD site 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 cytoplasmic 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 Ó FEBS 2004 CSD and PTB protein complexes on the VEGF mRNA (Eur J Biochem 271) 653 Balb/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, respectively) and these complexes were readily competed by a single-strand DNA oligonucleotide (CSDwt) from the GMCSF gene 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), suggesting 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 recombinant 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 complex on the 44 RNA probe (CC44a), Balb/c 3T3 fibroblast cytoplasmic extract was bound, in a gel shift assay, to a shorter 32 P-labeled RNA probe, containing the proposed CSD binding site (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 complex on 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 (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 complex had 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 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 complex was confirmed in a gel shift assay, by competition of the CCV1 complex with an unlabeled single-strand DNA oligonucleotide probe from the transferrin gene (PTB wt), that binds PTB [52] The CCV1 complex was 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 complex was blocked by the antiPTB Ig (PTB) but not by an irrelevant monoclonal antibody (anti-GMCSF; GM) (Fig 2E) Taken together, this data demonstrates that the CSDcontaining cytoplasmic complex forming 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 complex formation 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 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 complex formation Consistent with this, a double mutation (V19) abolished CCV1 complex formation (Fig 3B) To investigate the individual roles CSD and PTB proteins may play in directing CCV1 complex binding to the 5¢-ACCUCUU-3¢ and 5¢-UUUUCUU-3¢ sequences, recombinant CSD (GST-dbpB/YB-1) and PTB (GSTPTB) 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 complex formation 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 complex formation To confirm that PTB-containing complexes can form on the VEGF mRNA in vivo an RNA immunoprecipitation 654 L S Coles et al (Eur J Biochem 271) Ó FEBS 2004 assay 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 VEGF mRNA was readily detected by RT-PCR in samples immunoprecipitated with anti-PTB monoclonal Ig (PTB) VEGF mRNA was not, however, detected in immunoprecipitations 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/YB1 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 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 complex to that observed on the VEGF V1 probe (CCV1), and this complex was 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 complex revealed that it was identical to the VEGF 5¢-UTR CCV1 complex (Fig 4C) An identical complex from fibroblast extracts can therefore form on both the VEGF and IL-2 genes For the CCV1 complex to be of relevance to the regulation of expression of the IL-2 gene, it was important to determine if the complex could 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 complex therefore 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 RNA probes in a gel shift assay (Fig 5) CCV1 complex formation, binding to the V1 probe, was observed in fractions with an approximate molecular mass range of 400–490 kDa (fractions 6, 7) No other complexes were observed across the range of fractions analyzed (from 10 to 1000 kDa) (data not shown) CCV1 complex formation, in fractions and 7, was abolished by the V19 mutation, verifying the nature of these complexes (Fig 5) These data indicate that the CCV1 Fig Gel filtration fractionation of the VEGF 5¢-UTR CCV1 complex 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, determined by comparison to elution profiles of protein standards, is given in kDa The CCV1 complex is indicated complex is preformed in solution The presence of a higher order preformed complex suggests 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 posttranscriptional 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 complex binds 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 complex contains 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) Ó FEBS 2004 CSD and PTB protein complexes on the VEGF mRNA (Eur J Biochem 271) 655 Fig CSD and PTB proteins bind to the VEGF 3¢-UTR (A) Sequence of the VEGF 3¢-UTR CSD site 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 protein 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 Hence 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 The VEGF mRNA CSD/PTB binding sites play a role in VEGF 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 Deletion of the VEGF CSD/PTB sites affects VEGF mRNA stability in normoxic and hypoxic conditions (A) Diagrammatic representation 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 (+ 156 to + 179), (+ 650 to + 666) and (+ 1727 to + 1740) in the pfVEGFdel construct are indicated with dashes (B) Stable transfectants, containing pfVEGF or pfVEGFdel were serum stimulated at time 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 h time point The levels of pfVEGF and pfVEGFdel mRNAs were approximately 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 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 experiments 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 indicated (C) Presentation of data in (B) as a log plot 656 L S Coles et al (Eur J Biochem 271) sequences 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 transfectants 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 stimulation to determine the stability of respective mRNAs (Fig 7B,C) It can be seen in Fig 7(B,C) that degradation of the wildtype 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 presence 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 regulated 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 expression in the cytoplasm, in conjunction with another singlestrand RNA/DNA binding protein, PTB Conserved CSD/PTB binding sites in the VEGF 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 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, and [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- Ó FEBS 2004 Fig Comparison of CSD protein RNA binding sites (A) The sequences of the VEGF 5¢- and 3¢-UTR CSD site 1, and sequences are shown and consensus CSD (5¢) and PTB (3¢) protein binding sites are underlined Both sequences are required for cytoplasmic CSD/PTB complex formation 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 indicated in lower case The sequences are for chicken chkYB-1b/2 proteins [41], for Xenopus FRGY1/2 proteins 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 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 lapped the consensus CSD sequence in the VEGF 5¢-UTR site (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 complex formation The ability of both antibody and oligonucleotide competitors to reduce or abolish complex formation 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 complexes 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 Ó FEBS 2004 CSD and PTB protein complexes on the VEGF mRNA (Eur J Biochem 271) 657 sequences 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 conditions has not previously been reported Our data presented 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 VEGF mRNA 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 translation [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/PTBcontaining complexes are multiprotein complexes containing 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 complex is preformed in solution This indicates that the preformed CSD/PTB complex, with an approximate size of 400–490 kDa, may contain additional 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 complex may 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 complex formation with the binding of recombinant dbpB/ YB-1 and PTB, suggests that CSD proteins may direct the binding of the multiprotein complex to 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 complex has 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 stabilization [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 conditions 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 658 L S Coles et al (Eur J Biochem 271) could be replaced by alternative complexes under induced conditions other than hypoxia CSD/PTB complexes could also play a role in general insulin mRNA stability, as PTB has recently been implicated in insulin mRNA stabilization in normoxic conditions [62] Interestingly, we observed that the insulin mRNA PTB binding site has an overlapping consensus CSD binding site PTB, to date, has only been implicated in stability of insulin and VEGF (this report) mRNAs A broad role for CSD/PTB complexes in growth factor gene regulation, is supported by our finding of these complexes in a number of different mouse, rat and human cell lines (M A Bartley & L S Cole, unpublished observation), and is consistent with the ubiquitous expression of CSD and PTB proteins [23,52] The stability of another growth factor mRNA, that for GM-CSF, is regulated by CSD proteins [32] In contrast to the VEGF and IL-2 genes, CSD proteins were reported to contact AU-rich sequences in the GM-CSF 3¢-UTR Several AU-rich sequences are present in the VEGF 3¢-UTR, the possibility exists that they could also bind CSD proteins [59] Different types of CSD binding site may therefore be targets for alternative types of CSD complexes responding to different signaling Model for combined transcriptional and post-transcriptional regulation of VEGF expression We have previously reported that the VEGF and GM-CSF genes are transcriptionally regulated by CSD proteins [49–51] In both cases, CSD proteins act as transcriptional repressors and our data suggest that the CSD proteins are removed upon appropriate induction to allow full promoter activity Consistent with this, we have found that CSD proteins bind to a repressor element in the IL-2 gene (L S Cole, unpublished data) CSD proteins may therefore play a combined transcriptional and post-transcriptional role in regulation of certain growth factor genes It seems likely then that CSD proteins could bind to RNA in the nucleus at the time of transcription In support of this, CSD proteins have in fact been shown to bind to RNA concomitant with transcription [64] Furthermore, CSD proteins have been found to be involved in RNA packaging and transport to the cytoplasm [26,28,29] It is feasible that CSD proteins could be involved at every level of growth factor gene regulation, from transcription in the nucleus to transport to the cytoplasm, mRNA stability and translation Acknowledgements We thank Tom Cooper (Houston, Texas) and Doug Black (UCLA) for gifts of PTB expression plasmids and anti-PTB monoclonal antibodies, respectively This work was supported by a Heart Foundation Australia project grant (G J G.), a Cancer Council of South Australia project grant (L S C.), an RAH/IMVS project grant (L S C) and a National Health and Medical Research Committee program grant (M A V, G J G.) 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proteins, play diverse roles in both transcriptional and post-transcriptional regulation of growth. .. that CSD proteins form a cytoplasmic complex on VEGF mRNA that also contains the multifunctional singlestrand RNA/DNA binding protein, PTB [43–46,52–57], and that the binding of this complex may... and IRES-driven translation [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