A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor

Abstract

Four isoforms of human fibroblast growth factor 2 (FGF-2) result from alternative initiations of translation at three CUG start codons and one AUG start codon. Here we characterize a new 34-kDa FGF-2 isoform whose expression is initiated at a fifth initiation codon. This 34-kDa FGF-2 was identified in HeLa cells by using an N-terminal directed antibody. Its initiation codon was identified by site-directed mutagenesis as being a CUG codon located at 86 nucleotides (nt) from the FGF-2 mRNA 5' end. Both in vitro translation and COS-7 cell transfection using bicistronic RNAs demonstrated that the 34-kDa FGF-2 was exclusively expressed in a cap-dependent manner. This contrasted with the expression of the other FGF-2 isoforms of 18, 22, 22.5, and 24 kDa, which is controlled by an internal ribosome entry site (IRES). Strikingly, expression of the other FGF-2 isoforms became partly cap dependent in vitro in the presence of the 5,823-nt-long 3' untranslated region of FGF-2 mRNA. Thus, the FGF-2 mRNA can be translated both by cap-dependent and IRES-driven mechanisms, the balance between these two mechanisms modulating the ratio of the different FGF-2 isoforms. The function of the new FGF-2 was also investigated. We found that the 34-kDa FGF-2, in contrast to the other isoforms, permitted NIH 3T3 cell survival in low-serum conditions. A new arginine-rich nuclear localization sequence (NLS) in the N-terminal region of the 34-kDa FGF-2 was characterized and found to be similar to the NLS of human immunodeficiency virus type 1 Rev protein. These data suggest that the function of the 34-kDa FGF-2 is mediated by nuclear targets.

FIG. 1

Analysis of endogenous FGF-2 isoforms in HeLa and SK-Hep-1 cells. Aliquots (20 μg) of cell extracts from HeLa and SK-Hep-1 cells were analyzed by PAGE and transferred to nitrocellulose (see Materials and Methods). Immunoblotting was performed with anti-FGF-2 antibodies and chemiluminescence detection, immediately followed by autoradiography for 2 h. Positions of migration of size standards (right) and of the FGF-2 isoforms (left) are indicated.

FIG. 2

Identification of the 34-kDa FGF-2 with anti-N-terminal antibodies. (A) Schema representing the FGF-2 mRNA and the FGF-2 coding sequence, showing positions of the peptides used to prepare the anti-N23 and anti-FGF-2 polyclonal antibodies (see Materials and Methods). (B) Heparin-Sepharose-purified HeLa extract was analyzed by Western immunoblotting with either anti-N23 (αN23) or anti-FGF-2 (αFGF-2) antibodies (see Materials and Methods). Migration of size standards is indicated on the left; migration of FGF-2 isoforms is indicated on the right. The 16-kDa-migrating band revealed in lane 1 by anti-N23 probably corresponds to a cleavage product.

FIG. 3

Analysis of the ability of the FGF-2 mRNA 5′ UTR to promote translation initiation of the 34-kDa FGF-2. Expression of the 34-kDa FGF-2 was analyzed for three DNA constructs either by in vitro transcription and translation in RRL or by COS-7 cell transfection followed by Western immunoblotting (see Materials and Methods). (A) Schema of the mRNAs expressed from the different constructs (see Materials and Methods). WT5′-FGF corresponds to the FGF-2 mRNA with a complete 5′ leader; Δ1-257 corresponds to a deletion of nt 1 to 257 in the FGF-2 mRNA 5′ end; WT5′CAT corresponds to a fusion of FGF-2 cDNA nt 1 to 312 with the CAT sequence (devoid of an AUG start codon). (B) Analysis of mRNA expression either by in vitro translation (lanes 1 and 2) or by Western immunoblotting (lanes 3 to 6). Names of the antibodies (αFGF-2, αCAT, and αN23) are indicated at the bottom. Migration of size standards is indicated on the right; migration of FGF-2 isoforms is indicated on the left.

FIG. 4

Sequence of the FGF-2 mRNA leader. (A) The sequence of the FGF-2 cDNA 5′ region was obtained by using a DNA analyzer (see Materials and Methods) and compared to the two published sequences. Under the schema of the FGF-2 cDNA indicating the two HgaI sites at positions 121 and 224 downstream from the 5′ end are represented the three sequences of the region from 146 to 166. Lines 1 and 2 correspond to the published sequences of the cDNA and genomic DNA, respectively (41, 44). Line 3 is the sequence obtained in this study for both DNAs with the DNA analyzer. The new HgaI site is underlined (position 153). (B) The pFC1 plasmid with the 5′ region of the FGF-2 cDNA, its homolog with the corresponding genomic sequence (kindly provided by R. Florkiewicz), and a pFC1-derived plasmid lacking the HgaI site at position 153 (point mutations change nt 153-GACGCGG to 153-GATGGC; published for its in-frame ATG-154 [47]) were BssHII digested. The 291-nt-long resulting fragments were dephosphorylated, kinase treated in the presence of [³²P]ATP, and then digested with enzyme HgaI. Each step was followed by a G50 column purification. The restriction fragments were fractionated on a 15% polyacrylamide–Tris-borate-EDTA gel, which was dried and autoradiographed. Sizes of the expected fragments are (i) 121, 85, 51, and 34 nt in the presence of the new HgaI site and (ii) 121 nt plus a doublet at 85 nt in its absence. The DNA origin (cDNA or genomic) is indicated at the top; control corresponds to the plasmid lacking the HgaI site at position 153. (C) Representation of the new reading frame given by the DNA sequence shown in A (line 3). The sequence between nt 80 and 163 is shown. The two potential initiation codons are shown in boldface; the HgaI site (positions 153 to 157) is underlined.

FIG. 5

Identification of the start codon by site-directed mutagenesis. Several point mutations of CTG-86 or of ACG-122 were introduced into a chimeric FGF-CAT construct in which the first 539 nt of FGF-2 cDNA were fused to the CAT sequence (40) (see Materials and Methods). The mutant constructs were used either for in vitro transcription and translation in RRL or for COS-7 cell transfection followed by Western immunoblotting, as for Fig. 3. (A) Representation of nt 83 to 127 of the FGF-2 cDNA. CTG-86 and ACG-122 are represented in boldface; arrows indicate the mutations obtained. (B) Translation of the different in vitro-transcribed mRNAs in RRL. (C) Western immunoblotting of transfected COS-7 cell extracts with anti-CAT antibody. The names of the mutants are indicated above the lanes. WT corresponds to the wild-type FGF-CAT construct; the mutant m24 (lane 7) corresponds to a double mutant (m2 plus m4). Lane 1 corresponds to the control (C) assay without mRNA. Positions of migration of the size standards (left) and of FGF-CAT proteins (right) are indicated. The two bands corresponding to the largest FGF-CAT protein (42 and 40.5 kDa) are shown by arrows.

FIG. 6

Cap dependence of 34-kDa FGF-2 expression. Capped and uncapped FGF-CAT mRNAs, with or without the FGF-2 mRNA 3′ UTR, were transcribed in vitro 2, and different dilutions of each mRNA were translated in RRL in the presence of [³⁵S]methionine and analyzed as for Fig. 5. (A) Autoradiography after PAGE analysis of FGF-CAT mRNA expression. The mRNA was transcribed in vitro from plasmid p5′CAT-A1, in which the first 539 nt of FGF-2 cDNA were fused to the CAT sequence and the shortest FGF-2 3′ UTR (90 nt long). The results correspond to representative experiments that were repeated five times, either with a 90-nt-long 3′ UTR (p5′CAT-A1) or with no 3′ UTR (p5′CAT-A0). Migrations of the size standards and of FGF-CAT proteins are indicated (CUG 0 represents the largest isoform). C (lane 1) is the control without mRNA. FGF-CAT mRNA amounts used in each sample were 0.3, 0.9, 2.8, 8.3, and 25 μg/ml for lanes 2 to 6 (uncapped RNAs) and lanes 7 to 11 (capped RNAs), respectively. (B) FGF-CAT isoform expression using capped and uncapped mRNAs in which the 90-nt-long FGF-2 3′ UTR (−3′ UTR, corresponding to panel A) or the 5,823-nt-long FGF-2 3′ UTR (+3′ UTR) was quantified by PhosphorImager analysis (ImageQuant software). Expression of each isoform from capped or uncapped mRNA is shown in a separate plot. The data are from representative experiments that were repeated at least five times. (C) Ratio of 34-kDa FGF-2 isoform versus total FGF-2 expression in the presence or absence of a 3′ UTR, calculated for capped mRNAs from experiments shown in panel B (expressed as percentage of total FGF-2 expression).

FIG. 7

Expression of the 34-kDa FGF-2 from bicistronic vectors. Bicistronic vectors expressing CAT as first cistron (ORF1) and FGF-2 as second cistron (ORF2) were used to analyze the cap dependence of the 34-kDa FGF-2. (A) Representation of the two constructs with or without the presence of a 5′ hairpin. (B) Transfection of COS-7 cells with the bicistronic constructs. CAT expression was analyzed by measuring CAT activity (lanes 1 and 2). FGF-2 expression was analyzed by Western immunoblotting with anti-FGF-2 antibodies (lanes 3 and 4). The presence or absence of a hairpin is indicated by + or −, respectively. Migration of the different FGF-2 isoforms is shown on the right.

FIG. 8

Expression of the different FGF-2 isoforms in permanently transfected NIH 3T3 cells. NIH 3T3 cells were transfected with bicistronic vectors expressing the 24-kDa (pF24EN), the 18-kDa (pF18EN), or the 34-kDa (pF34EN) FGF-2 or no protein (pEN) from their first cistrons and the neomycin resistance gene from their second cistrons; 10 to 12 G418-resistant clones were kept for each transfection, and analyses were performed with at least three clones of each origin. Two clones expressing two different levels of 34-kDa FGF-2 (cl14 and cl19), and one clone expressing a medium level of FGF-2, chosen for 24- and 18-kDa FGF-2 expression, are shown as representative clones. (A) Western immunoblotting was performed with cell extracts of the different clones, using anti-FGF-2 antibody as for Fig. 1. Positions of size standards are represented on the right. (B) Phase-contrast microscopy examination (magnification, ×100) of the different cell clones after 14 days of cultivation in six-well plates in 1% CS (see Materials and Methods). The panels correspond to representative parts of the culture dishes. (C) Proliferation curves obtained by the crystal violet method for cells seeded in 96-well plates and cultivated for 17 days (see Materials and Methods). In this procedure, cell growth is measured by determining the optical density at 540 nm (OD 540). Experiments were done in quadruplicate; they were also reproduced several times in triplicate with the classical cell counting procedure, which always gave similar results (not shown).

FIG. 9

Characterization of an NLS in the N-terminal domain of the 34-kDa FGF-2. (A) Amino acid sequence of the NH2-terminal domain of the 34-kDa FGF-2 (78 amino acid residues). The boldface boxed characters represent the amino acids that were deleted in construct F34ΔNCAT. (B) Subcellular distribution of different chimeric CAT proteins was analyzed either by immunocytolocalization in transfected COS-7 cells (fluorescent staining micrographs in the middle) or by fractionation of transfected SK-Hep-1 cells into nuclear and cytosolic extracts (histograms on the right), as described in Materials and Methods. SVCAT fusion contains the minimal SV40 large-T antigen NLS (PKKKRKV [24]). F18- and F24CAT chimeras have N-terminal parts corresponding to the 20 and 75 N-terminal amino acid residues of 18- and 24-kDa FGF-2, respectively. The latter includes the 24-kDa FGF-2 NLS described previously (1). The F34CAT chimera contains the 78-amino-acid N-terminal domain of the 34-kDa FGF-2 shown in panel A. F34ΔNCAT has a deletion of the arginine-rich box shown in panel A, corresponding to residues 43 to 49. Results from immunocytolocalization and cell fractionation are presented at the right for each chimeric protein and the control (CAT). The subcellular distribution between nucleus and cytosol is represented as a percentage of the total CAT activity measured.

FIG. 10

Model of translational regulation of FGF-2 expression. The FGF-2 mRNA can be translated by two alternative mechanisms (see the text): either a cap-dependent mechanism dependent on eIF-4E activity or an IRES-dependent mechanism that is independent of eIF-4E activity. The initiation factors 4A and 4G, forming with eIF-4E the complex eIF-4F, are represented. The 43S corresponds to the 40S ribosome subunit associated with eIF2 and with the initiator tRNA. The two putative IRESes and the 3′ UTR are schematized. The start codons of the FGF-2 isoforms are indicated.