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. 2000 Jul 10;150(1):275-81.
doi: 10.1083/jcb.150.1.275.

Fibroblast growth factor 2 internal ribosome entry site (IRES) activity ex vivo and in transgenic mice reveals a stringent tissue-specific regulation

L Créancier  1 , D Morello, P Mercier, A C Prats
Affiliations

Fibroblast growth factor 2 internal ribosome entry site (IRES) activity ex vivo and in transgenic mice reveals a stringent tissue-specific regulation

L Créancier et al. J Cell Biol. .

Abstract

Fibroblast growth factor 2 (FGF-2) is a powerful mitogen involved in proliferation, differentiation, and survival of various cells including neurons. FGF-2 expression is translationally regulated; in particular, the FGF-2 mRNA contains an internal ribosome entry site (IRES) allowing cap-independent translation. Here, we have analyzed FGF-2 IRES tissue specificity ex vivo and in vivo by using a dual luciferase bicistronic vector. This IRES was active in most transiently transfected human and nonhuman cell types, with a higher activity in p53 -/- osteosarcoma and neuroblastoma cell lines. Transgenic mice were generated using bicistronic transgenes with FGF-2 IRES or encephalomyocarditis virus (EMCV) IRES. Measurements of luciferase activity revealed high FGF-2 IRES activity in 11-d-old embryos (E11) but not in the placenta; activity was high in the heart and brain of E16. FGF-2 IRES activity was low in most organs of the adult, but exceptionally high in the brain. Such spatiotemporal variations were not observed with the EMCV IRES. These data, demonstrating the strong tissue specificity of a mammalian IRES in vivo, suggest a pivotal role of translational IRES- dependent activation of FGF-2 expression during embryogenesis and in adult brain. FGF-2 IRES could constitute, thus, a powerful tool for gene transfer in the central nervous system.

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Figures

Figure 1
Figure 1
Schematic representation of the bicistronic LucR-I-LucF vectors. Vector construction is described in Materials and Methods. They contain the CMV promoter controlling the expression of a bicistronic LucR-LucF mRNA. A synthetic intron is present 5′ of LucR, and a poly(A) site is present 3′ of LucF (Huez et al. 1998). In the pCRFL construct, the complete FGF-2 5′ leader and beginning of coding sequence (up to nucleotide 539) has been fused to the firefly luciferase open reading frame. In the pCRHL construct, a hairpin has been introduced between the two luciferase genes. The pCREL construct contains the EMCV IRES between the two LUC genes.
Figure 2
Figure 2
Analysis of the FGF-2 IRES activity in transiently transfected cells. 14 different cell types were transiently transfected with pCRFL or pCRHL DNA. Cells were harvested 48 h after transfection, and the luciferase activities present in cell extracts were measured. FGF-2 IRES activity was determined by calculating the LucF/LucR ratio. To calibrate the data from the different cell types, the LucF/LucR ratio of pCRFL was divided by the ratio of the pCRHL negative control. Experiments were repeated 5–10 times, and the results are expressed as means ± SEM. The names of the different cell types (described in Materials and Methods) and the histograms values are indicated on the left.
Figure 3
Figure 3
FGF-2 and EMCV IRESes activities in transgenic mice embryos. Embryos from the transgenic mouse lines were recovered at 11 and 16 d postcoitum, and luciferase activities were measured in different organs (indicated on the left) as described in Materials and Methods. IRES activities are represented by histograms and were measured by calculating the LucF/LucR ratio (Table ). The results are shown for each of the two independent transgenic lines expressing one or other bicistronic construct pCRFL (FGF-2) or pCREL (EMCV), as means ± SEM of experiments that were performed 3–10 times. The lines expressing pCRFL (left) are RFLD (gray boxes) and RFL12 (black boxes). The lines expressing pCREL (right) are RELB (gray boxes) and RELA (black boxes). nd corresponds to experiments giving no detectable LucR activity.
Figure 4
Figure 4
FGF-2 and EMCV IRESes activities in transgenic adult mice. Different organs (indicated on the left) were prepared from adult transgenic mice and luciferase activities were measured as in Fig. 3. IRES activities are represented by histograms corresponding to the LucF/LucR activities (see Table ). The results are shown for each of the two independent transgenic lines, expressing one or other bicistronic construct pCRFL (FGF-2) or pCREL (EMCV), as means ± SEM of experiments that were performed 3–10 times. The lines expressing pCRFL (left) are RFLD (gray boxes) and RFL12 (black boxes). The lines expressing pCREL (on the right) are RELB (gray boxes) and RELA (black boxes). nd corresponds to experiments giving no detectable LucR activity.
Figure 5
Figure 5
Analysis of the bicistronic mRNA in transgenic tissues by RT-PCR. Total RNA was extracted from RFL12 or RELA transgenic mice brain (B), tongue (To), or testis (Te), and the level of LucF and LucR cistrons was analyzed by RT-PCR (as described in Materials and Methods). The negative control (C−) corresponds to a PCR experiment performed using RNA from the brain of a nontransgenic mouse. The positive control (C+) was performed with an in vitro–transcribed bicistronic RFL mRNA. The bands corresponding to amplification of LucR (465 nucleotide) or LucF (366 nucleotide) cDNAs are indicated by arrows. This experiment enabled us to check the LucF/LucR ratios at the RNA level in the different organs.
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