p53 directs conformational change and translation initiation blockade of human ®broblast growth factor 2 mRNA Bruno Galy 1,2 , Laurent CreÂancier 1 , Leonel Prado-LourencËo 1 , Anne-Catherine Prats* ,1 and Herve Prats 1 1 Institut National de la Sante  et de la Recherche Me Âdicale U397, Endocrinologie et Communication Cellulaire, Institut FeÂdeÂratif de Recherche Louis Bugnard, C.H.U. Rangueil, 31403 Toulouse Cedex 04, France Tumour suppressor p53 has been shown to inhibit ®broblast growth factor 2 expression post-transcription- ally in cultured cells. Here we have investigated the mechanism responsible for this post-transcriptional blockade. Deletion mutagenesis of the FGF-2 mRNA leader revealed the requirement of at least four RNA cis- acting elements to mediate the inhibitory eect of p53 in SK-Hep-1 transfected cells, suggesting the involvement of RNA secondary or tertiary structures. Recombinant wild-type, but not Ala 143 mutant p53, was able to speci®cally repress FGF-2 mRNA translation in rabbit reticulocyte lysate, in a dose dependent manner. Sucrose gradient experiments showed that p53 blocks translation initiation by preventing 80S ribosome formation on an mRNA bearing the FGF-2 mRNA leader sequence. Interaction of wild-type and mutant p53 with dierent RNAs showed no signi®cant correlation between p53 RNA binding activity and its translational inhibiting eect. However, by checking the accessibility of the FGF-2 mRNA leader to complementary oligonucleotide probes, we showed that the binding to RNA of wild-type, but not mutant p53, induced RNA conformational changes that might be responsible for the translational blockade. This strongly suggests that p53 represses FGF- 2 mRNA translation by a direct mechanism involving its nucleic acid unwinding ± annealing activity. Oncogene (2001) 20, 4613 ± 4620. Keywords: FGF; p53; translational control; RNA- protein interactions; RNA structure Introduction We have recently shown that the tumour suppressor p53 is able to inhibit the expression of chimeric FGF- CAT mRNAs by a post-transcriptional mechanism, independent of the activation of p53 target genes, which is speci®cally mediated by the FGF-2 mRNA 5' untranslated region (5' UTR; Galy et al., 2001). That study did not elucidate, however, whether p53 acts by modulating the activity of trans-acting factors involved in the control of mRNA translation, or by direct interaction with the FGF-2 mRNA. A few studies suggesting that p53 can repress translation by direct interactions are reported in the literature. p53 exhibits high anity for RNA (Ober- osler et al., 1993), and has been shown to repress translation of its own mRNA and that of the cdk4 mRNA, by a direct interaction (Miller et al., 2000; Mosner et al., 1995). p53 has also been detected in polysomes preparations (Fontoura et al., 1997). One can question the physiological relevance of the translational eect of a nuclear protein. However, some evidence has been reported that p53 can be transiently localized in the cytoplasm as a function of the cell cycle or cell dierentiation (Shaulsky et al., 1990; Moll et al., 1995). Moreover, p53 is now known to possess a nuclear export signal in its tetramerization domain (Stommel et al., 1999). Taken together these data favour the hypothesis that p53 might exhibit both nuclear and cytoplasmic functions. We show here by transient SK-Hep-1 cell transfec- tion that the p53 post-transcriptional inhibitory eect mediated by the FGF-2 mRNA leader requires at least four elements of the FGF-2 mRNA leader sequence, suggesting the involvement of RNA structures in the inhibitory mechanism. In vitro translation experiments were performed using recombinant wild-type (wt) or Ala 143 mutant p53, demonstrating a direct inhibition of FGF-2 translation by wt p53, occurring at the translation initiation step. The wt p53 enhanced the annealing of complementary oligonucleotides to the FGF-2 mRNA 5' UTR, thus revealing that it was able to induce an RNA conformational change. Results The post-transcriptional inhibitory effect of wt p53 requires at least four elements of the FGF-2 mRNA leader To characterize the cis-elements involved in the repressor eect of wt p53, deletions were performed in the FGF-2 mRNA leader on the basis of secondary structure prediction to remove putative structural Oncogene (2001) 20, 4613 ± 4620 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc *Correspondence: A-C Prats; E-mail: pratsac@rangueil.inserm.fr 2 Current address: Gene Expression Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany Received 26 February 2001; revised 4 April 2001; accepted 10 May 2001