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p53 directs conformational change and translation initiation blockade of human fibroblast growth factor 2 mRNA
B Galy1, L Créancier, L Prado-Lourenço
1Institut National de la Santé et de la Recherche Médicale U397, Endocrinologie et Communication Cellulaire, Institut Fédératif de Recherche Louis Bugnard, C.H.U. Rangueil, 31403 Toulouse Cedex 04, France.
Abstract:
Tumour suppressor p53 has been shown to inhibit fibroblast growth factor 2 expression post-transcriptionally 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 effect 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 specifically 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 different RNAs showed no significant correlation between p53 RNA binding activity and its translational inhibiting effect. 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.
Insights
Tumor suppressor p53 inhibits fibroblast growth factor 2 (FGF-2) mRNA translation by blocking ribosome formation. This repression involves p53-induced RNA structural changes in the FGF-2 mRNA leader sequence.
Area of Science:
- Molecular Biology
- RNA Biology
- Cancer Research
Background:
- Tumor suppressor p53 is known to inhibit fibroblast growth factor 2 (FGF-2) expression post-transcriptionally.
- The precise mechanism underlying this p53-mediated post-transcriptional regulation of FGF-2 remains to be fully elucidated.
Purpose of the Study:
- To investigate the molecular mechanism by which p53 represses FGF-2 mRNA translation.
- To identify the specific RNA elements and p53 activities involved in this translational blockade.
Main Methods:
- Deletion mutagenesis of the FGF-2 mRNA leader sequence.
- In vitro translation assays using rabbit reticulocyte lysate.
- Sucrose gradient centrifugation to analyze ribosome formation.
- RNA accessibility assays using complementary oligonucleotide probes.
Main Results:
- At least four cis-acting RNA elements within the FGF-2 mRNA leader are required for p53-mediated inhibition.
- Wild-type p53, but not a specific mutant (Ala143), repressed FGF-2 mRNA translation in a dose-dependent manner.
- p53 blocks translation initiation by preventing 80S ribosome complex formation.
- p53 binding induced conformational changes in the FGF-2 mRNA leader, correlating with translational repression, independent of direct RNA binding affinity.
Conclusions:
- p53 directly represses FGF-2 mRNA translation through a mechanism involving its nucleic acid unwinding-annealing activity.
- p53-induced structural alterations in the FGF-2 mRNA leader likely disrupt translation initiation.
- This study reveals a novel mechanism of translational control by p53 impacting FGF-2 expression.