LETTERS p53 mRNA controls p53 activity by managing Mdm2 functions Marco M. Candeias 1 , Laurence Malbert-Colas 1 , Darren J. Powell 1 , Chrysoula Daskalogianni 1 , Magda M. Maslon 1 , Nadia Naski 1 , Karima Bourougaa 1 , Fabien Calvo 1 and Robin Fåhraeus 1,2 The E3 ubiquitin ligase Mdm2 is a focal regulator of p53 tumour suppressor activity. It binds p53, promoting its polyubiquitination and degradation, and also controls p53 synthesis. However, it is not known how this dual function of Mdm2 on p53 synthesis and degradation is achieved. Here we show that the p53 mRNA region encoding the Mdm2- binding site interacts directly with the RING domain of Mdm2. This impairs the E3 ligase activity of Mdm2 and promotes p53 mRNA translation. We also show that introduction of cancer-derived single silent point-mutations in the p53 mRNA weakens its binding to Mdm2 and results in reduced p53 activity. These data are consistent with a mechanism by which changes in silent nucleotides can affect the function of the encoded protein, and indicate that Mdm2-mediated control of p53 synthesis and degradation has evolved in the p53 mRNA sequence and its encoded amino acids. Binding of Mdm2 to the transcriptional transactivation domain 1 (TD1; ref. 1) in the amino terminus of p53 inhibits the activity of p53 and tar- gets it for proteasomal degradation 2,3 . Mdm2 promotes translation of the full-length p53 (p53 FL ) and the alternative translation product p53/47, which is initiated 40 codons downstream 4 and lacks the Mdm2-binding domain (MBD, residues 15–26; ref. 5). Recently, it was shown that the MBD-encoding sequence (MBD-ES) of p53 mRNA can mediate control of p53 mRNA translation 6,7 . To inves- tigate whether this region is involved in Mdm2-mediated regulation of p53 translation, we introduced silent mutations in the third positions of codons 17, 18 and 19 of p53 (p53 TriM , Supplementary Information, Fig. S1). Expression of the TriM message in p53-negative H1299 cells result in lower wild-type p53 protein levels (Fig. 1a). However, in the presence of Mdm2, wild-type p53 expressed from the TriM message increased, whereas mRNA levels remained unchanged (Fig. 1a and data not shown). When studied separately, the rates of protein synthesis and degradation were both found to be enhanced in the TriM construct in the presence of Mdm2 (Fig. 1a; Supplementary Information, Fig. S2a). The capacity of p53 TriM mRNA to enhance Mdm2-dependent p53 synthesis was also observed using an Mdm2 protein that carries a point mutation (C464A), which causes abrogation of its E3 ubiquitin ligase activity 2,8 . This shows that the capacity of Mdm2 to regulate p53 mRNA translation is not dependent on its E3 ubiquitin ligase capacity (Fig. 1b). We next looked in data banks for naturally occurring silent mutations in the MBD-ES region of the p53 gene. The L22L (CTA>CTG) single silent mutation was identified in a human tumour 9 and generates p53 mRNA that translates p53 at a constant rate in the presence or absence of Mdm2 (Fig. 1c). These results highlight the importance of p53 mRNA in Mdm2- mediated control of p53 synthesis and degradation, and we wanted to determine whether the Mdm2–p53 protein interaction is also involved in Mdm2-dependent control of p53 synthesis. To address this question we mutated the first initiation codon in the TriM message (M1A). This construct only expresses the p53/47 protein, which lacks the MBD and thus does not interact with Mdm2. However, this mRNA retains the MBD-ES and its susceptibility to Mdm2-dependent induction of mRNA translation (Fig. 1d). In contrast, Mdm2 did not induce translation of p53 mRNA in which the first 120 encoding nucleotides had been deleted (Δ120; Fig. 1d). However, Mdm2 induced translation of a construct where the first 120 coding nucleotides of p53 had been fused in front of GFP, giving rise to the 120–GFP fusion product, as well as GFP itself (Fig. 1d). We also examined the effects of replacing the first 120 encod- ing nucleotides in the p53 mRNA with the 36 nucleotides that encode the core sequence of the MBD (amino acids 15–26, construct MBDp47). As shown by pulse label experiments (Fig. 1e), MBDp47 mRNA transla- tion could be induced by Mdm2 and the encoded protein was capable of binding Mdm2. The Mdm2 isoform p76 Mdm2 lacks the p53 binding domain and can- not target p53 for degradation. p76 Mdm2 can be generated either through alternative translation initiation or splicing of Mdm2 RNA, and has been shown to have a positive effect on p53 activity 10,11 . To test whether p76 Mdm2 promotes p53 synthesis, we expressed p76 Mdm2 either from an N-terminal deletion construct (Δ1–61, ΔNMdm2) or by substituting the first two AUG codons in the Mdm2 message with alanine (AA/MMdm2). Both constructs express p76 Mdm2 (data not shown) and induce a similar 3-fold 1 Inserm U716, Pharmacologie Expérimentale, Institut Génétique Moléculaire, Hôpital St Louis and Université Paris 7, 27 rue Juliette Dodu, 75010 Paris, France. 2 Correspondence should be addressed to R.F. (e-mail: robinfahraeus@yahoo.co.uk) Received 7 March 2008; accepted 4 July 2008; published online 10 August 2008; DOI: 10.1038/ncb1770 1098 NATURE CELL BIOLOGY VOLUME 10 | NUMBER 9 | SEPTEMBER 2008 © 2008 Macmillan Publishers Limited. All rights reserved.