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The p53 mRNA-Mdm2 interaction
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Nadia Naski, Madhavsai Gajjar, Karima Bourougaa, Laurence Malbert-Colas, Robin Fåhraeus and Marco M. Candeias* INSERM Unité 716; Institut de Génétique Moléculaire; Université Paris 7; Paris, France
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requires the interaction with the p53 mRNA; but neither its E3 ligase activity nor the binding to the p53 protein are needed. Hence, the two functions of Mdm2 to control p53 expression are disconnected and, therefore, the capacity of Mdm2 to promote p53 synthesis and degradation may not necessarily have to be coupled with a particular region in p53 or Mdm2. But in fact, the same p53 genomic sequence is at the origin of both the RNA (nt. 43–78) and the amino acid (aa. 15–26) sequences that bind respectively to the C- and N-terminus of Mdm2. This, together with the notion that the E3 ligase activity and the RNA binding domain of Mdm2 overlap, supports the idea that the two events that control the birth and death of p53 have evolved in parallel. It was first suggested that the capacity of Mdm2 to stimulate p53 synthesis was dependent on the p53-Mdm2 protein-protein interaction.8 This was based on the observation that the p53 point mutation phe19ala (F19A), which is located within the Mdm2 binding domain of p53 and is commonly used to generate a “non-Mdm2 binding” p53 protein, prevents Mdm2 from inducing p53 mRNA translation. Later results showed, however, that the N-terminal p53 proteinbinding domain of Mdm2, if anything, prevents Mdm2 binding to the p53 mRNA.7 In line with this, treatment of cells with Nutlin-3 that disrupts the p53-Mdm2 interaction does not prevent the binding of p53 mRNA to Mdm2 (Fig. 1D). So, what is the underlying reason why Mdm2 has no effect on the expression levels of the F19A p53? Contrary to the cancer-derived silent p53 mRNA mutants that were previously described to have reduced binding to Mdm2,7 the F19A p53 mRNA binds Mdm2 strongly. Indeed, when using transactivation dead R175H p53 mutants to minimize any influence of the p53 protein on Mdm2 expression, there is an increase of around 30% in R175H/F19A p53 double mutant mRNA binding to Mdm2 compared to R175H p53 mRNA (Fig. 1C). Another p53 mRNA, in which silent mutations were introduced in codons 17, 18 and 19 (Triple mutant RNA or TriM), was also shown to have increased binding to Mdm2.7 But in this case, the stronger binding is associated with an increase in the Mdm2-dependent rate of p53 synthesis. However, it turns out that the F19A p53 mRNA is translated at a higher basal rate compared to the wild type p53 and the TriM messages in the absence of Mdm2 (Fig. 1A, Pulse lane 1 and lane 3 and data not shown). In fact, overexpression of Mdm2 does not increase the level of translation of the wild type or TriM messages above that of the basal level of the F19A p53 mRNA. Interestingly, the high rate of F19A p53 synthesis does not reflect its steady state levels, indicating that the non-Mdm2-dependent turnover rate of this protein is increased (Fig. 1A and B).
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The E3 ligase Mdm2 is a key regulator of p53 activity via a complex regulatory feedback system that involves all levels of expression control including transcription, mRNA translation and protein degradation. Best known is the effect of p53 on Mdm2 transcription and the capacity of Mdm2 to target p53 for degradation, but more recently the role of Mdm2 as a positive regulator of p53 activity has also started to emerge. Mdm2 stimulates p53 mRNA translation by binding the p53 mRNA and, interestingly, this interaction also suppresses Mdm2’s capacity to promote p53 polyubiquitination and degradation. Another interesting aspect of the p53 mRNA-Mdm2 interaction is that the p53 mRNA sequence encoding the amino acids which bind the N-terminus of Mdm2 is the same that interacts with the Mdm2 RING domain. Indeed, the regulatory elements for controlling Mdm2-dependent expression of p53 are derived from the same p53 genomic sequence. In addition, the RNA binding and the E3 ligase domain of Mdm2 overlap, indicating that the two functions of Mdm2 to control p53 synthesis and degradation have co-evolved in parallel in both p53 and Mdm2. Here we illustrate how the p53-Mdm2 protein-protein and p53 mRNA-Mdm2 interactions affect Mdm2-mediated control of p53 expression using the Phe19Ala p53 mutant. We discuss how the new insights into the regulation of p53 expression levels can help to shed light on the origin of this elegant feedback system and on the function of Mdm2 isoforms.
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Key words: p53, Mdm2, mRNA translation, E3 ligase activity
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The expression of Mdm2 is essential for the development of the mouse embryo or it will die early on, while the cells undergo apoptosis. This can be avoided by knocking out p53, showing strong evidence of the importance of Mdm2 in controlling p53 activity during development.1 This, plus the fact that Mdm2 has the capacity to promote p53 degradation via the ubiquitin-dependent degradation pathway, has lead to the suggestion that Mdm2 is a negative regulator of p53 activity.2-6 However, if the effect of Mdm2 on p53 expression is studied more closely it becomes clear that Mdm2 also has the capacity to promote p53 synthesis and that the effect of Mdm2 on p53 expression is actually a balance between synthesis and degradation.7,8 Mdm2-mediated induction of p53 synthesis
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*Correspondence to: Marco M. Candeias; Inserm U716; Laboratoire de Pharmacologie; 27, rue Juliette Dodu; IGM; Paris 75010 France; Tel.: +33.142499269; Email:
[email protected] Submitted: 10/20/08; Accepted: 11/03/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/7326 www.landesbioscience.com
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The p53 mRNA-Mdm2 interaction
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Figure 1. Mdm2 binds to the F19A p53 mRNA but does not affect its rate of translation. (A) p53 null H1299 cells expressing wild type (wt) or F19A mutant p53 with or without additional Mdm2. The upper western blot (W.B.) using polyclonal CM1 antibody shows the steady state levels of full length (FL) and p53/47 proteins. The autoradiograph (bottom) shows the rate of p53 synthesis. p53 was immunoprecipitated using CM1 following a 20 min [35S]methionine pulse label in presence of the proteasome inhibitor MG132 (25 μM).7 (B) Lysates of H1299 cells transfected with R175H p53 (transactivation dead mutant) or R175H/F19A p53 double mutant with or without Mdm2 were blotted for p53 (CM1) after 1 h of treatment with the protein synthesis inhibitor cycloheximide (CHX 10 μg/ml) in order to look at the rate of degradation of the two p53 proteins and to minimize any potential influence of p53 downstream pathways. (C) The binding of Mdm2 to the F19A mRNA was tested by immunoprecipitating Mdm2 using the monoclonal 4B2 antibody from lysates of H1299 cells expressing R175H p53 or R175H/F19A p53. qRT-PCR was performed on RNA extracts using primers against p53 and control genes (TATA box-binding protein and βactin).7 (D) H1299 cells expressing Mdm2 and p53 mRNA (silent mutant with mutated AUG codons) were treated with Nutlin-3 which prevents p53 binding to Mdm2 for 8 h (5 μM). Mdm2-p53 mRNA binding was measured as descibed.7 (E) The hairpin secondary structure of the p53 mRNA sequence that encompasses codons 15 to 28 predicted by the RNA modelling programs (mFold version 3.2 web server) is significantly modified in the F19A p53 mutant in which a complete codon is changed (codon 19) leading to the formation of a new loop. The first 120 nt (40 aa) of the wt p53 mRNA are represented in the lower part of the figure. The p53 amino acids that interact with Mdm2 are indicated and codon 19 is boxed.
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The p53 mRNA-Mdm2 interaction
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interaction is weak and Mdm2 has no effect on p53 degradation (Figs. 1B WB and 2C). It might come as a surprise that a factor that can have a negative effect on p53 could also play a positive role. It is not fully clear yet why Mdm2 harbors this dual function towards p53 but it is interesting to speculate what would have happened if the capacity of Mdm2 to bind the p53 mRNA and to support p53 synthesis had been discovered prior to its capacity to promote p53 degradation. Would its capacity to act as a negative regulator during development have been looked at differently or would p53 be viewed as a regulator of Mdm2 and not vice versa? Mdm2, like p53, harbors many isoforms whose functions have not yet been investigated in detail. One of the most prominent forms is the p76Mdm2, sometimes also referred to as p75Mdm2, which is abundant in some tissues and upregulated in response to irradiation.14 It can be derived either through alternative translation or from alternative splicing, but since it lacks the N-terminal p53 binding motif, its function has remained obscure. In fact, it has been shown to act as a positive regulator on p53 but owing to a lack of potential mechanisms to explain how a non-p53 binding Mdm2 protein can affect p53 activity, these observations have not been pursued.14,15 However, the fact that p76Mdm2 binds the p53 mRNA and is an effective inducer of p53 mRNA translation offers a simple explanation of its capacity to promote p53 activity and suggests that expression of this isoform might play an important part in the regulation of p53 activity.7 In this context, it is interesting to point out that p53 also has isoforms, for example, the p53/47 which lacks the N-terminus and, hence, the capacity to interact with Mdm2.8,16,17,18 Considering that Mdm2 increases p53/47 expression, this leads to an interesting possibility that there might be a relationship between different p53 and Mdm2 isoforms, something which has not yet been explored. Not long before Mdm2 was shown to promote p53 degradation and to harbor E3 ligase activity, it was discovered that Mdm2 was associated with ribosomal RNA, ribosomal factor L5 and unspecific small RNA molecules.10,11 Later, other interactions between Mdm2 and ribosomal factors were also reported.11-13,19 It has been shown that the binding of the L23 and L11 ribosomal factors prevents Mdm2 from targeting p53 for degradation, and this has lead to the suggestion that the binding of Mdm2 to ribosomal proteins could act as a mechanism within the cell to detect ribosomal stress, which is an attractive idea, considering that most other types of cellular stresses involve p53 activation.12,19 The role of the interaction between Mdm2 and ribosomal factors in terms of p53 synthesis is still to be revealed but these interactions may encompass two purposes, both as a ribosomal stress response as well as a p53 mRNA translation support. It should be pointed out, however, that the difficulty of performing genetic studies on ribosomal components makes any conclusions regarding the function of these interactions difficult. And mutations within Mdm2 that prevent interactions with certain ribosomal factors could also affect the interaction with other cellular partners.
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The RNA modeling program predicts a secondary structure in the p53 mRNA sequence that includes the Mdm2 binding site. The previously reported cancer-derived mutation L22L9 is located in the head of this structure.7 It prevents Mdm2 binding but does not lead to any significant changes in the predicted secondary RNA structure and has a reduced level of basal rate of translation. The F19A p53, on the other hand, involves a complete change of nucleotides in codon 19 and its predicted effect on the hairpin structure is significant (Fig. 1E). Thus, these changes in sequence and/or structure are responsible for the increase in the basal rate of translation of the F19A p53 mRNA, suggesting that this hairpin structure normally mediates negative regulation of p53 synthesis. However, what this suppression consists on, or how binding of Mdm2 leads to an increase in mRNA translation, is not known yet. Mdm2 has been known for a long time to bind small RNA oligonucleotides non-specifically.10 Other than that, single nucleotide changes in the first 120 nt of the encoded sequence of the p53 mRNA impair Mdm2 binding. This suggests that Mdm2 harbors an RNA binding pocket which requires certain specific structures on the mRNA molecule so it can interact with it. This could explain why Mdm2 exhibits specificity for binding to mRNAs but not to shorter oligonucleotides. Fusion of the first 120 nt of the p53 mRNA to GFP is sufficient to induce mRNA translation of GFP, indicating that these 120 nucleotides are sufficient and necessary to bind Mdm2 and to allow Mdm2 to simulate mRNA translation. However, it is also possible that other factors, including RNA binding proteins, might help to mediate specificity to the p53 mRNA-Mdm2 interaction. Mdm2 is known to interact with several ribosomal factors including L5, L11 and L23,11-13 (see further below) and the interaction with one or several of these could assist in controlling Mdm2 binding to the p53 mRNA. The capacity of Mdm2 to bind the p53 mRNA can, as mentioned, be disrupted by introducing single point mutations, highlighting the specificity of the interaction. But it should be pointed out that it is not known yet if Mdm2 also interacts with other mRNAs. Not only does the TriM mRNA lead to an increase in Mdm2 binding and p53 synthesis, but p53 proteins expressed from this construct also have a higher rate of Mdm2-dependent turnover.7 How can silent changes in the p53 mRNA increase p53’s turnover rate? In light of the fact that binding of the p53 mRNA to Mmd2 actually shuts off Mdm2’s E3 ligase activity, this observation becomes even more puzzling. It is possible, however, that it reveals some important aspects of the p53-Mdm2 feedback loop. Since Mdm2 interacts with the p53 mRNA to induce p53 synthesis, a strong p53-Mdm2 interaction will increase the local concentration of Mdm2 at the p53 polysome and increase translation, which is similar to what happens when Mdm2 levels are increased by over expression. If one postulates that Mdm2 can interact with the nascent p53 peptide, a high p53 mRNA-Mdm2 interaction would lead to an increase in Mdm2 molecules becoming associated with the nascent p53. Once synthesis of p53 is completed and the p53-Mdm2 complex is released from the polysome, Mdm2 would regain its E3 ligase activity and target p53 for degradation. Hence, this model could explain why a strong p53 mRNA-Mdm2 interaction would have the same net effect as an increase in Mdm2 expression levels and explain why there is an increase in both synthesis and degradation of the TriM p53 message (Fig. 2A and B). In the case of the F19A p53, the interaction between the p53 mRNA and Mdm2 is retained, while the nascent p53-Mdm2 www.landesbioscience.com
Acknowledgements
This work was supported by la Ligue Nationale Contre le Cancer and the Indo French centre for the promotion of Advanced Research (IFCPAR). Anti-p53 and anti-Mdm2 antibodies were a gift from B. Vojtesek.
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Figure 2. A model for how silent and non-silent mutations in the p53 mRNA affect Mdm2-dependent regulation of p53 expression. (A, Part 1) Mdm2 RING domain binds wt p53 mRNA and promotes its translation and shuts off Mdm2 E3 ligase activity (Red Mdm2). (A, Part 2) Mdm2 binds the nascent p53 protein and when the Mdm2-p53 protein complex is released from the polysome, Mdm2 E3 ligase activity is restored (Green Mdm2) and it can target p53 to proteasomal degradation. (B, Part 1) Increased affinity between Mdm2 and p53 mRNA leads to a higher local concentration of Mdm2 at the p53 polysome and enhanced p53 synthesis. (B, Part 2) As more Mdm2-p53 complexes are released from the polysome, the turnover is also enhanced. (C, Part 1) Mdm2 binds F19A p53 mRNA better than the wt mRNA but has no effect on its rate of synthesis as the basal rate of F19A translation is already high (C, Part 2) The F19A p53 mutant does not bind Mdm2 (white Mdm2) which protects it from Mdm2-mediated ubiquitination and proteasomal degradation.
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