The two murine double minute (MDM) family members, MDM2 and MDMX, are a well-established negative regulator of p53 activity. Under DNA damage conditions, MDM2 and MDMX are phosphorylated near their RING domains (serine 395 at MDM2 and serine 403 at MDMX) and switch to act as p53 positive regulators. MDMX binds to TP53 mRNA and acts as a chaperone for RNA structure, enabling MDM2 to bind. This interaction enhances TP53 mRNA translation, leading to increased p53 protein production. While the biological significance of this interaction has been described, the specific features of the MDMX-RNA interaction remain poorly understood. We used various MDMX protein constructs to characterize binding to TP53 mRNA and identified that the interaction mediated by the RING domain is modulated by the presence of other domains. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and binding assays in high salt conditions and various pH demonstrate that the whole protein participates in RNA interaction, with the C-terminal domain likely providing the contact with RNA by electrostatic forces. We show that protein structural changes induced by the chelating agent EDTA or the reducing agent TCEP enhance RNA binding by promoting partial structural destabilization of the protein. Our findings suggest that the MDMX/TP53 mRNA interaction is complex, with the RING domain binding to RNA and being supported by the entire protein, which acts as a scaffold for the RNA interaction. These results contribute to a better understanding of MDMX's role in TP53 mRNA binding and provide valuable insights for future investigation of the MDM2-MDMX-TP53 mRNA complex, which is crucial for p53 stabilization and activation under DNA-damaging conditions.

Department of Experimental Biology Faculty of Science Masaryk University Brno 625 00 Czechia

Department of Medical Biosciences Umea University Umea 901 87 Sweden

Inserm UMRS1131 Institut de Genetique Moleculaire Universite Paris Cite Hopital St Louis Paris 75010 France

Instituto de Fisica Universidad Autonoma de San Luis Potosi San Luis Potosi 78290 Mexico

Laboratory of Growth Regulators Institute of Experimental Botany The Czech Academy of Sciences Olomouc 779 00 Czechia

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno 625 00 Czechia

RECAMO Masaryk Memorial Cancer Institute Brno 602 00 Czechia

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