Fig. 1.

The ATM kinase interacts with the p53 mRNA in an MDM2-dependant and DNA damage dependant manner. a Co-immunoprecipitation coupled with qPCR (RNA binding) testing the ATM—p53 mRNA interaction, of H1299 cells (p53-null) transfected with ATM (FLAG-ATM) and either p53wt or p53(Δ120) lacking the 5’CDS and box-I sequence, and of endogenous targets in A549 and HCT116 cells. Treatments include: “- “: dmso; “d”: doxorubicin; “e”: etoposide; “a”: actinomycin D; "m”: mitoxantrone. b-i Images of Proximity Ligation Assay (PLA) on H1299 cells, transfected with silenced p53 sequences of either wt, or p53(Δ120) lacking the 5’CDS and box-I sequence, or p53(L22L) expressing a modified secondary structure of the box-I. The y-axis represents the PLA signal per cell (amount of puncta per cell) (b-ii). c PLA on AT5 BiVA cells (ATM-null), transfected with combinations of ATM (FLAG-ATM) or silenced p53wt, as indicated; and on A549 cells expressing endogenous ATM and p53. d PLA on H1299 cells, transfected with either p53wt and gfp or p53-ms2 and ms2-gfp. A PLA signal is exclusively seen with the ms2 transfected constructs, using anti-ATM and anti-GFP antibodies, confirming that the ATM interacts with the p53 mRNA. e PLA on H1299 cells, transfected with silenced p53 and FLAG-ATM and either MDMX or the phosphor-mimetic mutant MDMX-403D that activates its interaction with the p53 mRNA. In normal conditions, the PLA signal is decreased in the presence of MDMX(403D), showing that the ATM-p53 mRNA interaction is competed by MDMX and that activated MDMX develops an affinity for the p53 mRNA. f PLA on H1299 cells, transfected with either FLAG-ATM or silenced p53 and FLAG-ATM, showing that the p53 mRNA promotes the association of ATM with NBS1 of the MRN complex in the nucleus during normal conditions and in the cytoplasm following DNA damage. g PLA on AT5 cells (ATM-null) and H1299 cells (p53-null) testing the effect of ATM activity on the NBS1—p53 mRNA interaction. ATM is required for the association of NBS1 with the p53 mRNA in the nucleus and inhibition of ATM activity prevents the association in the nucleus. The asterisks in the PLAs represent t-test p values of three independent experiments, as follows: ‘n/s’ for P > 0.05; ‘*’ for values P ≤ 0.05; ‘**’ for values P ≤ 0.01; and ‘***’ for values P ≤ 0.001. The statistical analyses of the PLAs are based on results obtained by at least 50 cells of each experiment. The cell lines, hybridization probes and antibodies used for each PLA are indicated