Fig 2: AKT1 interacts with UPF1 and UPF3X. A proximity ligation assay was performed to assess interactions between AKT1 and UPF1, UPF2, UPF3X or MDM2 (positive control). As a negative control, interactions between UPF1 and eIF4E were assessed. The white arrows indicate typical interaction points. The white squares correspond to a magnification of the background image. The bar plot at the bottom of the figure shows for each condition the average number of interaction points per cell as determined on more than 200 cells for each condition. Error bar = S.D., P-values were calculated with Student's t-test: *<0.05, **<0.01. All the results of this figure are representative of two experiments.

Fig 3: Screening of the PKIS library. (A) Schematic representation of the screening system used to identify NMD inhibitors. HeLa cells are transfected with constructs expressing the cDNA encoding the firefly luciferase carrying MS2 binding sites in the 3′UTR and expressing an MS2/UPF1 fusion protein. If a tested molecule inhibits NMD, the firefly luciferase mRNA is stabilized and translated to a functional firefly luciferase, the activity of which is measurable. (B) Results of the screening of the two plates containing the molecules promoting the highest luciferase activity. (C) Molecules and target identification.

Fig 4: Model of UPF1 phosphorylation by AKT1. UPF3X is loaded on mRNP by the exon junction complex (EJC). UPF3X would recruit AKT1 before recruiting UPF1 and phosphorylating it. These events occur before or during the pioneer round of translation, since the cap structure is bound by CBP80 and not eIF4E.

Fig 5: AKT1 phosphorylates NMD factors. (A) In vitro phosphorylation of UPF1 and UPF2 (761-1227) but not UPF3X by AKT1. Error bar = S.D., P-values were calculated with Student's t-test: *<0.05. The right panel is a Coomassie staining gel showing the UPF proteins used in the assay. (B) UPF1 was immunoprecipitated from HEK293FT WT and HEK293FT ΔAKT1 cells to assess the level of UPF1 phosphorylation. The three leftmost lanes of each panel of the figure correspond to serial dilutions of HEK293FT whole cell extract. The right panel shows quantification of UPF1 phosphorylation in both cell lines after immunoprecipitation of UPF1. (C) Absence of any additive effect of AKT1 and SMG1 on NMD efficiency. SMG1 was downregulated with siRNA in HEK293FT WT and HEK293FT ΔAKT1 cells expressing Globin Norm or Globin Ter in order to evaluate the NMD efficiency. The NMD efficiency was measured by RT-PCR. The left panel shows a representative gel analysis after quantitative PCR. MUP mRNA was used as a loading and transfection control. The three leftmost lanes correspond to serial dilutions of the Norm control plasmid sample. The bar plot on the right side of the gel shows the NMD efficiencies measured under the different tested conditions. (D) Measurement of UPF1 phosphorylation in HEK293FT WT and HEK293FT ΔAKT1 cells in the presence and absence of SMG1. The left panel shows a representative gel analysis and the bar plot on the right side of the gel is a graphic illustration of the quantification of two independent experiments. The three leftmost lanes of each panel of the figure correspond to serial dilutions of HEK293FT whole cell extract. Error bar = S.D., P-values were calculated with Student's t-test: *<0.05. The in vitro phosphorylation and immunoprecipitation results are representative of two and three experiments, respectively. The RT-PCR results showing the effects of SMG1 and AKT1 kinases are representative of four experiments.