Fig. 1: Luciferase activity of an EJC-NanoBiT.
a The 3D structure of an in vitro assembled EJC PDB2J0S24 shows that MAGOH N-terminus is close to Y14 C-terminus and that Magoh C-terminus is close to eIF4A3 C-terminus; b MAGOH stably binds Y14, SmBiT fused to C-terminus (C) of Y14 interacts with LgBiT fused to N-terminus (N) of MAGOH thereby recapitulating NanoBiT luciferase activity; c MAGOH and eIF4A3 interact only when belonging to an assembled EJC, luciferase activity is recapitulated if LgBiT and SmBiT are fused to their respective C-termini (C); d, e relative luciferase activities (RLUs) obtained following transient transfection in HEK 293 cells; f, g HEK293 derived cell lines stably expressing eIF4A3-SmBiT (OB2 cells) or MAGOH-LgBiT (OB3 cells) are transiently transfected with plasmids expressing either wild-type or mutant HA-MAGOH-LgBiT or Flag-eIF4A3-SmBiT; data for d–g are from at least two biological replicates. Western blots probe the expression of tagged transfected proteins, GAPDH is a control for equal amount of cells loaded in every lane. GAPDH, eIF4A3, MAGOH and Y14 proteins are detected on Western blots with specific antibodies diluted 1/1000. Source data are provided in the Source Data file.
