Fig. 2: Fractionation of Exon Junction Complex on sucrose gradients.

a Lysates from cells coexpressing MAGOH-LgBiT and eIF4A3-SmBiT (OB9 cells) are fractionated on sucrose gradients. Fractions (48 × 0.25 ml) from HEK 293 EJC-NanoBiT expressing cells were collected after a sucrose gradient (black curve - mean of 3 biological replicates normalized by their total luciferase activity). Mean values +/- standard deviation are plot. b Fractions from genuine HEK 293 cell lysates (12 × 1 ml fractions) were collected and analysed by Western blot. Fractions 5 to 9 in 2b correspond to fractions 16 to 36 in 2a, 2d and 2e. eIF4A3, Y14 and MAGOH are detected by Western blot in input fractions and in eIF4A3 immunoprecipitates. c Fragment Analyzer profiles of RNAs isolated from precipitates with (red curve) or without (black curve) anti-HA antibodies from pooled 3–12 fractions (12 fractions) of ultracentrifuged HA-eIF4A3 cell lysates. eIF4A3, MAGOH and Y14 proteins are detected on Western blots with specific antibodies diluted 1/1000. d Ultracentrifugation profiles of EJC-NanoBiT expressing cells treated (pink) or not (black) with cycloheximide (100 μg/ml) for 60 min. e Ultracentrifugation profiles of EJC-NanoBiT expressing cells treated (green) or not (black) with harringtonine (2 μg/ml) for 60 min. All lysates are fractionated on 11–54% sucrose gradients. RNA in 2b is detected by SYBR green fluorescence. RNA content in 2a, 2d and 2e in 48 × 0.25 ml fractions, is monitored by optical densities at 254 nm (dotted curves) while NanoBiT luciferase activities are shown as continuous lines. Positions of 40 S, 60 S, 80 S disomes (D) and trisomes (T) are indicated. f The increased EJC particle size upon treatment with translation inhibitors is attributed to the additional loading of ribosomes arrested at weak non canonical initiation codon or frozen just after initiation and preribosomes queuing upstream. Source data are provided in the Source Data file.