Fig. 4: Kinetics of Exon Junction Complexes assembly and disassembly.

Time-courses for luciferase activities in lysates from cells stably expressing both eIF4A3-SmBiT and MAGOH-LgBiT (OB9 cells) exposed to a inhibitors of transcription; 5,6-dichloro-l-β-ribofuranosylbenzimidazole (DRB) and actinomycin D (ActD). The black doted curve corresponds to the slow exponential decrease. The black continuous curve corresponds to the best fit of experimental points by two exponentials; b cells exposed simultaneously to inhibitors of transcription (ActD or DRB) and translation (CHX) (pink dots); the continuous black curve corresponds to transcription inhibition as in B; c Amplitudes (A) and time constants (τ) of the exponentials describing the decrease in EJC-NanoBiT activity following an arrest in transcription and/or translation. Amplitudes (A1 or A2) and time constants in minutes (τ1 or τ2) correspond to the best fits obtained using indicated data sets shown in a, b. d Scheme illustrating why translation-dependent disassembly accounts for only 30% EJC at steady state yet 85% of newly deposited EJCs are disassembled by such pathway. Assembly rates (R_ass) and disassembly (k_diss) rate constants for EJCs prone to fast translation dependent (TD) and slow translation independent disassembly (TinD), respectively. As shown in panel c, k^TinD_diss = 1/τ1 and k^TD_diss = 1/τ2 and k^TD_diss = 17 x k^TinD_diss, Therefore R^TD_{ass =} 7 x R^TinD_ass. e Increase in luciferase activity in lysates from cells exposed to inhibitors of translation; cycloheximide (CHX) or harringtonine f Recovery of luciferase activity when DRB containing media are replaced after 2 h of incubation by fresh media with (pink circles) or without (black dots) cycloheximide (CHX). Yellow triangles correspond to cells treated with DRB until lysis. The yellow dotted curve corresponds to the best fit determined in b. Each point corresponds to technical replicates normalized by NanoBiT activity of untreated cells from the same 12-well plate. Initial points of recovery are fit by straight lines. Mean values±standard deviation are plot in a, b, e. Source data are provided in the Source Data file.