Fig 1.

A high-throughput assay (EJIPT) utilizing EJC binding to spliced mRNAs to measure splicing in vitro. Splicing reaction mixtures containing biotin-labeled pre-mRNA, 20 μM each compound, splicing extract, and 0.5 mM ATP are assembled by liquid handler in 384-well plates and incubated for 1.5 h at 30°C. Reaction mixtures are then transferred to a NeutrAvidin-coated plate, which captures biotin-labeled RNAs/RNPs postsplicing. Anti-eIF4AIII (3F1) antibody is added to detect splicing-dependent EJC formation on spliced mRNAs. Following a washing step, incubation with an HRP-conjugated anti-mouse IgG secondary antibody, and then addition of chemiluminescent substrate, signals representing eIF4AIII/mRNA complexes are obtained by an automatic plate reader.