Assembly of exon junction complexes on spliceosomes begins before spliceosome activation.

Despite the importance of exon junction complexes (EJCs) to co- and post-transcriptional gene control, how and when the EJC core of EIF4A3, RBM8A, and MAGOH, and the peripheral factors of the alternative RNPS1-EJC and CASC3-EJC, assemble on spliceosomes remain unclear. Existing characterizations that order EJC assembly on spliceosomes offer different conclusions. Using immunoprecipitations (IPs) of EIF4A3, RNPS1, or CASC3 in the presence of RNase I followed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) analyses of human HEK293T cells, we expand upon those splicing factors that associate with EJC constituents. Referencing these data against existing cryogenic electron microscopy (cryo-EM) spliceosome structures at different stages of pre-messenger RNA (pre-mRNA) splicing, coupled with data from a series of western blots of nuclear fractions before and after IP, we report that EIF4A3, RBM8A, MAGOH, and RNPS1 associate with assembled but inactive spliceosomes. This is followed by CASC3 joining to late activated spliceosomes. Our work bridges long-standing gaps in understanding EJC assembly on spliceosomes.