The Exon Junction Complex coordinates the co-transcriptional inclusion of blocks of neighboring exons

The Exon Junction Complex (EJC) has roles in mRNA export and cytoplasmic quality control. Yet EJC is recruited to pre-mRNA by the spliceosome, prior to the second catalytic step of splicing. Because splicing often occurs co-transcriptionally, EJC can be deposited on nascent RNA almost immediately after introns are synthesized and excised, raising the question of whether EJC regulates downstream RNA processing decisions. Here, we show that degron-mediated depletion of EJC component EIF4A3 leads to substantial skipping of neighboring pairs of two or more exons. These exon skipping events occurred on the same molecule of mRNA, detected by long read sequencing, suggesting that “exon blocks” require EJC for inclusion. Introns flanking EJC-dependent exon blocks were longer and spliced after internal introns. Thus, block exons form a larger EJC-marked exon prior to surrounding splicing events. These findings identify a previously unknown nuclear function of EJC that coordinates co-transcriptional inclusion of exon blocks. Overall design: To investigate a role of EIF4A3 in co-transcriptional splicing, we genereated HEK293T cells with an EIF4A3-SMASh degron tag and an EIF4A3 overexpressing HEK293 Flp-In T-REx cell line. We extracted mRNA and chromatin bound nascent RNA (nRNA) and conducted Illumina paired end sequencing and PacBio long-read sequencing.