Splicing dependency between EIF4G2 introns is mediated by exon definition and relies on a downstream splicing event

In mammalian cells, pre-mRNA splicing requires the excision of multiple introns within the same nascent transcript through the coordinated action of the spliceosome and accessory factors. We and others recently demonstrated that multi-intron splicing follows a predetermined order, and that removal of proximal introns is coordinated. Notably, specific introns that are removed later within a transcript depend on their neighboring introns for efficient excision, but the underlying mechanisms remain unknown. Here, we investigated the determinants of splicing order and coordination using a multi-intron minigene splicing reporter based on the human gene EIF4G2. We found that dependency between a later-excised intron and its neighbor is partially mediated through the downstream 5' splice site, consistent with the exon definition model, but is independent from intron length. Importantly, splicing coordination also relies on a prior downstream act of splicing, but not on specific cis regulatory elements in neighboring introns. Together, these findings improve our understanding of how distinct splicing reactions are connected and coordinated to produce mature RNA molecules.