Retention of spliceosomal components along ligated exons ensures efficient removal of multiple introns.

The majority of mammalian pre-mRNAs contains multiple introns that are excised prior to export and translation. After intron excision, ligated exon intermediates participate in subsequent intron excisions. However, exon ligation generates an exon of increased size, a feature of pre-mRNA splicing that can interfere with downstream splicing events. These considerations raise the question of whether unique mechanisms exist that permit efficient removal of introns neighboring ligated exons. Kinetic analyses of multiple intron-containing pre-mRNAs revealed that splicing is more efficient following an initial intron removal event, suggesting that either the recruitment of the exon junction complex (EJC) to ligated exons increases the efficiency of multiple intron excisions or that the initial definition of splice sites is sufficient to permit efficient splicing of introns neighboring ligated exons. Knockdown experiments show that the deposition of the EJC does not affect subsequent splicing kinetics. Instead, spliceosomal components that are not involved in the initial splicing event remain associated with the pre-mRNA to ensure efficient removal of neighboring introns. Thus, ligated exons do not require redefinition, providing an additional kinetic advantage for exon defined splice sites.