Interactions between introns via exon definition in plant pre-mRNA splicing

Abstract

Summary Exon definition is a mechanism whereby splice sites are selected initially via interactions between splicing factors across an exon, prior to spliceosome assembly and intron removal. It occurs in the splicing of vertebrate pre-mRNAs and, recently, evidence for exon definition and the role of exon sequences has been obtained in plant intron splicing. Here we demonstrate that interactions between plant introns influence splicing efficiency and that these interactions are consistent with an exon definition process. The splicing efficiency of a UA-poor, inefficiently spliced intron (wheat amylase) increases 3.5- to 4.4-fold when placed in tandem with a UA-rich, well spliced, intron (legumin). Enhanced splicing is also observed with partial pea legumin intron sequences. However, mutation of splice sites in the partial UA-rich intron sequences abolished the enhanced splicing effect such that intact splice sites at the 5′ and 3′ ends of the exon were required, thus pointing to exon definition. This was further supported by reducing the size of the intervening exon or replacing with a UA-rich sequence which leads to loss of splicing of the UA-poor intron. Finally, the results support UA-rich sequences functioning early in the splicing process in plants.