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

Exon definition is a mechanism whereby splice sites are selected initially via interactions between splicing factors across an exon, prior to spliceos ome assembly and intron removal. It occurs in the splicing of vertebrate pr e-mRNAs and, recently, evidence for exon definition and the role of exon se quences has been obtained in plant intron splicing. Here we demonstrate tha t interactions between plant introns influence splicing efficiency and that these interactions are consistent with an exon definition process. The spl icing 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 spli ced, 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 i ntact splice sites at the 5' and 3' ends of the exon were required, thus po inting 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-ri ch sequences functioning early in the splicing process in plants.