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.