SummaryThe removal of introns from pre-mRNA requires accurate recognition and selection of the intron splice sites. Mutations which alter splice site selection and which lead to skipping of specific exons are indicative of intron/ exon recognition mechanisms involving an exon definition process. In this paper, three independent mutants to the COP1 gene in Arabidopsis which show exon skipping were identified and the mutations which alter the normal splicing pattern were characterized. The mutation in cop1-1 was a G→A change 4 nt upstream from the 3Ј splice site of intron 5, while the mutation in cop1-2 was a G→A at the first nucleotide of intron 6, abolishing the conserved G within the 5Ј splice site consensus. The effect of these mutations was skipping of exon 6. The mutation in cop1-8 was G→A in the final nucleotide of intron 10 abolishing the conserved G within the 3Ј splice site consensus and leading to skipping of exon 11. The splicing patterns surrounding exons 6 and 11 of COP1 in these three mutant lines of Arabidopsis provide evidence for exon definition mechanisms operating in plant splicing.

SummaryExon 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 5Ј and 3Ј 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.

A reverse transcriptase-polymerase chain reaction (RT-PCR) where one oligonucleotide primer is end-labelled has been used to analyse expression in transgenic plants carrying antisense gene constructs. Specific detection of both sense and antisense RNA transcripts of the spliceosomal protein gene, U2B'', was achieved using the same pair of oligonucleotide primers. To maintain specificity, a reaction step in which reverse transcriptase was inactivated and RNA digested was found to be essential.