Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 39 splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 39 and 59 splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint /U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint /U-rich region and at the 59 splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.

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.