Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants

Invertases are responsible for the breakdown of sucrose to fructose and glu cose. 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 ail invertases of plant origin. Sequences responsible f or normal splicing (inclusion) of exon 2 have been investigated in vivo usi ng 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 pu tative branchpoint sequence and an adjacent U-rich region. Both are recogni zed plant intron splicing signals, The branchpoint sequence lies further up stream from the 3' splice site of intron 1 than is normally seen in plant i ntrons. All dicotyledonous plant invertase genes contain this arrangement o f sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequence s 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 a bsent. in addition, mutation of the 3' and 5' splice sites flanking the min i-exon cause skipping of the mini-exon, suggesting that both splice sites a re 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 appe ar to function via interactions between factors bound at the branchpoint/U- rich region and at the 5' splice site of intron 2, activating removal of th is intron followed by removal of intron 1. This first example of splicing o f 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.