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 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 3' 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 3' and 5' 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 5' 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.
Simpson
C G
CG
Division of Genetics, Scottish Crop Research Institute, Dundee, United Kingdom.
Hedley
P E
PE
Watters
J A
JA
Clark
G P
GP
McQuade
C
C
Machray
G C
GC
Brown
J W
JW
D001483:Base Sequence; D017124:Conserved Sequence; D005091:Exons / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D018749:RNA, Plant / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics*; D043324:beta-Fructofuranosidase
D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase