Factors Affecting Authentic 5' Splice Site selection in Plant Nuclei

To define elements critical for 5' splice selection in dicot plant nuclei, wild-type and mutant transcripts containing the first intron of the pea rbcS3A gene were expressed in vivo by using an autonomously replicating plant expression vector. Mutations within the normal 5' splice site (+1) of this intron demonstrate that 5' splice sites at the normal exon-intron boundary having only limited agreement with a 5' splice site consensus sequence can be spliced quite effectively in dicot nuclei. Inactivation of the normal 5' splice site occurs only by point mutations of the G at position +1 of the intron (+1G) or +2U or by multiple mutations at other positions and results in the activation of three cryptic 5' splice sites in the adjacent exon and intron. cis competition of cryptic sites having consensus 5' splice site sequences with the normal 5' splice site demonstrates that cryptic splice sites in the exon, but not the intron, can compete to some extent with the normal site. Replacement of the sequences between the cryptic and normal 5' splice sites with heterologous exon or intron sequences demonstrates that the 5' boundary of this plant intron is defined by its position relative to the AU transition point between exon and intron. These results suggest that potential 5' splice sites upstream of the AU transition point are accessible for recognition by the plant pre-mRNA splicing machinery and that those downstream in the AU-rich intron are masked from recognition.