INTERACTIONS ACROSS EXONS CAN INFLUENCE SPLICE-SITE RECOGNITION IN PLANT NUCLEI

In vivo analyses of cis-acting sequence requirements for pre-mRNA spli cing in tobacco nuclei have previously demonstrated that the 5' splice sites are selected by their position relative to AU-rich elements wit hin plant introns and by their degree of complementarity to the U1 sma ll nuclear RNA, To determine whether the presence of adjacent introns effects 5' splice site recognition in plant nuclei, we have analyzed t he in vivo splicing patterns of two-intron constructs containing 5' sp lice site mutations in the second intron. These experiments indicated that the splice site selection patterns in plant nuclei are defined pr imarily by sequences within the intron (intron definition) and seconda rily by weak interactions across exons (exon definition), The effects of these secondary interactions became evident only when mutations in the downstream 5' splice site decreased its functionality and differed depending on the availability of cryptic splice sites close to the mu tant site. In beta-conglycinin chimeric transcripts containing multipl e cryptic 5' splice sites, the presence of an intact upstream intron s ignificantly increased splicing at the downstream 5' splice sites in a polar fashion without activating exon skipping, In a natural beta-con glycinin transcript, which does not contain cryptic 5' splice sites, m utation of the first nucleotide of the downstream intron activated an array of noncanonical 5' and 3' splice sites and some exon skipping.