Structural analysis of elements contributing to 5' splice site selection in plant pre-mRNA transcripts.

In vivo analyses of the cis-acting sequence requirements for pre-mRNA splicing in tobacco nuclei have previously demonstrated that 5' splice sites are selected by their position relative to AU-rich sequences within plant introns and by their degree of complementarity to the 5' end of U1 snRNA. To identify the specific nucleotide sequences promoting recognition of the 5' exon-intron boundary, multiple mutations have been introduced into a 22 nt AU-rich block positioned between two competing 5' splice sites in a derivative of pea rbcS3A intron 1. Transient expression of these mutant transcripts has delineated a 9 nt element positioned 30-38 nt downstream from the 5' splice site whose sequence composition affects 5' splice site choice. Uridine substitutions within this element do not alter 5' splice selection patterns, and, in fact, enhance recognition of the upstream +1wt 5' splice site slightly. Guanosine substitutions in this region, specifically creating an AG-rich AAAGGAGAGGCAGA motif (mutations shown underlined), reduce recognition of the upstream +1wt 5' splice site and enhance recognition of the downstream +106E 5' splice site. Cytosine substitutions at homologous positions marginally reduce recognition of the upstream 5' splice site. Additional mutations in this 9 nt element suggest that the AAAGGAGAGGCAGA motif acts as an specific 5' exon-defining element promoting recognition of downstream 5' splice sites, while AU-rich motifs promote recognition of upstream 5' splice sites. Mutations in an adjacent AU-rich region attenuate the effects of mutations in this short element but are not in themselves sufficient to alter 5' splice site selection. It is concluded that specific elements on both sides of the exon-intron boundary define the 5' splice site in this plant transcript.