Pre-mRNA transcripts in a variety of organisms, including plants, Drosophila and Caenorhabiditis elegans, contain introns which are significantly richer in adenosine and uridine residues than their flanking exons. Previous analyses using exonic and intronic replacements between two nonequivalent 5′ splice sites in the 469 nt long rbcS3A intron 1 provided the first evidence indicating that, in both tobacco and Drosophila nuclei, 5′ splice site selection is strongly influenced by the position of that site relative to the AU transition point between exon and intron. To differentiate between two potential models for 5′ splice site recognition, we have expressed a completely different set of intronic and exonic replacement constructs containing identical 5′ splice sites upstream of β-conglycinin intron 4 (115 nt). Mutagenesis and deletion of the upstream 5′ splice site demonstrate that intronic AU-rich sequences function by promoting recognition of the most upstream 5′ splice site rather than by masking the downstream 5′ splice site. Sequence insertions define a role for AG-rich exonic sequences in plant pre-mRNA splicing by demonstrating that an AG-rich element is capable of promoting downstream 5′ splice site recognition. We conclude that AU-rich intronic sequences, AG-rich exonic sequences and the 5′ splice site itself collectively define 5′ intron boundaries in dicot nuclei.
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