ADDITION OF A-RICH AND U-RICH SEQUENCE INCREASES THE SPLICING EFFICIENCY OF A DELETED FORM OF A MAIZE INTRON

Plant introns are generally short (< 200 nt) and AU-rich, and an eleva ted AU content is necessary for efficient splicing. Further, an intron in some plant genes enhances gene expression by a post-transcriptiona l mechanism that results in an increase of cytoplasmic mRNA. The speci fic intron features responsible for efficient splicing and enhancement are not well characterized in plants. Internal deletions of up to 80% of two maize introns, Adh1 intron 1 and maize actin intron 3, indicat e that large segments of these introns are dispensable for normal func tion. However, extensive deletion (> 75%) of Adh1 intron 1 diminishes both intron enhancement and splicing efficiency. This finding suggests that there are internal sequence motifs required for intron function, and that these motifs are redundant. We attempted to repair a deletio n-impaired Adh1 intron 1 variant by adding back either oligomers of de fined sequence content or fragments of maize internal intron sequence. The addition of AU-rich oligomers improved splicing efficiency and in one example, a U-rich oligomer activated a cryptic 3' splice acceptor . We also found that replacing the region proximal to the Adh1 intron 1 3' acceptor with U-rich sequence improved splicing. We found that ad ding G- and C-rich oligomers did not improve intron function, but a C- rich oligomer activated a cryptic 3' acceptor. The addition ofinternal intron sequence to an impaired intron improved splicing, and in one c ase, resulted in the activation of a cryptic 3' acceptor. We present e vidence that U-rich sequence immediately upstream of the 3' splice jun ction increases splicing efficiency and contributes to, but does not u niquely specify, 3' acceptor AG choice.