Addition of A- and U-rich sequence increases the splicing efficiency of a deleted form of a maize intron

Abstract

Plant introns are generally short (<200 nt) and AU-rich, and an elevated AU content is necessary for efficient splicing. Further, an intron in some plant genes enhances gene expression by a post-transcriptional mechanism that results in an increase of cytoplasmic mRNA. The specific 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, indicate that large segments of these introns are dispensable for normal function. 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 deletion-impaired Adh1 intron 1 variant by adding back either oligomers of defined 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 adding G- and C-rich oligomers did not improve intron function, but a C-rich oligomer activated a cryptic 3′ acceptor. The addition of internal intron sequence to an impaired intron improved splicing, and in one case, resulted in the activation of a cryptic 3′ acceptor. We present evidence that U-rich sequence immediately upstream of the 3′ splice junction increases splicing efficiency and contributes to, but does not uniquely specify, 3′ acceptor AG choice.