AU-Rich Intronic Elements Affect Pre-mRNA 5′ Splice Site Selection in Drosophila melanogaster

Abstract

cis-spliced nuclear pre-mRNA introns found in a variety of organisms, including Tetrahymena thermophila, Drosophila melanogaster, Caenorhabditis elegans, and plants, are significantly richer in adenosine and uridine residues than their flanking exons are. The functional significance of this intronic AU richness, however, has been demonstrated only in plant nuclei. In these nuclei, 5′ and 3′ splice sites are selected in part by their positions relative to AU-rich elements spread throughout the length of an intron. Because of this position-dependent selection scheme, a 5′ splice site at the normal (+1) exon-intron boundary having only three contiguous consensus nucleotides can compete effectively with an enhanced exonic site (-57E) having nine consensus nucleotides and outcompete an enhanced site (+106E) embedded within the AU-rich intron. To determine whether transitions from AU-poor exonic sequences to AU-rich intronic sequences influence 5′ splice site selection in other organisms, alleles of the pea rbcS3A1 intron were expressed in Drosophila Schneider 2 cells, and their splicing patterns were compared with those in tobacco nuclei. We demonstrate that this heterologous transcript can be accurately spliced in transfected Drosophila nuclei and that a +1 G-to-A knockout mutation at the normal splice site activates the same three cryptic 5′ splice sites as in tobacco. Enhancement of the exonic (-57) and intronic (+106) sites to consensus splice sites indicates that potential splice sites located in the upstream exon or at the 5′ exon-intron boundary are preferred in Drosophila cells over those embedded within AU-rich intronic sequences. In contrast to tobacco, in which the activities of two competing 5′ splice sites upstream of the AU-rich intron are modulated by their proximity to the AU transition point, D. melanogaster utilizes the upstream site which has a higher proportion of consensus nucleotides. The enhanced version of the cryptic intronic site is efficiently selected in D. melanogaster when the normal +1 site is weakened or discrete AU-rich elements upstream of the +106E site are disrupted. Selection of this internal site in tobacco requires more drastic disruption of these motifs. We conclude that 5′ splice site selection in Drosophila nuclei is influenced by the intrinsic strengths of competing sites and by the presence of AU-rich intronic elements but to a different extent than in tobacco.