AU-RICH INTRONIC ELEMENTS AFFECT PREMESSENGER RNA 5' SPLICE-SITE SELECTION IN DROSOPHILA-MELANOGASTER

cis-spliced nuclear pre-mRNA introns found in a variety of organisms, including Tetrahymena thermophila, Drosophila melanogaster, Caenorhabd itis elegans, and plants, are significantly richer in adenosine and ur idine residues than their flanking exons are. The functional significa nce 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 through out the length of an intron. Because of this position-dependent select ion scheme, a 5' splice site at the normal (+1) exon-intron boundary h aving only three contiguous consensus nucleotides can compete effectiv ely with an enhanced exonic site (-57E) having nine consensus nucleoti des and outcompete an enhanced site (+106E) embedded within the AU-ric h intron. To determine whether transitions from AU-poor exonic sequenc es 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 compare d 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 acti vates the same three cryptic 5' splice sites as in tobacco. Enhancemen t of the exonic (-57) and intronic (+106) sites to consensus splice si tes indicates that potential splice sites located in the upstream exon or at the 5' exon-intron boundary are preferred in Drosophila cells o ver those embedded within AU-rich intronic sequences. In contrast to t obacco, in which the activities of two competing 5' splice sites upstr eam of the AU-rich intron are modulated by their proximity to the AU t ransition 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 w hen the normal +1 site is weakened or discrete AU-rich elements upstre am 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 t he intrinsic strengths of competing sites and by the presence of AU-ri ch intronic elements but to a different extent than in tobacco.