Approximately half of the introns in Drosophila melanogaster are too small to function in a vertebrate and often lack the pyrimidine tract associated with vertebrate 3' splice sites. Here, we report the splicing and spliceosome assembly properties of two such introns: one with a pyrimidine-poor 3' splice site and one with a pyrimidine-rich 3' splice site. The pyrimidine-poor intron was absolutely dependent on its small size for in vivo and in vitro splicing and assembly. As such, it had properties reminiscent of those of yeast introns. The pyrimidine-rich intron had properties intermediate between those of yeasts and vertebrates. This 3' splice site directed assembly of ATP-dependent complexes when present as either an intron or exon and supported low levels of in vivo splicing of a moderate-length intron. We propose that splice sites can be recognized as pairs across either exons or introns, depending on which distance is shorter, and that a pyrimidine-rich region upstream of the 3' splice site facilitates the exon mode.Pairing of individual splice sites during early recognition of pre-mRNAs is an attractive model for the orchestration of splicing in genes with multiple introns. Indeed, in Saccharomyces cerevisiae, direct support exists for the pairing of splice sites across introns during the first step of spliceosome assembly (4,8,30,31). In vertebrate pre-mRNAs, however, initial interaction between factors recognizing both ends of an intron is problematic because of the large size of many vertebrate introns. We have recently suggested that the exon is the unit of recognition during early spliceosome assembly in vertebrates with the concomitant pairing of splice sites across exons (29,34). Interactive binding of factors across exons is feasible in vertebrates because internal exons are quite small, rarely exceeding 300 nucleotides (10).Exon-intron architecture varies widely among eukaryotes. Although vertebrate exons are quite small and cannot be expanded without loss of recognition (29), many genes in lower eukaryotes have large exons (10). Most of these genes with large exons have small introns; however, some have large introns. If splice sites are paired, these genes have an awkward array of large and small distances between pairs of sites, making the mechanism of splice site recognition in these pre-mRNAs unclear. To address this problem, we have turned to the study of splice site recognition in Drosophila melanogaster. The majority of Drosophila exons are 100 to 180 nucleotides in length; however, 15% are more than 550 nucleotides, which is predicted to be too large to function in a vertebrate (10). This difference suggests that pairing of splice sites across exons will not work for recognition of these large exons.A comparison of intron sizes between vertebrates and Drosophila species also reveals another obvious difference from vertebrates. More First, Drosophila introns tend not to have a G in the position preceding the branched nucleotide (24). G is the most common nucleotide at this position in ma...
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