Enhancer elements activate the weak 3 ' splice site of alpha-tropomyosin exon 2

We have identified four purine-rich sequences that act as splicing enhancer elements to activate the weak 3' splice site of alpha-tropomyosin exon 2. These elements also activate the splicing of heterologous substrates contai ning weak 3' splice sites or mutated 5' splice sites. However, they are uni que in that they can activate splicing whether they are placed in an upstre am or downstream exon, and the two central elements can function regardless of their position relative to one another. The presence of excess RNAs containing these enhancers could effectively in hibit in vitro pre-mRNA splicing reactions in a substrate-dependent manner and, at lower concentrations of competitor RNA, the addition of SR proteins could relieve the inhibition. However, when extracts were depleted by incu bation with biotinylated exon 2 RNAs followed by passage over streptavidin agarose, SR proteins were not sufficient to restore splicing. Instead, both SR proteins and fractions containing a 110-kD protein were necessary to re scue splicing. Using gel mobility shift assays, we show that formation of s table enhancer-specific complexes on alpha-tropomyosin exon 2 requires the presence of both SR proteins and the 110-kD protein. By analogy to the daub lesex exon enhancer elements in Drosophila, our results suggest that assemb ly of mammalian exon enhancer complexes requires both SR and non-SR protein s to activate selection of weak splice sites.