We have identified four purine-rich sequences that act as
splicing enhancer elements to activate the weak 3′
splice site of α-tropomyosin exon 2. These elements
also activate the splicing of heterologous substrates containing
weak 3′ splice sites or mutated 5′ splice sites.
However, they are unique in that they can activate splicing
whether they are placed in an upstream 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 inhibit 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
incubation 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 rescue splicing.
Using gel mobility shift assays, we show that formation
of stable enhancer-specific complexes on α-tropomyosin
exon 2 requires the presence of both SR proteins and the
110-kD protein. By analogy to the doublesex exon
enhancer elements in Drosophila, our results suggest
that assembly of mammalian exon enhancer complexes requires
both SR and non-SR proteins to activate selection of weak
splice sites.