The rat β-tropomyosin gene encodes two tissue-specific
isoforms that contain the internal, mutually exclusive
exons 6 (nonmuscle/smooth muscle) and 7 (skeletal muscle).
We previously demonstrated that the 3′ splice site
of exon 6 can be activated by introducing a 9-nt polyuridine
tract at its 3′ splice site, or by strengthening
the 5′ splice site to a U1 consensus binding site,
or by joining exon 6 to the downstream common exon 8. Examination
of sequences within exons 6 and 8 revealed the presence
of two purine-rich motifs in exon 6 and three purine-rich
motifs in exon 8 that could potentially represent exonic
splicing enhancers (ESEs). In this report we carried out
substitution mutagenesis of these elements and show that
some of them play a critical role in the splice site usage
of exon 6 in vitro and in vivo. Using UV crosslinking,
we have identified SF2/ASF as one of the cellular factors
that binds to these motifs. Furthermore, we show that substrates
that have mutated ESEs are blocked prior to A-complex formation,
supporting a role for SF2/ASF binding to the ESEs during
the commitment step in splicing. Using pre-mRNA substrates
containing exons 5 through 8, we show that the ESEs within
exon 6 also play a role in cooperation between the 3′
and 5′ splice sites flanking this exon. The splicing
of exon 6 to 8 (i.e., 5′ splice site usage of exon
6) was enhanced with pre-mRNAs containing either the polyuridine
tract in the 3′ splice site or consensus sequence
in the 5′ splice site around exon 6. We show that
the ESEs in exon 6 are required for this effect. However,
the ESEs are not required when both the polyuridine and
consensus splice site sequences around exon 6 were present
in the same pre-mRNA. These results support and extend
the exon-definition hypothesis and demonstrate that sequences
at the 3′ splice site can facilitate use of a downstream
5′ splice site. In addition, the data support the
hypothesis that ESEs can compensate for weak splice sites,
such as those found in alternatively spliced exons, thereby
providing a target for regulation.