Accurate selection of a 5′ splice site requires sequences within fibronectin alternative exon B

Inclusion of fibronectin alternative exon B in mRNA is developmentally regulated. Here we demonstrate that exon B contains two unique purine-rich sequence tracts, PRE1 and PRE2, that are important for proper 5′ splice site selection both in vivo and in vitro. Targeted mutations of both PREs decreased the inclusion of exon B in the mRNA by 50% in vivo. Deletion or mutation of the PREs reduced removal of the downstream intron, but not the upstream intron, and induced the activation of cryptic 5′ splice sites in vitro. PRE-mediated 5′ splice selection activity appears sensitive to position and sequence context. A well characterized exon sequence enhancer that normally acts on the upstream 3′ splice site can partially rescue proper exon B 5′ splice site selection. In addition, we found that PRE 5′ splice selection activity was preserved when exon B was inserted into a heterologous pre-mRNA substrate. Possible roles of these unique activities in modulating exon B splicing are considered.