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

Inclusion of fibronectin alternative exon B in mRNA is developmentally regu lated. Here we demonstrate that exon B contains two unique purine-rich sequ ence tracts, PRE1 and PRE2, that are important for proper 5' splice site se lection both in vivo and in vitro. Targeted mutations of both PREs decrease d 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 se quence context. A well characterized exon sequence enhancer that normally a cts on the upstream 3' splice site can partially rescue proper exon B 5' sp lice site selection. In addition, we found that PRE 5' splice selection act ivity was preserved when exon B was inserted into a heterologous pre-mRNA s ubstrate. Possible roles of these unique activities in modulating exon B sp licing are considered.