Multiple distinct splicing enhancers in the protein-coding sequences of a constitutively spliced pre-mRNA

We have identified multiple distinct splicing enhancer elements within prot ein-coding sequences of the constitutively spliced human beta-globin pre-mR NA. Each of these highly conserved sequences is sufficient to activate the splicing of a heterologous enhancer-dependent pre-mRNA One of these enhance rs is activated by and binds to the SR protein SC35, whereas at least two o thers are activated by the SR protein SF2/ASF. A single base mutation withi n another enhancer element inactivates the enhancer but does not change the encoded amino acid. Thus, overlapping protein coding and RNA recognition e lements may be coselected during evolution. These studies provide the first direct evidence that SR protein-specific splicing enhancers are located wi thin the coding regions of constitutively spliced pre-mRNAs. We propose tha t these enhancers function as multisite splicing enhancers to specify 3' sp lice-site selection.