A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA

The mammalian thyroid hormone receptor gene c-erbA alpha gives rise to two mRNAs that code for distinct isoforms, TR alpha1 and TR alpha2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TR alpha1-specific polyadenylation site or TR alpha2-specific 5' splice site. A previous investigation of TR alpha minigene expression defined a critical role for the TR alpha2 5' splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SE alpha2, enhance splicing of TR alpha2 in vitro as well as in vivo. Although SE alpha2 is located within the intron of TR alpha2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SE alpha2 functions by binding trans-acting factors in HeLa nuclear extract. Protein-RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SE alpha2. SE alpha2 also includes an element resembling a 5' splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5' splice site sequence have a dramatic effect on splicing and protein binding. Thus SE alpha2 and its associated factors are required for splicing of TR alpha2 pre-mRNA.