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 antago nistic functions. Alternative processing of these mRNAs involves the mutual ly exclusive use of a TR alpha1-specific polyadenylation site or TR alpha2- specific 5' splice site. A previous investigation of TR alpha minigene expr ession defined a critical role for the TR alpha2 5' splice site in directin g alternative processing. Mutational analysis reported here shows that puri ne 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 lo cated within the intron of TR alpha2 mRNA, it activates splicing of a heter ologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SE alpha2 functions by binding tra ns-acting factors in HeLa nuclear extract. Protein-RNA crosslinking identif ies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SE alpha2. SE alpha2 also includes an element resembling a 5' splice si te consensus sequence that is critical for splicing enhancer activity. Muta tions 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.