The mammalian thyroid hormone receptor gene c-erbAα
gives rise to two mRNAs that code for distinct isoforms,
TRα1 and TRα2, with antagonistic functions. Alternative
processing of these mRNAs involves the mutually exclusive
use of a TRα1-specific polyadenylation site or TRα2-specific
5′ splice site. A previous investigation of TRα
minigene expression defined a critical role for the TRα2
5′ splice site in directing alternative processing.
Mutational analysis reported here shows that purine residues
within a highly conserved intronic element, SEα2, enhance
splicing of TRα2 in vitro as well as in vivo. Although
SEα2 is located within the intron of TRα2 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 SEα2 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
SEα2. SEα2 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 SEα2 and its associated factors
are required for splicing of TRα2 pre-mRNA.