A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA.
The mammalian thyroid hormone receptor gene c-erbAalpha gives rise to two mRNAs that code for distinct isoforms, TRalpha1 and TRalpha2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRalpha1-specific polyadenylation site or TRalpha2-specific 5' splice site. A previous investigation of TRalpha minigene expression defined a critical role for the TRalpha2 5' splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEa2, enhance splicing of TRalpha2 in vitro as well as in vivo. Although SEalpha2 is located within the intron of TRalpha2 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 SEalpha2 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 SEalpha2. SEalpha2 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 SEa2 and its associated factors are required for splicing of TRalpha2 pre-mRNA.
Hastings
M L
ML
Department of Biology, Marquette University, Milwaukee, Wisconsin 53201, USA.
Wilson
C M
CM
Munroe
S H
SH
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004330:Drosophila; D004742:Enhancer Elements, Genetic; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D011687:Purines / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone / Q000235:genetics*; D012261:Ribonucleoproteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D011687:Purines; D012333:RNA, Messenger; D011988:Receptors, Thyroid Hormone; D012261:Ribonucleoproteins