Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erb alpha and Characterization of an Exonic G-Rich Element That Regulates Splicing of TR alpha 2 mRNA: e0137893

The alpha -thyroid hormone receptor gene (TR alpha ) codes for two functionally distinct proteins: TR alpha 1, the alpha -thyroid hormone receptor; and TR alpha 2, a non-hormone-binding variant. The final exon of TR alpha 2 mRNA overlaps the 3' end of Rev-erb alpha mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TR alpha /Rev-erb alpha locus lacks elements essential for expression of TR alpha 2. Comparative analysis suggests that alternative splicing of TR alpha 2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30) located downstream of the alternative 3'splice site of TR alpha 2 mRNA and antisense to the 3'UTR of Rev-erb alpha plays an important role in regulating TR alpha 2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TR alpha 2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TR alpha 2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing.