Insights into the evolution and regulation of hybrid internal-terminal exons from tropomyosin exon 9A in Xenopus laevis.

Hybrid internal-terminal exons function as either internal or terminal exons. Their evolutionary origins remain unclear. Here, we investigate the phylogenetic origin and regulation of a hybrid exon, 9A9', in the TPM1 gene encoding tropomyosin alpha-1. We demonstrate that exon 9A9' was originally terminal in non-vertebrate deuterostomes and switched to internal in vertebrates through the exonization of a downstream exon, 9B. While the terminal nature of exon 9A9' was lost in most vertebrates, it was conserved in amphibians and coelacanths where it behaves as a hybrid internal-terminal exon. Using Xenopus laevis as a model, we show that the preservation of terminal exon 9A9' in the tpm1 gene likely arose from evolutionary pressures to mitigate the developmental toxicity linked to exon 9B inclusion during neurulation. We identify two peculiarities of terminal exon 9A9': it lies downstream of an AG-independent intron, and its definition is supported by an intronic cis-regulatory element, the UTE, which enhances recognition of the weak cleavage-polyadenylation site. Our findings characterize the molecular mechanisms underlying the regulation of hybrid internal-terminal exons and reveal how evolutionary pressures can reactivate vestigial traits to resolve developmental challenges. This work broadens our understanding of alternative splicing evolution and its significance in vertebrate development.