Localization of an exonic splicing enhancer responsible for mammalian natural trans-splicing

Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and transsplicing reactions, which may lead to the synthe sis of two proteins. Generation of the three COT transcripts in rat does no t depend on sex, development, fat feeding, the inclusion of the peroxisome proliferator diethylhexyl phthalate in the diet or hyper-insulinemia. In ad dition, trans-splicing was not detected in COT of other mammals, such as hu man, pig, cow and mouse, or in Cos7 cells from monkey. Rat COT exon 2 conta ins two purine-rich sequences. Mutation of the rat COT exon 2 upstream box does not affect the trans-splicing in vitro between two truncated construct s containing exon 2 and its adjacent intron boundaries. In contrast, mutati on of the downstream box from the rat sequence (GAAGAAG) to a random sequen ce or the sequence observed in the other mammals (AAAAAAA) decreased trans- splicing in vitro. In contrast, mutation of the AAAAAAA box of human COT ex on 2 to GAAGAAG increases trans-splicing. Heterologous reactions between CO T exon 2 from rat and human do not produce trans-splicing. HeLa cells trans fected with minigenes of rat COT sequences produced cis- and trans-spliced bands. Mutation of the GAAGAAG box to AAAAAAA abolished trans-splicing and decreased cis-splicing in vivo. We conclude that GAAGAAG is an exonic splic ing enhancer that could induce natural trans-splicing in rat COT.