Translation initiation of a cardiac voltage-gated potassium channel by internal ribosome entry

The mammalian Kv1.4 voltage-gated potassium channel mRNA contains an unusually long (1.2 kilobases) 5'-untranslated region (UTR) and includes 18 AUG codons upstream of the authentic site of translation initiation. Computer-predicted secondary structures of this region reveal complex stem-loop structures that would serve as barriers to 5' --> 3' ribosomal scanning. These features suggested that translation initiation in Kv1.4 might occur by the mechanism of internal ribosome entry, a mode of initiation employed by a variety of RNA viruses but only a limited number of vertebrate genes. To test this possibility we introduced the 5'-UTR of mouse Kv1.4 mRNA into the intercistronic region of a bicistronic vector containing two tandem reporter genes, chloramphenicol acetyltransferase and luciferase. The control construct translated only the upstream chloramphenicol cistron in transiently transfected mammalian cells. In contrast, the construct containing the mKv1.4 UTR efficiently translated the luciferase cistron as well, demonstrating the presence of an internal ribosome entry segment. Progressive 5' --> 3' deletions localized the activity to a 3'-proximal 200-nucleotide fragment. Suppression of cap-dependent translation by extracts from poliovirus-infected HeLa cells in an in vitro translation assay eliminated translation of the upstream cistron while allowing translation of the downstream cistron. Our results indicate that the 5'-untranslated region of mKv1.4 contains a functional internal ribosome entry segment that may contribute to unusual and physiologically important modes of translation regulation for this and other potassium channel genes.