Regulation of hERG C-Terminal Isoform Expression by Modified U1 Small Nuclear RNA

Abstract

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. The expression of C-terminal isoforms is directed by the alternative splicing and polyadenylation of intron 9. Splicing of intron 9 leads to the formation of a functional, full-length hERGa channel and polyadenylation of intron 9 results in the production of a non-functional, C-terminally truncated hERGaUSO isoform. The relative expression of hERGa and hERGaUSO plays an important role in regulating hERG channel function. In the heart only one-third of hERG pre-mRNA is processed to hERGa due to the weak 5' splice site of intron 9. We previously showed that the weak splice site is caused by sequence deviation from consensus and that strengthening the splice site with mutations at the +4 and +6 positions toward consensus increased the efficiency of intron 9 splicing. It is well established that 5' splice sites are recognized by complementary base-paring with U1 small nuclear RNA (U1 snRNA). In this study, we modified the sequence of U1 snRNA to increase its complementarity to the 5' splice site of hERG intron 9 at the +4 and +6 positions. Using modified U1 snRNA, we observed a significant increase in the efficiency of intron 9 splicing. RNase protection assay and western blot analysis show that modified U1 snRNA increased the expression of the functional hERGa isoform and concomitantly decreased the expression of the non-functional hERGaUSO isoform. In patch-clamp experiment, modified U1 snRNA significantly increased the hERG current. Our findings suggest that relative expression of hERG C-terminal isoforms can be regulated by modified U1 snRNA.