Upregulation of functional Kv11.1a isoform expression by modified U1 small nuclear RNA.

The KCNH2 or human ether-a go-go-related gene (hERG) encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier potassium current in the heart. The expression of Kv11.1 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 Kv11.1a isoform and polyadenylation of intron 9 results in the production of a non-functional, C-terminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO plays an important role in regulating Kv11.1 channel function. In the heart, only one-third of KCNH2 pre-mRNA is processed to Kv11.1a due to the weak 5' splice site of intron 9. We previously showed that the weak 5' splice site is caused by sequence deviation from the consensus, and that mutations toward the consensus sequence 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 KCNH2 intron 9 and observed a significant increase in the efficiency of intron 9 splicing. RNase protection assay and western blot analysis showed that modified U1 snRNA increased the expression of the functional Kv11.1a isoform and concomitantly decreased the expression of the non-functional Kv11.1a-USO isoform. In patch-clamp experiments, modified U1 snRNA significantly increased Kv11.1 current. Our findings suggest that relative expression of Kv11.1 C-terminal isoforms can be regulated by modified U1 snRNA.

Nonsense-mediated mRNA decay caused by a frameshift mutation in a large kindred of type 2 long QT syndrome.

BACKGROUND: Nonsense and frameshift mutations are common in congenital long QT syndrome type 2 (LQT2). We previously demonstrated that hERG nonsense mutations cause degradation of mutant mRNA by nonsense-mediated mRNA decay (NMD) and are associated with mild clinical phenotypes. The impact of NMD on the expression of hERG frameshift mutations and their phenotypic severity is not clear. OBJECTIVE: The purpose of this study was to examine the role of NMD in the pathogenesis of a hERG frameshift mutation, P926AfsX14, identified in a large LQT2 kindred and characterize genotype-phenotype correlations. METHODS: Genetic screening was performed among family members. Phenotyping was performed by assessment of ECGs and LQTS-related cardiac events. The functional effect of P926AfsX14 was studied using hERG cDNA and minigene constructs expressed in HEK293 cells. RESULTS: Significant cardiac events occurred in carriers of the P926AfsX14 mutation. When expressed from cDNA, the P926AfsX14 mutant channel was only mildly defective. However, when expressed from a minigene, the P926AfsX14 mutation caused a significant reduction in mutant mRNA, protein, and hERG current. Inhibition of NMD by RNA interference knockdown of up-frameshift protein 1 partially restored expression of mutant mRNA and protein and led to a significant increase in hERG current in the mutant cells. These results suggest that NMD is involved in the pathogenic mechanism of the P926AfsX14 mutation. CONCLUSION: Our findings suggest that the hERG frameshift mutation P926AfsX14 primarily results in degradation of mutant mRNA by the NMD pathway rather than production of truncated proteins. When combined with environmental triggers and genetic modifiers, LQT2 frameshift mutations associated with NMD can manifest with a severe clinical phenotype.