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

Abstract

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.

Keywords: Alternative polyadenylation; Arrhythmia; Long QT syndrome; Splicing; U1 snRNA; hERG.

Fig. 1

Effect of modified U1 snRNA on splicing of KCNH2 intron 9 using a luciferase reporter construct. (A) Diagram of the KCNH2 minigene luciferase reporter construct. (B) Diagram of the modified U1 snRNA that increases its complementarity to the 5′ splice site of KCNH2 intron 9 at the +4 and +6 positions. (C) Histogram showing the effect of modified U1 snRNA on luciferase activity (n=6-7, ***P < 0.001).

Fig. 2

Regulation of Kv11.1 isoform expression by modified U1 snRNA in the full-length KCNH2 splicing-competent construct. (A) The structure of the full-length KCNH2 splicing-competent construct. (B) RPA analysis of the effect of modified U1 snRNA on Kv11.1 isoform expression. Flp-In CV-1 cells stably expressing the full-length KCNH2 splicing-competent construct were treated with WT or modified U1 snRNA adenovirus (500 MOI) for 48 hours. (C) RPA signals were quantified, normalized to hygromycin resistance gene (Hygro), and plotted as the relative expression of the total uninfected (1a+1a-USO) Kv11.1 mRNA (n=3, **P < 0.01).

Fig. 3

Regulation of Kv11.1 protein expression by modified U1 snRNA. (A) Flp-In CV-1 cells stably expressing the full-length KCNH2 splicing-competent construct were treated with WT or modified U1 snRNA adenovirus (250 or 500 MOI) for 48 hours. The expression level of hygromycin B phosphotransferase (HPT) encoded by the hygromycin B resistant gene served as a loading control. Cell lysates were subjected to SDS-PAGE and probed with antibodies against the N-terminus of Kv11.1 and against HPT. (B) The level of protein bands was quantified, normalized to HPT, and plotted as the relative expression of the total uninfected (1a+1a-OSU) Kv11.1 protein (n=3, *P < 0.05, ***P < 0.001).

Fig. 4

Effect of modified U1snRNA on Kv11.1 channel current. (A) Representative currents recorded from Flp-In CV-1 cells stably expressing following treatment with WT or modified U1 snRNA adenovirus (500 MOI) for 48 hours. (B) I-V plot of tail current density measured at –50 mV following test voltages from –70 to +50 mV for WT (triangle, n=6) and modified U1 snRNA (3A>C/5U>A) (circle, n=8) adenoviruses.

Fig. 5

Regulation of Kv11.1 isoform expression by modified U1 snRNA in canonical poly(A) signal construct. (A) RPA analysis of mRNA from Flp-In CV-1 cells stably expressing the full-length KCNH2 splicing-competent construct containing the canonical poly(A) signal following the treatment with WT or modified U1 snRNA adenovirus. (B) RPA signals were quantified, normalized to hygromycin resistance gene (Hygro), and plotted as the relative expression of the total uninfected (1a+1a-USO) Kv11.1 mRNA (n=3, *P < 0.05, **P < 0.01).

Fig. 6

Effect of modified U1 snRNA on Kv11.1 channel current in canonical poly(A) signal construct. (A) Representative currents recorded from Flp-In CV-1 cells stably expressing the full-length KCNH2 splicing-competent construct containing the canonical poly(A) signal following treatment with WT or modified U1 snRNA adenovirus (500 MOI) for 48 h. (B) I-V plot of tail current density measured at –50 mV following test voltages from –70 to +50 mV for WT (triangle, n=7) and modified U1 snRNA (3A>C/5U>A) (circle, n=8) adenoviruses.