Abstract
The potassium voltage-gated channel subfamily H member 2 (KCNH2) gene encodes the Kv11.1 potassium channel, which conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation and forms a functional, full-length Kv11.1a isoform if exon 15 is polyadenylated or a nonfunctional, C-terminally truncated Kv11.1a-USO isoform if intron 9 is polyadenylated. The molecular mechanisms that regulate Kv11.1 isoform expression are poorly understood. In this study, using HEK293 cells and reporter gene expression, pulldown assays, and RNase protection assays, we identified the RNA-binding proteins Hu antigen R (HuR) and Hu antigen D (HuD) as regulators of Kv11.1 isoform expression. We show that HuR and HuD inhibit activity at the intron 9 polyadenylation site. When co-expressed with the KCNH2 gene, HuR and HuD increased levels of the Kv11.1a isoform and decreased the Kv11.1a-USO isoform in the RNase protection assays and immunoblot analyses. In patch clamp experiments, HuR and HuD significantly increased the Kv11.1 current. siRNA-mediated knockdown of HuR protein decreased levels of the Kv11.1a isoform and increased those of the Kv11.1a-USO isoform. Our findings suggest that the relative expression levels of Kv11.1 C-terminal isoforms are regulated by the RNA-binding HuR and HuD proteins.
Highlights
The potassium voltage-gated channel subfamily H member 2 (KCNH2) gene encodes the Kv11.1 potassium channel, which conducts the rapidly activating delayed rectifier current in the heart
As a first step in demonstrating whether Hu proteins can regulate KCNH2 intron 9 alternative polyadenylation, we used a reporter construct containing the Renilla luciferase gene downstream of a splicing competent minigene composed of human KCNH2 genomic DNA from exon 8 to exon 11 [21, 22]
Our present experiments reveal that Hu antigen R (HuR) and Hu antigen D (HuD) inhibit the poly(A) signal in KCNH2 intron 9 and modulate relative expression of Kv11.1 C-terminal isoforms
Summary
As a first step in demonstrating whether Hu proteins can regulate KCNH2 intron 9 alternative polyadenylation, we used a reporter construct containing the Renilla luciferase gene downstream of a splicing competent minigene composed of human KCNH2 genomic DNA from exon 8 to exon 11 [21, 22]. Expression of HuR, HuD, Sam, and AUF1 in transfected cells was confirmed by immunoblot analysis (Fig. S1) These results suggest that HuR and HuD, but not Sam and AUF1, may inhibit intron 9 polyadenylation and promote intron 9 splicing, leading to an increase in the luciferase activity. RPA analysis showed that transfection of HuR or HuD resulted in an increase in the Kv11.1a transcript and a decrease in the Kv11.1a-USO transcript (Fig. 4, B and C) This result indicates that relative expression of Kv11.1 C-terminal isoforms can be regulated by HuR and HuD. Transient transfection of HuR or HuD into Flp-In HEK293 cells stably expressing the short KCNH2 gene significantly increased the level of Kv11.1a protein and decreased the Kv11.1a-USO protein level (Fig. 5, A and B).
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