Regulation of Kv11.1 Isoform Expression by Polyadenylate Binding Protein Nuclear 1.

Matthew R Stump,Delaney Search,Zhengfeng Zhou,Qiuming Gong,Rachel T Nguyen,Rachel H Drgastin

doi:10.3390/ijms22020863

Abstract

The Kv11.1 voltage-gated potassium channel, encoded by the KCNH2 gene, conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation to generate two C-terminal Kv11.1 isoforms in the heart. Utilization of a poly(A) signal in exon 15 produces the full-length, functional Kv11.1a isoform, while intron 9 polyadenylation generates the C-terminally truncated, nonfunctional Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO isoforms plays an important role in the regulation of Kv11.1 channel function. In this study, we tested the hypothesis that the RNA polyadenylate binding protein nuclear 1 (PABPN1) interacts with a unique 22 nt adenosine stretch adjacent to the intron 9 poly(A) signal and regulates KCNH2 pre-mRNA alternative polyadenylation and the relative expression of Kv11.1a C-terminal isoforms. We showed that PABPN1 inhibited intron 9 poly(A) activity using luciferase reporter assays, tandem poly(A) reporter assays, and RNA pulldown assays. We also showed that PABPN1 increased the relative expression level of the functional Kv11.1a isoform using RNase protection assays, immunoblot analyses, and patch clamp recordings. Our present findings suggest a novel role for the RNA-binding protein PABPN1 in the regulation of functional and nonfunctional Kv11.1 isoform expression.

Highlights

The Kv11.1 voltage-gated potassium channel conducts the rapidly activating delayed rectifier current (IKr ) in the heart and contributes to the repolarization of the cardiac action potential [1,2,3]
We have shown that the RNA polyadenylate binding protein nuclear 1 modulates the relative expression of Kv11.1 C-terminal isoforms by suppressing
The increase in the relative expression of the Kv11.1a isoform was determined at the RNA, protein, and functional levels using short KCNH2 gene constructs in RNase protection assay (RPA), immunoblot and patch clamp experiments

Summary

Introduction

The Kv11.1 voltage-gated potassium channel conducts the rapidly activating delayed rectifier current (IKr ) in the heart and contributes to the repolarization of the cardiac action potential [1,2,3]. KCNH2 pre-mRNA undergoes alternative processing to generate two C-terminal isoforms with distinct functional properties [7]. The Kv11.1a isoform represents the full-length functional channel and is generated following the splicing of intron 9 and polyadenylation in exon 15. The Kv11.1a-USO isoform is produced following alternative polyadenylation in intron 9 and lacks 359 C-terminal amino acids present in the Kv11.1a isoform. Kv11.1a-USO isoforms do not form functional channels when expressed in mammalian cells [7,8,9]. Because formation of Kv11.1 C-terminal isoforms from KCNH2 pre-mRNA is mutually exclusive, the nonfunctional Kv11.1a-USO isoform is generated at the expense of the functional

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Conclusion