Mutations underlying Episodic Ataxia type-1 antagonize Kv1.1 RNA editing

Elizabeth A Ferrick-Kiddie,Joshua J C Rosenthal,Ronald B Emeson,Gregory D Ayers

doi:10.1038/srep41095

Abstract

Adenosine-to-inosine RNA editing in transcripts encoding the voltage-gated potassium channel Kv1.1 converts an isoleucine to valine codon for amino acid 400, speeding channel recovery from inactivation. Numerous Kv1.1 mutations have been associated with the human disorder Episodic Ataxia Type-1 (EA1), characterized by stress-induced ataxia, myokymia, and increased prevalence of seizures. Three EA1 mutations, V404I, I407M, and V408A, are located within the RNA duplex structure required for RNA editing. Each mutation decreased RNA editing both in vitro and using an in vivo mouse model bearing the V408A allele. Editing of transcripts encoding mutant channels affects numerous biophysical properties including channel opening, closing, and inactivation. Thus EA1 symptoms could be influenced not only by the direct effects of the mutations on channel properties, but also by their influence on RNA editing. These studies provide the first evidence that mutations associated with human genetic disorders can affect cis-regulatory elements to alter RNA editing.

Highlights

The Kv1.1 voltage-gated potassium (Kv) channel α-subunit plays an important role in regulating neuronal excitability
Three known Episodic ataxia type-1 (EA1)-associated mutations, V404I, I407M, and V408A, lie within the predicted 114-bp RNA duplex, which represents the minimum sequence required for site-specific editing of Kv1.1 transcripts by ADAR2 (Fig. 1a)[15]
Using an RNA-folding algorithm[30], we examined whether any of these mutations were predicted to grossly alter the structure of the duplex region. Results from this analysis revealed that each individual mutation predicted a single-nucleotide mismatch within the duplex at each mutation site, with no further perturbations to the predicted RNA secondary structure and only minimal alterations in the free-energy (ΔG) calculations for each duplex[31]. To test whether these mutations affected the rate of editing for Kv1.1 RNAs, each of the EA1-associated mutations was incorporated separately into constructs encompassing a 463-bp region centered on the known editing site

Summary

Introduction

The Kv1.1 voltage-gated potassium (Kv) channel α-subunit plays an important role in regulating neuronal excitability. RNA transcripts encoding Kv1.1 are modified by a site-specific adenosine-to-inosine (A-to-I) RNA editing event in which a genomically-encoded isoleucine (AUU) is converted to a valine (IUU) codon at amino acid position 400 of the protein[14]. This amino acid lies within the S6 transmembrane domain predicted to line the ion-conducting pore of the channel. Editing of Kv1.1 transcripts is dependent upon a region of double-stranded RNA (dsRNA) formed by intramolecular base-pairing interactions between imperfect, inverted repeat elements surrounding the targeted adenosine moiety[15] This process is catalyzed by ADAR2, a member of a family of dsRNA-specific adenosine deaminases (ADARs)[15,16]. Do our studies demonstrate that EA1 mutations can impede RNA editing and alter resulting protein function, and represent the first examples of existing disease-associated human mutations which act to disrupt cis-regulatory elements required for RNA editing

Methods

Results

Conclusion