Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD

Qiuming Gong, Matthew R. Stump, Zhengfeng Zhou

    Research output: Contribution to journalArticle

    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.

    Original languageEnglish (US)
    Pages (from-to)19624-19632
    Number of pages9
    JournalJournal of Biological Chemistry
    Volume293
    Issue number51
    DOIs
    StatePublished - Jan 1 2018

    Fingerprint

    ELAV Proteins
    RNA-Binding Proteins
    Potassium Channels
    Protein Isoforms
    Assays
    Polyadenylation
    Ribonucleases
    Introns
    Genes
    RNA Isoforms
    Voltage-Gated Potassium Channels
    HEK293 Cells
    RNA Precursors
    Clamping devices
    Reporter Genes
    Gene expression
    Small Interfering RNA

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Cite this

    Regulation of Kv11.1 potassium channel C-terminal isoform expression by the RNA-binding proteins HuR and HuD. / Gong, Qiuming; Stump, Matthew R.; Zhou, Zhengfeng.

    In: Journal of Biological Chemistry, Vol. 293, No. 51, 01.01.2018, p. 19624-19632.

    Research output: Contribution to journalArticle

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    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.",
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