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Post-transcriptional regulation of Kv11.1 (hERG) channel expression by alternative splicing and polyadenylation

通过选择性剪接和多聚腺苷酸化对 Kv11.1 (hERG) 通道表达进行转录后调控

基本信息

批准号：
10626127
负责人：
ZHENGFENG ZHOU
金额：
$ 46.09万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10626127
关键词：
5&apos Splice Site Action Potentials Adult Alternative Splicing Antineoplastic Agents Arrhythmia Cardiac Cardiac Myocytes Clinical Trials Development Disease Enhancers Ethers Exons Genes Goals Heart Heart Diseases Human Inherited Introns Knock-in Mouse Length Long QT Syndrome Membrane Transport Proteins Messenger RNA Molecular Mus Mutation Myocardial dysfunction Pathogenicity Pharmaceutical Preparations Play Poly A Polyadenylation Post-Transcriptional Regulation Potassium Channel Process Protein Isoforms Proteins RNA RNA Splicing RNA-Binding Proteins Regulation Role Signal Transduction Site Testing Trans-Activators Type 2 Long QT syndrome cardiogenesis cis acting element drug development heart rhythm inhibitor mRNA Precursor mouse model mutant novel posttranscriptional potassium ion protein expression sudden cardiac death

项目摘要

The KCNH2 gene (human ether-a-go-go-related gene, hERG) encodes the Kv11.1 K+ channel that conducts the rapidly activating delayed rectifier K+ current (IKr) in the heart. The Kv11.1 channel contributes to the repolarization of cardiac action potentials and plays an important role in both inherited and drug-induced forms of long QT syndrome. Several alternatively processed mRNA isoforms have been identified in KCNH2. Two of these isoforms, Kv11.1a and Kv11.1a-USO, are expressed at high levels in the heart. We have shown that expression of Kv11.1a and Kv11.1a-USO are generated by alternative splicing and polyadenylation of KCNH2 pre-mRNA. The full-length Kv11.1a isoform is produced by the splicing of intron 9 and polyadenylation at a poly(A) site in exon 15, whereas Kv11.1a-USO is generated by polyadenylation at a poly(A) site in intron 9. Because only the Kv11.1a isoform is functional, the alternative processing of KCNH2 pre-mRNA represents a post-transcriptional mechanism that regulates Kv11.1 channel function. More importantly, disruption of this regulation leads to long QT syndrome. The relative expression of Kv11.1a and Kv11.1a-USO is developmentally regulated in the heart. The mechanisms underlying the regulation of Kv11.1 isoform expression are not fully understood. In the present application, we propose to study the molecular mechanisms underlying alternative splicing and polyadenylation of KCNH2 pre-mRNA and the role of alternative splicing and polyadenylation in regulation of Kv11.1 isoform expression during heart development and in inherited and drug-induced long QT syndrome. The specific aims are: Aim 1: To identify the cis-acting elements that regulate alternative splicing and polyadenylation of KCNH2 pre-mRNA and to develop strategies to restore expression of the functional Kv11.1a isoform disrupted by a long QT syndrome-associated mutation. Aim 2: To study effects of RNA binding proteins CELF1, CELF2 and SF3B1 on regulation of Kv11.1 isoform expression. Aim 3: To study mechanisms of developmental regulation of Kv11.1 isoform expression in a humanized knock-in mouse model. Aim 4: To study effects of SF3B1 inhibitors, a new class of anticancer drugs, on Kv11.1 isoform expression. The results from these studies will provide detailed information about the molecular mechanisms of alternative splicing and polyadenylation of KCNH2 pre-mRNA and how this process is regulated in normal and disease conditions. Elucidating these mechanisms will strengthen our understanding of post-transcriptional regulation of Kv11.1 channel expression and have important implications in the assessment of arrhythmogenic liability during drug development.

KCNH2 基因（人类 ether-a-go-go 相关基因，hERG）编码 Kv11.1 K+ 通道，在心脏中传导快速激活的延迟整流 K+ 电流 (IKr)。 Kv11.1 通道有助于心脏动作电位的复极化，在遗传性和药物诱导性中发挥重要作用长 QT 综合征的形式。 KCNH2 中已鉴定出几种可替代加工的 mRNA 亚型。其中两种亚型 Kv11.1a 和 Kv11.1a-USO 在心脏中高水平表达。我们已经展示了 Kv11.1a 和 Kv11.1a-USO 的表达是通过选择性剪接和聚腺苷酸化产生的 KCNH2 前 mRNA。全长 Kv11.1a 同工型由内含子 9 和多腺苷酸化剪接产生位于外显子 15 的 Poly(A) 位点，而 Kv11.1a-USO 是通过内含子 9 的 Poly(A) 位点多聚腺苷酸化生成的。因为只有 Kv11.1a 同工型具有功能，所以 KCNH2 前体 mRNA 的替代加工代表了调节 Kv11.1 通道功能的转录后机制。更重要的是，这种破坏调节导致长 QT 综合征。 Kv11.1a和Kv11.1a-USO的相对表达量为心脏的发育调节。 Kv11.1 同工型的调控机制表达没有完全理解。在本申请中，我们建议研究分子机制 KCNH2 pre-mRNA 的潜在选择性剪接和多聚腺苷酸化以及选择性剪接的作用和多聚腺苷酸化在心脏发育过程中以及遗传和遗传中 Kv11.1 同工型表达的调节中药物引起的长 QT 综合征。具体目标是：目标 1：确定调节的顺式作用元件 KCNH2 前体 mRNA 的选择性剪接和聚腺苷酸化并制定恢复表达的策略功能性 Kv11.1a 亚型被长 QT 综合征相关突变破坏。目标2：学习 RNA 结合蛋白 CELF1、CELF2 和 SF3B1 对 Kv11.1 亚型表达调节的影响。目标 3：研究人源化敲入中 Kv11.1 亚型表达的发育调控机制鼠标模型。目标 4：研究新型抗癌药物 SF3B1 抑制剂对 Kv11.1 亚型的影响表达。这些研究的结果将提供有关分子机制的详细信息 KCNH2 前体 mRNA 的选择性剪接和聚腺苷酸化以及该过程在正常和疾病情况。阐明这些机制将加强我们对转录后的理解 Kv11.1 通道表达的调节并对致心律失常的评估具有重要意义药物开发期间的责任。