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

• 批准号: 10442308
• 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
• 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/10442308
• 关键词: 5' Splice Site; Action Potentials; Adult; Alternative Splicing; Antineoplastic Agents; Arrhythmia; Cardiac; Cardiac Myocytes; Clinical Trials; Development; Disease; Enhancers; Ethers; Exons; Genes; Genetic Transcription; 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; cardiogenesis; cis acting element; drug development; heart rhythm; inhibitor; mRNA Precursor; mouse model; mutant; novel; potassium ion; protein expression; sudden cardiac death

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.

KCNH 2基因（人类ether-a-go-go相关基因，hERG）编码Kv11.1 K+通道， 传导心脏中快速激活的延迟整流钾电流（IKr）。Kv11.1频道有助于 心脏动作电位的复极，并在遗传和药物诱导的 QT间期延长综合征在KCNH 2中已经鉴定出几种可选择加工的mRNA亚型。 其中两种亚型Kv11.1a和Kv 11.1a-USO在心脏中高水平表达。我们已经表明 Kv11.1a和Kv 11.1a-USO的表达是通过选择性剪接和多聚腺苷酸化 KCNH 2前mRNA。全长Kv11.1a同种型通过内含子9的剪接和多聚腺苷酸化产生 在外显子15的poly（A）位点，而Kv 11.1a-USO通过在内含子9的poly（A）位点的聚腺苷酸化产生。 因为只有Kv11.1a亚型是有功能的，所以KCNH 2前体mRNA的替代加工代表了一种新的表达方式。 调节Kv11.1通道功能的转录后机制。更重要的是， 调节导致长QT综合征。Kv 11. 1a和Kv 11.1a-USO的相对表达量分别为： 在心脏中发育调节。Kv11.1亚型的调控机制 表达不完全理解。在本申请中，我们建议研究分子机制 KCNH 2前体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Kv11.1亚型表达的调节以及遗传和 药物引起的长QT综合征具体目标是：目标1：确定调节细胞凋亡的顺式作用元件。 KCNH 2前体mRNA的选择性剪接和多聚腺苷酸化，并开发恢复表达的策略 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CELF 1、CELF 2和SF 3B 1对Kv11.1亚型表达的调节作用。目标三： 研究Kv11.1亚型在人源化基因敲入中表达的发育调控机制， 小鼠模型目的4：研究新型抗肿瘤药物SF 3B 1抑制剂对Kv11.1亚型的影响 表情这些研究的结果将提供有关的分子机制， KCNH 2前mRNA的选择性剪接和多聚腺苷酸化，以及这一过程如何在正常和 疾病状况。阐明这些机制将加强我们对转录后的理解 调节Kv11.1通道的表达，并在评估促癌基因的表达方面具有重要意义。 药物开发过程中的责任。