Alternative pre-mRNA splicing of mu opioid receptor gene and mu opioid actions

mu阿片受体基因的选择性前mRNA剪接和mu阿片作用

• 批准号: 10257279
• 负责人: YING-XIAN PAN
• 金额: $ 30.79万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10257279
• 关键词: Alternative; pre; mRNA; splicing; mu

Project Summary/Abstract Mu opioid receptors play a fundamental role in mediating the actions of morphine and most clinical analgesics, as well as drugs of abuse, such as heroin. The single-copy mu opioid receptor gene (OPRM1) creates an array of splicing variants by undergoing extensive alternative pre-mRNA splicing (AS), that is conserved from rodents to humans. These splice variants are categorized into three types based on receptor structure: 1) Full- length carboxyl (C-) terminal variants with 7-transmembrane (TM) domains; 2) Truncated variants containing 6- TM domains; and 3) Truncated variants containing single TM. Increasing evidence suggests that the OPRM1 AS variants are pharmacologically important. Several C-terminal variants display marked differences in region- specific expression, mu agonist-induced G protein coupling, phosphorylation, internalization and post- endocytic sorting. MOR-1D is responsible for morphine-induced itch. Dysregulation of several variant mRNA expressions has been observed in a number of cell and animal models, as well as human diseases. Evidence from our three knockout mouse models suggest that individual C-terminal sequences generated through alternative 3' splicing play distinct roles in various morphine actions, including tolerance, physical dependence and reward. Additionally, intracerebroventricular (i.c.v.) administration of an antisense vivo-morpholino antisense oligo, which blocks 3' splicing from exon 3 to exon 7, significantly attenuates morphine tolerance in mice. Together, these studies not only strongly support our hypothesis that OPRM1 alternative splicing contributes to the regulation of complex opioid actions in animals and humans, but also provide a compelling rationale and scientific premise for further study of the molecular mechanisms controlling OPRM1 alternative splicing and assessing the impact of modulating OPRM1 alternative splicing using antisense vivo-morpholino oligos on morphine actions, as proposed in this application. The primary goal of this application is to further investigate mechanisms and functions of OPRM1 gene alternative splicing by using a variety of in vitro and in vivo approaches. The specific aims include: 1) Decoding molecular mechanisms underlying OPRM1 3' splicing by identifying cis-acting elements and trans-acting factors that regulate the 3' splicing; 2) Investigating the role of the OPRM1 3' splicing in mu opioid actions in both Be(2)C cells and mice using an antisense vivo- morpholino oligo approach. The proposed studies promise to generate significant insights into the mechanism and function of OPRM1 3' splicing, and may have the potential for developing new therapeutics for controlling pain and alleviating the detrimental side-effects of mu opioids.

项目摘要/摘要 MU阿片受体在介导吗啡和大多数临床镇痛药的作用中起着基本作用， 以及海洛因等滥用药物。单拷贝MU阿片受体基因（OPRM1）创建阵列 通过进行广泛的替代前MRNA剪接（AS）来保守的剪接变体 啮齿动物到人类。这些剪接变体根据受体结构分为三种类型：1） 长度羧基（C-）末端变体具有7跨膜（TM）域； 2）包含6-的截短变体 TM域； 3）包含单个TM的截短变体。越来越多的证据表明OPRM1 由于变体在药理学上很重要。几种C末端变体显示区域的明显差异 特定表达，MU激动剂诱导的G蛋白偶联，磷酸化，内在化和后 内吞分类。 MOR-1D负责吗啡引起的瘙痒。几个变体mRNA的失调 在许多细胞和动物模型以及人类疾病中都观察到表达。证据 从我们的三个敲除鼠标模型中，表明通过 替代3'剪接在各种吗啡作用中起着不同的作用，包括耐受性，身体依赖性 和奖励。另外，脑室内（i.c.v.）对反义 - 摩尔磷脂的给药 反义寡核，从外显子3到外显子7的剪接3'显着减弱吗啡的耐受性 老鼠。这些研究不仅强烈支持我们的假设，即OPRM1替代剪接 有助于调节动物和人类中复杂的阿片类药物作用，但也提供了令人信服的 基本原理和科学前提，用于进一步研究控制OPRM1替代方案的分子机制 使用反义 - 莫尔菲利诺调制OPRM1替代剪接的剪接和评估的影响 如本应用中提出的那样，对吗啡作用的寡素。该应用的主要目标是进一步 研究OPRM1基因替代剪接的机制和功能，通过使用多种体外和 体内方法。具体目的包括：1）解码OPRM1 3'剪接的分子机制 通过识别调节3'剪接的顺式作用元件和跨作用因子； 2）调查角色 在Be（2）C细胞和小鼠中使用反义的体内，在MU阿片类药物作用中的OPRM1 3'剪接中 Morpholino Oligo方法。拟议的研究有望产生对机制的重要见解 OPRM1 3'拼接的功能，并且可能有可能开发用于控制的新疗法 疼痛并减轻MU阿片类药物的有害副作用。