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

项目总结/摘要 μ阿片受体在介导吗啡和大多数临床镇痛药的作用中起着重要作用， 以及滥用药物，如海洛因。单拷贝μ阿片受体基因（OPRM 1）创建一个阵列 剪接变异体通过经历广泛的选择性前mRNA剪接（AS），这是保守的， 从啮齿动物到人类这些剪接变体基于受体结构分为三种类型：1）全剪接变体， 具有7-跨膜（TM）结构域的长度羧基（C-）末端变体; 2）含有6-跨膜（TM）结构域的截短变体; TM结构域;和3）含有单个TM的截短变体。越来越多的证据表明，OPRM 1 AS变体非常重要。几种C-末端变体在区域上显示出明显的差异- 特异性表达，mu激动剂诱导的G蛋白偶联，磷酸化，内化和后 内吞分选莫尔-1D负责吗啡诱导的瘙痒。几种变体mRNA的失调 已经在许多细胞和动物模型以及人类疾病中观察到表达。证据 从我们的三个敲除小鼠模型中发现， 选择性3'剪接在吗啡的多种作用中起着不同的作用，包括耐受性、身体依赖性 和奖励此外，脑室内（i. c. v.）施用反义活体吗啉代 反义寡核苷酸阻断外显子3至外显子7的3'剪接，显著减弱吗啡耐受。 小鼠总之，这些研究不仅有力地支持了我们的假设，即OPRM 1选择性剪接 有助于调节动物和人类复杂的阿片类药物作用，但也提供了一个令人信服的 为进一步研究OPRM 1替代物的分子调控机制提供了理论基础和科学前提 剪接和评估使用反义体内吗啉代调节OPRM 1选择性剪接的影响 寡核苷酸对吗啡作用的影响，如本申请中所提出的。该应用程序的主要目标是进一步 利用多种体外和体内方法研究OPRM 1基因选择性剪接的机制和功能 体内方法。具体目标包括：1）解码OPRM 1 3'剪接的分子机制 通过鉴定调节3'剪接的顺式作用元件和反式作用因子; 2）调查3'剪接的作用， 在Be（2）C细胞和小鼠中使用反义体内- 吗啉代寡聚物方法。拟议中的研究有望对该机制产生重要的见解 和OPRM 1 3'剪接的功能，并可能有潜力开发新的治疗方法，以控制 疼痛和减轻μ阿片类药物的有害副作用。

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.