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

项目摘要/摘要 MU阿片受体在介导吗啡和大多数临床镇痛剂的作用中起着基础性作用， 以及滥用毒品，如海洛因。单拷贝u阿片受体基因(OPRM1)创建了一个阵列 通过经历广泛的选择性前mRNA剪接(AS)，这是保守的 啮齿动物对人类。根据受体结构，这些剪接变异体可分为三种类型：1)全长- 具有7-跨膜(TM)结构域的长度羧基(C-)末端变体；2)含有6- TM结构域；以及3)包含单个TM的截短变体。越来越多的证据表明，OPRM1 因为变种在药理上很重要。几个C-末端的变异体在区域- 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。吗啡诱导的瘙痒与MOR-1D有关。几种变异型信使核糖核酸的失调 在许多细胞和动物模型以及人类疾病中都观察到了这种表达。证据 从我们的三个基因敲除小鼠模型来看，单个C末端序列通过 替代3‘剪接在吗啡的各种行为中扮演着不同的角色，包括耐受、身体依赖 和奖励。此外，脑室内(i.c.v.)一种反义生物体--吗啉的给药 阻断外显子3至外显子7 3‘剪接的反义寡核苷酸显著降低小鼠对吗啡的耐受性 老鼠。总之，这些研究不仅有力地支持了我们的假设，即OPRM1的选择性剪接 有助于调节动物和人类的复杂阿片类药物行为，但也提供了一种令人信服的 进一步研究控制OPRM1替代的分子机制的理论基础和科学前提 利用反义体内吗啡进行剪接及对OPRM1选择性剪接影响的评估 对吗啡作用的寡聚，如本申请中所建议的。此应用程序的主要目标是进一步 用多种体外和体外实验研究OPRM1基因选择性剪接的机制和功能 活体接近了。具体目标包括：1)破译OPRM1 3‘剪接的分子机制 通过确定调控3‘剪接的顺式作用元件和反式作用因子；2)研究其作用 使用反义活体在BE(2)C细胞和小鼠中的MU阿片类药物作用中OPRM1 3‘剪接的研究- 吗啡寡核苷酸方法。拟议中的研究有望对这一机制产生重要的见解 和OPRM1 3‘剪接的功能，并有可能开发新的治疗方法来控制 止痛和减轻Mu阿片类药物的不良副作用。

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.