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

基本信息

批准号：
9550957
负责人：
YING-XIAN PAN
金额：
$ 37.52万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9550957
关键词：
Adverse effects Affinity Chromatography Agonist Alternative Splicing Analgesics Animal Model Animals Antisense Oligonucleotides Artificial Chromosomes Attenuated Bacteria C-terminal Cell Count Cell model Cells Clinical Complex Coupled Coupling Dependence Drug abuse Elements Event Exons GTP-Binding Proteins Genes Goals Heroin Human In Vitro Individual Knockout Mice Knowledge Length Mediating Molecular Molecular Chaperones Monitor Morphine Mus Neuroblastoma Oligonucleotides Opioid Opioid Analgesics Pain Pain management Pharmacology Phosphorylation Physical Dependence Play Protein Isoforms Proteins Pruritus RNA RNA Splicing RNA-Protein Interaction Receptor Gene Regulation Reporter Research Proposals Rewards Rodent Role Scientist Small Interfering RNA Sorting - Cell Movement Structure Structure of thyroid parafollicular cell Tail Trans-Activators Transmembrane Domain Variant base cis acting element deletion analysis drug of abuse high throughput screening human disease in vivo insight mRNA Expression mRNA Precursor morphine tolerance mouse model mu opioid receptors novel novel therapeutics receptor

项目摘要

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.

项目概要/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发挥着重要作用，以及滥用药物，例如海洛因。单拷贝 mu 阿片受体基因 (OPRM1) 创建阵列通过进行广泛的选择性前 mRNA 剪接 (AS) 来产生剪接变体，这是保守的啮齿动物对人类。根据受体结构，这些剪接变体分为三种类型：1) 全剪接变体具有 7 次跨膜 (TM) 结构域的长度羧基 (C-) 末端变体； 2) 包含 6- 的截短变体 TM 域； 3) 包含单个 TM 的截断变体。越来越多的证据表明 OPRM1 AS 变体具有重要的药理学意义。几个 C 端变体在区域上显示出显着差异特异性表达、mu 激动剂诱导的 G 蛋白偶联、磷酸化、内化和后处理内吞分选。 MOR-1D 负责吗啡引起的瘙痒。几种变异 mRNA 的失调已在许多细胞和动物模型以及人类疾病中观察到表达。证据从我们的三个基因敲除小鼠模型中可以看出，单个 C 端序列是通过选择性 3' 剪接在吗啡的各种作用中发挥着不同的作用，包括耐受性、身体依赖性和奖励。此外，脑室内（i.c.v.）给予反义体内吗啉反义寡核苷酸可阻断从外显子 3 到外显子 7 的 3' 剪接，显着减弱吗啡耐受性老鼠。总之，这些研究不仅有力地支持了我们的假设，即 OPRM1 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，同时也提供了令人信服的进一步研究控制OPRM1替代的分子机制的基本原理和科学前提使用反义体内吗啉进行剪接并评估调节 OPRM1 选择性剪接的影响如本申请中所提出的，寡核苷酸对吗啡作用。该应用程序的主要目标是进一步通过使用各种体外和体内研究研究 OPRM1 基因选择性剪接的机制和功能体内逼近。具体目标包括：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反式作用因子； 2）调查角色 OPRM1 3' 剪接在 Be(2)C 细胞和小鼠中 mu 阿片类药物作用中使用反义体内- 吗啉寡核苷酸方法。拟议的研究有望对该机制产生重要的见解 OPRM1 3'剪接的功能，并可能有潜力开发新的疗法来控制疼痛并减轻 mu 阿片类药物的有害副作用。