How do untranslated regions of cannabinoid receptor type 1 mRNA determine receptor subcellular localisation and function?

1 型大麻素受体 mRNA 的非翻译区如何决定受体亚细胞定位和功能？

• 批准号: 2744317
• 金额: --
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2744317
• 关键词: How; do; untranslated; regions; cannabinoid

The endocannabinoid system (ECS) is a homeostatic mechanism that plays key roles in a wide range of brain functions including cognition, learning, appetite, and pain. One way it does this is by reducing the amount of neurotransmitter released at synapses to prevent excessive neuronal activity. This modulation of synaptic function is mediated by the very precise localisation of the primary endocannabinoid receptor, CB1R, at the presynaptic membrane. However, the ECS also has different, and sometimes opposing, functions mediated by CB1Rs located at different subcellular compartments, particularly at mitochondria. How these different pools of CB1R are established and maintained, and how their differential functions are controlled, is poorly understood. CB1R transcription can generate mRNA isoforms with different untranslated regions (UTRs). UTR-mediated mechanisms are intimately involved in the regulation of mRNA trafficking and its subsequent local translation. For example, RNA binding protein (RBP) interactions with UTRs of BDNF mRNA determine activity-dependent translation at distinct cellular locations in response to specific stimuli. We hypothesise that analogous mechanisms control CB1R localisation and function.The aim of this project is to explore how different UTR variants direct trafficking and translation of CB1R mRNAs to different cellular CB1R pools that serve different functions.Techniques: The project will involve genetic manipulation of primary neurons to express and monitor the subcellular localisation and functions of CB1Rs with alternative UTRs. To achieve this the student will be trained in a wide variety of molecular, protein, and cell biology techniques, including in silico analysis; cloning; virus production; cell culture; Western blotting; co-immunoprecipitation; live and fixed cell confocal imaging; neurotransmitter release, electrophysiology etc in culture and in vivo.Objectives:1) Identify of novel CB1R-UTRs through in silico analysis and experimentation and map RNA-binding protein (RBP) sites in the alternative CB1R-UTRs.2) Investigate the trafficking of CB1R mRNAs with alternative UTRs.3) Define the effects of neuronal activity on alternative CB1R-UTR selection.4) Determine how UTR mediated mechanisms drive CB1R to interact with distinct proteins, such as in homo/heterodimerization.5) Understand the consequences of alternative CB1R-UTRs on ECS signalling.Outcomes: This integrated multidisciplinary approach and combination of techniques will provide excellent PhD training. The results obtained will define the roles of CB1R UTRs and provide important new insights into how the ECS is regulated, and if these pathways could be manipulated for beneficial effect.

内源性大麻素系统（ECS）是一种稳态机制，在认知、学习、食欲和疼痛等广泛的大脑功能中发挥关键作用。其中一种方法是通过减少突触释放的神经递质的数量来防止过度的神经元活动。突触功能的这种调节是由突触前膜上的主要内源性大麻素受体CB 1 R的非常精确的定位介导的。然而，ECS也有不同的，有时相反的，位于不同的亚细胞区室，特别是在线粒体的CB 1 R介导的功能。这些不同的CB 1 R库是如何建立和维持的，以及它们的差异功能是如何控制的，目前还知之甚少。CB 1 R转录可以产生具有不同非翻译区（UTR）的mRNA同种型。UTR介导的机制密切参与mRNA运输及其随后的局部翻译的调节。例如，RNA结合蛋白（RBP）与BDNF mRNA UTR的相互作用决定了在不同细胞位置响应特定刺激的活性依赖性翻译。我们假设类似的机制控制CB 1 R的定位和功能。该项目的目的是探索不同的UTR变体如何指导CB 1 R mRNA的运输和翻译到具有不同功能的不同细胞CB 1 R库。技术：该项目将涉及原代神经元的遗传操作，以表达和监测具有替代UTR的CB 1 R的亚细胞定位和功能。为了实现这一目标，学生将接受各种分子，蛋白质和细胞生物学技术的培训，包括计算机分析;克隆;病毒生产;细胞培养;蛋白质印迹;免疫共沉淀;活细胞和固定细胞共聚焦成像;培养和体内神经递质释放，电生理学等。1）通过计算机模拟分析和实验鉴定新的CB 1 R-UTR，并绘制替代CB 1 R-UTR中的RNA结合蛋白（RBP）位点。研究CB 1 R mRNA与替代UTR的运输。3）确定神经元活性对替代CB 1 R-UTR选择的影响。4）确定UTR介导的机制如何驱动CB 1 R与不同的蛋白质相互作用，例如同源/异源二聚化。5）了解替代CB 1 R-UTR对ECS信号传导的影响。这种综合的多学科方法和技术组合将提供优秀的博士培训。所获得的结果将定义CB 1 R UTR的作用，并提供重要的新见解如何调节ECS，以及是否可以操纵这些途径以产生有益的效果。