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

内源性大麻素系统(ECS)是一种动态平衡机制，在包括认知、学习、食欲和疼痛在内的一系列大脑功能中发挥关键作用。它做到这一点的一种方式是减少突触释放的神经递质数量，以防止神经元过度活动。这种突触功能的调节是通过初级内源性大麻素受体CB1R在突触前膜非常精确的定位来介导的。然而，ECS也有不同的功能，有时是相反的，由位于不同亚细胞间的CB1Rs介导，特别是在线粒体上。这些不同的CB1R池是如何建立和维护的，以及它们的不同功能是如何控制的，人们对此知之甚少。CB1R转录可产生具有不同非翻译区(UTRs)的mRNA亚型。非编码区介导的机制密切参与了信使核糖核酸的转运及其随后的局部翻译的调控。例如，RNA结合蛋白(RBP)与BDNF mRNA的UTRs的相互作用决定了不同细胞位置对特定刺激的反应依赖于活性的翻译。我们假设CB1R的定位和功能是由相似的机制控制的。这个项目的目的是探索不同的UTR变体如何将CB1R mRNAs的运输和翻译引导到不同的细胞CB1R池中，以服务于不同的功能。技术：该项目将涉及对初级神经元的遗传操作，以表达和监测具有替代UTRs的CB1R的亚细胞定位和功能。为了达到这一目的，学生将接受各种各样的分子、蛋白质和细胞生物学技术的培训，包括电子分析、克隆、病毒生产、细胞培养、免疫印迹、免疫共沉淀、活细胞和固定细胞共聚焦成像；目标：1)通过电子分析和实验鉴定新的CB1R-UTRs，并定位替代CB1R-UTRs中的RNA结合蛋白(RBP)位点。2)研究具有替代CB1R-UTRs的CB1R mRNAs的运输。3)确定神经元活动对替代CB1R-UTRs选择的影响。4)确定UTR介导的机制如何驱动CB1R与不同的蛋白质相互作用，如同源/异源二聚体。5)了解替代CB1R-UTRs对ECS信号的影响。结果：这种多学科的综合方法和技术组合将提供极好的博士培训。所获得的结果将确定CB1R UTRs的作用，并为ECS如何调控以及这些途径是否可以被操纵以实现有益效果提供重要的新见解。

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.