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）是一种内稳态机制，在认知、学习、食欲和疼痛等广泛的大脑功能中起着关键作用。其中一种方法是减少突触释放的神经递质数量，以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初级内源性大麻素受体CB1R的精确定位介导的。然而，ECS也有不同的，有时是相反的功能，由位于不同亚细胞区室的CB1Rs介导，特别是在线粒体。这些不同的CB1R库是如何建立和维持的，以及它们的不同功能是如何控制的，人们知之甚少。CB1R转录可产生具有不同非翻译区（UTRs）的mRNA亚型。utr介导的机制密切参与mRNA转运及其随后的局部翻译的调节。例如，RNA结合蛋白（RBP）与BDNF mRNA的utr相互作用决定了特定刺激下不同细胞位置的活性依赖性翻译。我们假设类似的机制控制着CB1R的定位和功能。该项目的目的是探索不同的UTR变体如何将CB1R mrna直接运输和翻译到具有不同功能的不同细胞CB1R库。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CB1Rs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，包括硅分析；克隆;病毒生产;细胞培养;西方墨点法;co-immunoprecipitation;活细胞和固定细胞共聚焦成像；神经递质释放，电生理等在培养和体内。目的：1)通过计算机分析和实验鉴定新型cb1r - utr，并在替代cb1r - utr中定位rna结合蛋白（RBP）位点。2)研究CB1R mrna与替代utr的交易。3)确定神经元活动对备选CB1R-UTR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，例如在同源/异源二聚化中。5)了解备选cb1r - utr对ECS信号的影响。结果：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CB1R utr的作用，并为如何调节ECS以及这些途径是否可以被操纵以产生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.