Role of regulated mRNA degradation in the control of axonal mRNA localization and translation.

 描述（由申请人提供）：轴突内蛋白质合成与神经系统的正确布线有关，并可对神经损伤和神经退行性刺激产生恢复或致病作用。轴突合成的蛋白质组，轴突翻译体，通过控制mRNA的定位和翻译来调节。靶向轴突translatome可能会导致新的治疗神经系统疾病的治疗方法的发展。然而，调节轴突mRNA定位和翻译特异性的基本机制需要更详细的研究。我们发现轴突mRNA在翻译后立即降解。此外，我们发现Pumilio-1/2靶mRNA在轴突中耗尽，Pumilio-1在轴突中表达并在生长锥中富集。这一点，连同已知的Pumilio-1/2作为翻译抑制因子和mRNA去腺苷化的启动子的作用表明，Pumilio同源物通过排除机制调节轴突mRNA定位。这个项目的目标是测试我们的假设，mRNA降解是一个主要的机制，调节mRNA定位和翻译的本地化转录本。为此，我们设计了以下目标：i）确定mRNA降解在定位转录本的翻译控制中的作用，ii）确定Pumilio-1/2在建立转录本中的作用。 轴突mRNA定位通过排除机制。我们将在微流控装置中使用神经元培养物来分离纯轴突材料，并伴随高通量测序来进行无偏的轴突mRNA定量。我们将通过RNAi干扰Pumilio-1/2功能，并使用显微镜分析与轴突生长相关的神经发育缺陷。这项研究的完成将大大推进我们对轴突内蛋白质合成调控的理解，从而支持增加对生物过程的理解的使命，为疾病诊断、治疗和预防的进步奠定基础。

 DESCRIPTION (provided by applicant): Intra-axonal protein synthesis is involved with the proper wiring of the nervous system and can have restorative or pathogenic effects in response to nerve injury and neurodegenerative stimuli. The set of axonally synthesized proteins, the axonal translatome, is regulated through the control of mRNA localization and translation. Targeting the axonal translatome could result in the development of novel therapies for the treatment of neurological disorders. Yet, the basic mechanisms regulating the specificity of axonal mRNA localization and translation need to be studied in more detail. We have found that axonal mRNAs are immediately degraded after being translated. Additionally, we have found depletion in axons of Pumilio-1/2 target mRNAs, with Pumilio-1 being expressed in axons and enriched in growth cones. This, together with the known role of Pumilio-1/2 as translational repressors and promoters of mRNA deadenylation suggests that Pumilio homologues regulate axonal mRNA localization through an exclusion mechanism. The goal of this project is to test our hypothesis that mRNA degradation is a major mechanism regulating both mRNA localization and translation of localized transcripts. For this purpose we have designed the following aims: i) to establish the role of mRNA degradation in the translational control of localized transcripts and ii) to determine the contributions of Pumilio-1/2 in the establishment of axonal mRNA localization through an exclusion mechanism. We will be using neuronal cultures in microfluidic devices for the isolation of pure axonal material, accompanied by high-throughput sequencing for an unbiased axonal mRNA quantification. We will interfere with Pumilio-1/2 function by RNAi and use microscopy for the analysis of neurodevelopmental defects associated with axon growth. The completion of this study will significantly advance our understanding of the regulation of intra-axonal protein synthesis and thus support the mission to increase understanding of biological processes that lay the foundation for advances in disease diagnosis, treatment, and prevention.