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

 描述(申请人提供)：轴突内蛋白质合成与神经系统的适当连接有关，并可对神经损伤和神经退行性刺激产生恢复或致病作用。轴突合成的一组蛋白质，轴突翻译组，通过控制mRNA的定位和翻译来调节。以轴突翻译组为靶点可能会导致开发治疗神经疾病的新疗法。然而，调控轴突mRNA定位和翻译特异性的基本机制还需要更详细的研究。我们发现轴突mRNAs在翻译后立即降解。此外，我们还发现Pumilio-1/2靶基因的轴突缺失，其中Pumilio-1在轴突中表达，并在生长锥体中丰富。这一点，再加上Pumilio-1/2作为mRNA去烯化的翻译抑制和促进剂的已知作用，表明Pumilio同系物通过排斥机制调节轴突mRNA的定位。这个项目的目标是检验我们的假设，即mRNA降解是调节mRNA本地化和本地化转录本翻译的主要机制。为此，我们设计了以下目标：i)确定mRNA降解在本地化转录本的翻译控制中的作用；ii)确定Pumilio-1/2在建立 轴突信使核糖核酸通过排斥机制定位。我们将使用微流控设备中的神经元培养分离纯轴突材料，同时进行高通量测序，以进行无偏倚的轴突mRNA定量。我们将通过RNAi干扰Pumilio-1/2的功能，并使用显微镜分析与轴突生长相关的神经发育缺陷。这项研究的完成将极大地促进我们对轴突内蛋白质合成调节的理解，从而支持增加对生物学过程的理解的使命，为疾病诊断、治疗和预防的进展奠定基础。

 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.