Transcriptome-wide analysis of cytosolic polyadenylation events that mediate axon guidance: RNA processing helps wiring the brain

对介导轴突引导的胞质多腺苷酸化事件进行全转录组分析：RNA 处理有助于连接大脑

• 批准号: 253180514
• 负责人: Dr. Bastian Linder
• 金额: --
• 依托单位: Europäisches Laboratorium für Molekularbiologie (EMBL)
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/253180514?language=en
• 关键词: Transcriptome; wide; analysis; cytosolic; polyadenylation

To create the neural pathways that underlie brain function, dendrites and axons of billions of neurons have to be connected precisely. In order to route the growth cone that is situated at the tip of developing axons, guidance cues such as NGF and Sema3A require the translation of axonally localized mRNAs. How the local translation of these mRNAs is regulated is not fully understood but their polyadenylation by non-canonical poly(A) polymerases is thought to be critically important. Although the pool of mRNAs available for translation in axons is surprisingly large, only a few have been identified to undergo translation in axons and none are known to be polyadenylated. This discrepancy represents a major knowledge gap in our understanding of axonal biology. One reason for this is the lack of a method for the unbiased detection of mRNAs that are polyadenylated and translated in axons. At present, these mRNAs are studied by a relatively slow candidate gene approach testing only known effectors of individual signaling pathways. As a consequence, the full set of mRNAs translated in axons in response to guidance cues is unknown. Here, I propose to identify these mRNAs by a novel chemical genetic approach and to investigate their role in axon guidance. This approach is based on the labeling of polyadenylated mRNAs with 2 alkynyl-adenosine, a novel analog of adenosine that has been developed and synthesized in the laboratory of S. Jaffrey. This molecule is a substrate for non-canonical poly(A) polymerases and can be conjugated to biotin by click-chemistry. Thus, it can be used to capture and identify newly polyadenylated mRNAs.The first aim of this project is to use 2-alkynyl-adenosine to identify mRNAs that are polyadenylated in axons in response to NGF and Sema3A. These mRNAs will be captured from guidance-cue treated primary neurons and identified by deep-sequencing. In an independent approach, a compartmentalized cell culture system will be used to directly capture newly polyadenylated mRNAs from axons of primary neurons.The second aim is to investigate the role of these mRNAs in axon guidance. For this, their intra-axonal translation will be confirmed by immunofluorescence-based protein quantification. Then, their function in NGF-mediated axon outgrowth and Sema3A-mediated growth cone collapse will be tested by axon-specific RNA interference. These experiments will identify novel signaling pathways that underlie the function of NGF and Sema3A and are expected to provide unprecedented insights into the polyadenylation networks that govern axon guidance. As polyadenylation is a fundamental mechanism controlling gene expression, the techniques established in this project will be of broad relevance also to other cellular contexts. In addition, these findings might uncover novel mechanisms that link aberrant translational regulation to neuropsychiatric disease.

为了创造支撑大脑功能的神经通路，几十亿个神经元的树突和轴突必须精确地连接起来。为了引导位于发育轴突顶端的生长锥，诸如NGF和Sema3A等引导线索需要轴突定位mrna的翻译。这些mrna的局部翻译是如何被调控的尚不完全清楚，但它们的非规范聚(A)聚合酶的聚腺苷化被认为是至关重要的。尽管可用于轴突翻译的mrna数量惊人地多，但只有少数已被确定在轴突中进行翻译，而且没有一个已知是聚腺苷化的。这种差异代表了我们对轴突生物学理解的一个主要知识缺口。其中一个原因是缺乏一种方法来无偏地检测在轴突中被聚腺苷化和翻译的mrna。目前，这些mrna是通过一种相对缓慢的候选基因方法来研究的，只测试单个信号通路的已知效应物。因此，在轴突中翻译的全套mrna响应引导线索是未知的。在这里，我建议通过一种新的化学遗传方法来鉴定这些mrna，并研究它们在轴突引导中的作用。这种方法是基于用2烷基腺苷标记多腺苷化mrna，这是一种在S. Jaffrey实验室开发和合成的新型腺苷类似物。该分子是非典型聚(a)聚合酶的底物，可以通过点击化学偶联到生物素上。因此，它可以用来捕获和鉴定新的聚腺苷化mrna。该项目的第一个目标是使用2-炔基腺苷来鉴定轴突中响应NGF和Sema3A的聚腺苷化mrna。这些mrna将从引导线索处理的初级神经元中捕获，并通过深度测序进行鉴定。在一种独立的方法中，一个区隔化的细胞培养系统将被用来直接从原代神经元的轴突捕获新的聚腺苷化mrna。第二个目的是研究这些mrna在轴突引导中的作用。为此，它们的轴突内翻译将通过基于免疫荧光的蛋白质定量来证实。然后，它们在ngf介导的轴突生长和sema3a介导的生长锥塌陷中的功能将通过轴突特异性RNA干扰进行测试。这些实验将确定NGF和Sema3A功能背后的新信号通路，并有望为控制轴突引导的聚腺苷化网络提供前所未有的见解。由于聚腺苷酸化是控制基因表达的基本机制，本项目建立的技术也将与其他细胞环境具有广泛的相关性。此外，这些发现可能揭示将异常翻译调节与神经精神疾病联系起来的新机制。