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

为了创造大脑功能的神经通路，数十亿神经元的树突和轴突必须精确连接。为了引导位于发育中的轴突尖端的生长锥，诸如NGF和Sema 3A的引导线索需要轴突定位的mRNA的翻译。这些mRNA的局部翻译是如何被调节的还不完全清楚，但它们被非典型的poly（A）聚合酶的聚腺苷酸化被认为是至关重要的。尽管可用于在轴突中翻译的mRNA库惊人地大，但只有少数已被鉴定为在轴突中进行翻译，并且已知没有一个是聚腺苷酸化的。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无偏检测多聚腺苷酸化并在轴突中翻译的mRNA的方法。目前，这些mRNA的研究是通过一个相对缓慢的候选基因的方法测试只有个别信号通路的已知效应。因此，轴突中响应引导线索而翻译的全套mRNA尚不清楚。在这里，我建议通过一种新的化学遗传学方法来识别这些mRNA，并研究它们在轴突引导中的作用。该方法基于用2-炔基-腺苷标记多聚腺苷酸化的mRNA，2-炔基-腺苷是一种新的腺苷类似物，已在S.杰弗瑞该分子是非典型聚（A）聚合酶的底物，并且可以通过点击化学与生物素缀合。因此，它可以用来捕获和鉴定新的多聚腺苷酸化的mRNA.The第一个目标，本项目是使用2-炔基-腺苷，以确定在轴突中的多聚腺苷酸化的mRNAs响应于NGF和Sema 3A。这些mRNA将从引导线索处理的原代神经元中捕获，并通过深度测序鉴定。在一个独立的方法中，一个区室化的细胞培养系统将被用来直接捕获新的多聚腺苷酸化的mRNA从原代神经元的轴突。第二个目的是调查这些mRNA在轴突导向的作用。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确认。然后，将通过轴突特异性RNA干扰来测试它们在NGF介导的轴突生长和Sema 3A介导的生长锥塌陷中的功能。这些实验将确定神经生长因子和Sema 3A的功能基础的新的信号通路，并有望提供前所未有的见解到多聚腺苷酸化网络，管理轴突的指导。由于多聚腺苷酸化是控制基因表达的基本机制，因此本项目中建立的技术也将与其他细胞环境具有广泛的相关性。此外，这些发现可能揭示了将异常翻译调节与神经精神疾病联系起来的新机制。

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.