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

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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.