The objective of the proposed research is to identify and characterize signaling mechanisms that lead to local mRNA translation in growth cones and developing axons. Studies of local translation have been hampered because the identity of specific mRNAs that are regulated by guidance cues are unknown. Furthermore the signaling mechanisms that couple receptor activation to mRNA translation are poorly understood. One mechanism to regulate local translation is polyadenylation of axonally localized transcripts, but this process is highly difficult to study. We have developed a chemical genetic approach to selectively label and recover transcripts that become polyadenylated. To do this, we synthesized a novel adenosine nucleotide that can be incorporated into polyA tails by cellular PAPs. Using this analog, we developed a deep sequencing approach to profile polyadenylation across the transcriptome. We have also sought to identify the polyA polymerase (PAP) that catalyzes polyadenylation in response to axonal signaling. By screening axons using antibodies to known PAPs, we identified a specific PAP isoform that is enriched in axons and colocalizes with actin in growth cones. As part of our overall goal to identify the intracellular signaling pathways that regulate the translation of mRNAs in axons and growth cones, the specific aims of this proposal are: (1) To profile polyadenylation events that mediate the effects of nerve growth factor (NGF) and Semaphorin 3A (Sema3A). In this aim, we will selectively label, identify, and test the functional roles of mRNAs polyadenylated in response to NGF and Sema3A in sensory neurons. (2) To identify polyadenylation and deadenylation pathways that regulate local translation in axons. In this aim, we use the polyadenylation tagging approach to identify the network of polyadenylation events induced by NGF and Sema3A within axons. Furthermore, we explore the role of deadenylation in repressing axonal translation, and will identify the axonal deadenylase that regulates axonal mRNA translation. (3) To establish the PAPs that regulate intra-axonal translation. In this aim, we characterize a novel axonally enriched PAP, and determine its roles in local translation in axons. These studies will characterize the role of a novel axonal PAP, potentially uncovering the "missing" regulator of polyadenylation in axons. The experiments proposed here provide a comprehensive analysis of polyadenylation networks that are elicited by axonal guidance cues, and will identify new proteins that regulate polyadenylation in axons. Since polyadenylation is a major mechanism controlling protein expression, we anticipate that the methods and translational regulators that will be characterized here will have broad relevance to signaling pathways in diverse cellular contexts.
Regulated protein synthesis is important for synaptic plasticity, axon guidance, and regeneration, and mutations in proteins that regulate protein synthesis or RNA processing have been linked to numerous neurological disorders. Although polyadenylation of mRNA is known to be a major mechanism by which axon guidance cues and neurotrophins activate protein synthesis, methods to explore polyadenylation on a transcriptome-wide scale are not available, and the enzymes that mediate polyadenylation are poorly understood. This project will, for the first time, identify the polyadenylation pathways induced in response to neuronal stimuli, elucidate the signaling pathways that regulate the polyadenylation pathways that control local translation in axons, and identify a novel enzyme regulator of polyadenylation that may have critical roles in local translation pathways that influence axon pathfinding and synaptic plasticity.
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