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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Special Emphasis Panel (ZRG1 (91))
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Mamounas, Laura
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Weill Medical College of Cornell University
Schools of Medicine
New York
United States
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Lundquist, Mark R; Storaska, Andrew J; Liu, Ting-Chun et al. (2014) Redox modification of nuclear actin by MICAL-2 regulates SRF signaling. Cell 156:563-76
Harkcom, William T; Ghosh, Ananda K; Sung, Matthew S et al. (2014) NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents. Proc Natl Acad Sci U S A 111:E2443-52
Ji, Sheng-Jian; Jaffrey, Samie R (2014) Axonal transcription factors: novel regulators of growth cone-to-nucleus signaling. Dev Neurobiol 74:245-58
Colak, Dilek; Ji, Sheng-Jian; Porse, Bo T et al. (2013) Regulation of axon guidance by compartmentalized nonsense-mediated mRNA decay. Cell 153:1252-65
Curanovic, Dusica; Cohen, Michael; Singh, Irtisha et al. (2013) Global profiling of stimulus-induced polyadenylation in cells using a poly(A) trap. Nat Chem Biol 9:671-3
Hess, Martin E; Hess, Simon; Meyer, Kate D et al. (2013) The fat mass and obesity associated gene (Fto) regulates activity of the dopaminergic midbrain circuitry. Nat Neurosci 16:1042-8
Walker, B A; Hengst, U; Kim, H J et al. (2012) Reprogramming axonal behavior by axon-specific viral transduction. Gene Ther 19:947-55
Cohen, Michael S; Ghosh, Ananda K; Kim, Hyung Joon et al. (2012) Chemical genetic-mediated spatial regulation of protein expression in neurons reveals an axonal function for wld(s). Chem Biol 19:179-87
Cohen, Michael S; Bas Orth, Carlos; Kim, Hyung Joon et al. (2011) Neurotrophin-mediated dendrite-to-nucleus signaling revealed by microfluidic compartmentalization of dendrites. Proc Natl Acad Sci U S A 108:11246-51
Rivieccio, Mark A; Brochier, Camille; Willis, Dianna E et al. (2009) HDAC6 is a target for protection and regeneration following injury in the nervous system. Proc Natl Acad Sci U S A 106:19599-604

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