The project aims to characterise the mRNAs that are translated in response to different guidance cues in axons. Previous work, using a candidate approach, has shown that different guidance cues induce the de novo synthesis of cytoskeletal and regulatory proteins in axons. For example, netrin-1 (attractive cue) stimulates beta-actin synthesis whereas Semaphorin 3A (repulsive cue) stimulates RhoA synthesis. These cue-specific induced changes in the cytoskeletal proteome contribute to the directional responses of growth cones. Recent genome-wide studies have revealed that hundreds of mRNAs are present in growing axons suggesting the possibility that different guidance cues may elicit the translation of multiple functionally related mRNAs. Our group has recently developed the Axon-TRAP (translating ribosome affinity purification) technology using an eye-specific Ribo-Tag (HA-tagged-ribosome) mouse line to conduct a global analysis of translating mRNAs in axons in vivo. Our analysis has revealed a highly complex axonal translatome and we now aim to use this technology to conduct an unbiased global analysis of changes in translatome induced by different guidance cues. The project will involve treating RiboTag retinal axons in culture with different cues followed by Axon-TRAP and comparative RNA-Seq analysis. This highly sensitive approach should enable us to test whether different cues have distinct signatures of mRNA translation. Once we have characterised the cue-induced translatome signatures, we will be interested to learn whether they represent functionally related subsets of mRNAs (e.g. mRNAs the encode proteins that assemble or disassemble the cytoskeleton) and investigate questions focused on how subsets of mRNAs are co-ordinately regulated in axons.
- Yoon BC, H Jung, A Dwivedy, CM O’Hare, KH Zivraj and CE Holt Local translation of extranuclear lamin B promotes axon maintenance. Cell Feb 17;148(4):752-64. 2012.
- Leung LC, Urbančič V, Baudet ML, Dwivedy A, Bayley TG, Lee AC, Harris WA, Holt CE. Coupling of NF-protocadherin signaling to axon guidance by cue-induced translation. Nature Neurosci. Jan 6;16(2):166-73. 2013
- Zivraj, K. L. Tung, M. Piper, L. Gumy, J. Fawcett, G. Yeo, and CE. Holt. Subcellular profiling reveals distinct and developmentally regulated repertoire of growth cone mRNAs. J. Neurosci. 30(46):15464-78. 2010