Abstract
Translating ribosome affinity purification (TRAP) is a widely used technique to analyze ribosome-bound mRNAs in particular target cells that express a tagged ribosomal protein. We developed axon-TRAP-RiboTag, a TRAP-based method that allows purification and identification of translated mRNAs from distal neuronal axons in mouse, and identified more than 2000 of translated mRNAs in retinal ganglion cell (RGC) axons in vivo. The use of Cre-negative littermate control to filter out false-positive signals allows unbiased detection, and combining TRAP with in vitro ribosome run-off enables identification of actively translated mRNAs. Here, we describe a detailed protocol to identify translated mRNAs in RGC axons in mouse in vivo. This method can be applied to any neurons whose cell bodies and distal axons are anatomically separated.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schwanhausser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M (2011) Global quantification of mammalian gene expression control. Nature 473(7347):337–342. doi: nature10098 [pii]10.1038/nature10098
Heiman M, Schaefer A, Gong S, Peterson JD, Day M, Ramsey KE, Suarez-Farinas M, Schwarz C, Stephan DA, Surmeier DJ, Greengard P, Heintz N (2008) A translational profiling approach for the molecular characterization of CNS cell types. Cell 135(4):738–748. doi:10.1016/j.cell.2008.10.028
Dougherty JD, Schmidt EF, Nakajima M, Heintz N (2010) Analytical approaches to RNA profiling data for the identification of genes enriched in specific cells. Nucleic Acids Res 38(13):4218–4230. doi:10.1093/nar/gkq130
Yoon BC, Jung H, Dwivedy A, O’Hare CM, Zivraj KH, Holt CE (2012) Local translation of extranuclear lamin B promotes axon maintenance. Cell 148(4):752–764. doi:10.1016/j.cell.2011.11.064
Shigeoka T, Jung H, Jung J, Turner-Bridger B, Ohk J, Lin JQ, Amieux PS, Holt CE (2016) Dynamic axonal translation in developing and mature visual circuits. Cell 166(1):181–192. doi:10.1016/j.cell.2016.05.029
Sanz E, Yang L, Su T, Morris DR, McKnight GS, Amieux PS (2009) Cell-type-specific isolation of ribosome-associated mRNA from complex tissues. Proc Natl Acad Sci U S A 106(33):13939–13944. doi:10.1073/pnas.0907143106
Doyle JP, Dougherty JD, Heiman M, Schmidt EF, Stevens TR, Ma G, Bupp S, Shrestha P, Shah RD, Doughty ML, Gong S, Greengard P, Heintz N (2008) Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell 135(4):749–762. doi:10.1016/j.cell.2008.10.029
Fang Y, Gupta V, Karra R, Holdway JE, Kikuchi K, Poss KD (2013) Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration. Proc Natl Acad Sci U S A 110(33):13416–13421. doi:10.1073/pnas.1309810110
Marquardt T, Ashery-Padan R, Andrejewski N, Scardigli R, Guillemot F, Gruss P (2001) Pax6 is required for the multipotent state of retinal progenitor cells. Cell 105(1):43–55
Darnell JC, Van Driesche SJ, Zhang C, Hung KY, Mele A, Fraser CE, Stone EF, Chen C, Fak JJ, Chi SW, Licatalosi DD, Richter JD, Darnell RB (2011) FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146(2):247–261. doi:10.1016/j.cell.2011.06.013
Tang F, Barbacioru C, Nordman E, Li B, Xu N, Bashkirov VI, Lao K, Surani MA (2010) RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nat Protoc 5(3):516–535. doi:10.1038/nprot.2009.236
Cencic R, Hall DR, Robert F, Du Y, Min J, Li L, Qui M, Lewis I, Kurtkaya S, Dingledine R, Fu H, Kozakov D, Vajda S, Pelletier J (2011) Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F. Proc Natl Acad Sci U S A 108(3):1046–1051. doi:10.1073/pnas.1011477108
Acknowledgments
This work was supported by Samsung Science and Technology Foundation under Project Number SSTF-BA1602-13 (to H.J.) and by Wellcome Trust Programme Grant (085314/Z/08/Z) (to C.E.H.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Shigeoka, T., Jung, J., Holt, C.E., Jung, H. (2018). Axon-TRAP-RiboTag: Affinity Purification of Translated mRNAs from Neuronal Axons in Mouse In Vivo. In: Gaspar, I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7213-5_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7213-5_5
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7212-8
Online ISBN: 978-1-4939-7213-5
eBook Packages: Springer Protocols