RNA Detection

Chapter 5

Axon-TRAP-RiboTag: Affinity Purification of Translated

mRNAs from Neuronal Axons in Mouse In Vivo

Toshiaki Shigeoka, Jane Jung, Christine E. Holt, and Hosung Jung

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.

Key wordsRibosome, Translation, mRNAs, Immunoprecipitation, Axon, Neuron

1 Introduction

Translation is a key step that controls the abundance of proteins in

the cell [1], and therefore it is crucial to profile translated mRNAs

in a specific cell population in vivo to understand how cells regulate

their gene expression within an organism. Translating ribosome

affinity purification (TRAP), affinity purification of ribosomes car-

rying an epitope-tagged ribosomal protein (RP) from a specific cell

population, is a useful approach to obtain cell type-specific profiles

of ribosome-bound mRNAs [2–8]. This technique utilizes a genet-

ically engineered animal that expresses an epitope-tagged RP, such

as EGFP (enhanced green fluorescence protein)-Rpl10a and

Rpl22-HA (hemagglutinin), in a genetically defined population of

cells. Using a cell type-specific promoter that drives the expression

of the tagged RP, ribosome–mRNA complexes can be specifically

purified from the cells where the promoter is active in a tissue, an

organ, or an organism. Compared to RNase protection-based ribo-

some profiling, TRAP has the advantage that it generates sequence

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_5,©Springer Science+Business Media LLC 2018

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