Translating Ribosome Affinity Purification (TRAP) uses an antigen-tagged ribosome subunit produced under the control of a tissue-specific promoter to immunoprecipitate mRNA bound polysomes in specific cell types (1).
Using a similar strategy to TRAP, RiboTag is a genetically bred line of mice which selectively express a hemagglutinin A (HA)-tagged ribosomal protein in a target cell type (2). Immunoprecipitation of polysomes with a monoclonal antibody against HA allows for rapid and efficient isolation of ribosome-associated mRNA transcripts from any cell type in vivo.
TRAP-seq/RiboTag specifically enriches transcripts in a sample that are difficult to isolate for subsequent analysis. Unlike methods of monosome enrichment used in RIBO-seq, the purified ribosomes are not contaminated with non-ribosomal proteins that might co-sediment with ribosomes, providing a more accurate measure of translational levels (3).
Overview: Ribosomes from the cell type of interest are antigen tagged (orange circle) while ribosomes from other cell types are not. Nucleases digest any unprotected RNA before cell specific monosomes are affinity purified from the ribosomes of the other cell types. Ribosome protected fragments are purified and size selected on Urea PAGE gels. Fragments are converted into a cDNA library which undergoes deep sequencing.
- Identify altered patterns of translational efficiency in specific cell types within a heterogenous background in vivo.
- Investigate the effects cell specific targeted drug therapies exert on mechanisms of translational control.
- Explore translatomic annotations to help pinpoint cell specific therapeutic targets and biomarkers.
- Detection of short open reading frames (ORFs)
- Investigation of translational regulation in disease and development.
1. Inada, T., Winstall, E., Turin Jr, S.Z., Yates 3rd, J.R., Schieltz, D. and Sachs, A.B. (2002). One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs. RNA. 8(7):948-958.
2. Sanz, E., Yang, L., Su, T., Morris, D.R., McKnight, G.S. and Amieux, P.S. (2009). Cell-type-specific isolation of ribosome-associated mRNA from complex tissues. Proc Natl Acad Sci USA. 106(33):13939-13944.
3. Heiman, M., Kulicke, R., Fenster, R.J., Greengard, P. and Heintz, N. (2014). Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nat Protoc. 9(6):1282-1291.