Abstract
RNA–protein interaction can be detected by RNA pull-down and immunoblotting methods. Here, we describe a method to detect RNA–protein interaction using RNA pull down and to identify the proteins that are pulled-down by the RNA using immunoblotting. In this protocol, RNAs with specific sequences are biotinylated and immobilized onto Streptavidin beads, which are then used to pull down interacting proteins from cellular extracts. The presence of a specific protein is subsequently verified by SDS- polyacrylamide gel electrophoresis and immunoblotting with antibodies. Interactions between the SMN RNA and the PSF protein and between the caspase-2 RNA and the SRSF3 protein (SRp20) in nuclear extract prepared from HeLa cells are illustrated as examples.
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
Ascano M, Hafner M, Cekan P, Gerstberger S, Tuschl T (2012) Identification of RNA-protein interaction networks using PAR-CLIP. Wiley Interdiscip Rev RNA 3(2):159–177. doi:10.1002/wrna.1103
Shen H, Zheng X, Shen J, Zhang L, Zhao R, Green MR (2008) Distinct activities of the DExD/H-box splicing factor hUAP56 facilitate stepwise assembly of the spliceosome. Genes Dev 22(13):1796–1803
Kyung Oh H, Lee E, Jang HN, Lee J, Moon H, Sheng Z, Jun Y, Loh TJ, Cho S, Zhou J (2013) hnRNP A1 contacts exon 5 to promote exon 6 inclusion of apoptotic Fas gene. Apoptosis 18(7):825–835
Moon H, Cho S, Loh TJ, Oh HK, Jang HN, Zhou J, Kwon YS, Liao DJ, Jun Y, Eom S, Ghigna C, Biamonti G, Green MR, Zheng X, Shen H (2014) SRSF2 promotes splicing and transcription of exon 11 included isoform in Ron proto-oncogene. Biochim Biophys Acta 1839(11):1132–1140. doi:10.1016/j.bbagrm.2014.09.003
Moritz B, Wahle E (2014) Simple methods for the 3' biotinylation of RNA. RNA 20(3):421–427. doi:10.1261/rna.042986.113
Shi M, Han W, Spivack SD (2013) A quantitative method to identify microRNAs targeting a messenger RNA using a 3'UTR RNA affinity technique. Anal Biochem 443(1):1–12. doi:10.1016/j.ab.2013.08.002
Li H, Chen W, Zhou Y, Abidi P, Sharpe O, Robinson WH, Kraemer FB, Liu J (2009) Identification of mRNA binding proteins that regulate the stability of LDL receptor mRNA through AU-rich elements. J Lipid Res 50(5):820–831. doi:10.1194/jlr.M800375-JLR200
Lopez de Silanes I, Galban S, Martindale JL, Yang X, Mazan-Mamczarz K, Indig FE, Falco G, Zhan M, Gorospe M (2005) Identification and functional outcome of mRNAs associated with RNA-binding protein TIA-1. Mol Cell Biol 25(21):9520–9531. doi:10.1128/MCB.25.21.9520-9531.2005
Hall-Pogar T, Liang S, Hague LK, Lutz CS (2007) Specific trans-acting proteins interact with auxiliary RNA polyadenylation elements in the COX-2 3'-UTR. RNA 13(7):1103–1115. doi:10.1261/rna.577707
Butter F, Scheibe M, Morl M, Mann M (2009) Unbiased RNA-protein interaction screen by quantitative proteomics. Proc Natl Acad Sci U S A 106(26):10626–10631. doi:10.1073/pnas.0812099106
Cho S, Moon H, Loh TJ, Oh HK, Williams DR, Liao DJ, Zhou J, Green MR, Zheng X, Shen H (2014) PSF contacts exon 7 of SMN2 pre-mRNA to promote exon 7 inclusion. Biochim Biophys Acta 1839(6):517–525. doi:10.1016/j.bbagrm.2014.03.003
Jang HN, Lee M, Loh TJ, Choi SW, Oh HK, Moon H, Cho S, Hong SE, Kim do H, Sheng Z, Green MR, Park D, Zheng X, Shen H (2014) Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8. Biochim Biophys Acta 1839(1):25–32. doi:10.1016/j.bbagrm.2013.11.006
Bollag DM, Edelstein SJ, Rozycki MD (1996) Protein methods, 2nd edn. Wiley-Liss, New York
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Penna A, Cahalan M (2007) Western blotting using the invitrogen NuPage Novex Bis Tris minigels. J Vis Exp 7:264. doi:10.3791/264
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Zheng, X., Cho, S., Moon, H., Loh, T.J., Jang, H.N., Shen, H. (2016). Detecting RNA–Protein Interaction Using End-Labeled Biotinylated RNA Oligonucleotides and Immunoblotting. In: Lin, RJ. (eds) RNA-Protein Complexes and Interactions. Methods in Molecular Biology, vol 1421. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3591-8_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3591-8_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3589-5
Online ISBN: 978-1-4939-3591-8
eBook Packages: Springer Protocols