Abstract
This chapter describes the technique of RNA affinity chromatography, which is a powerful approach for isolating RNA-binding proteins. This method takes advantage of the fact that sequence-specific RNA-binding proteins often bind their targets with high affinity. Here we outline a protocol for purifying Apobec-1 complementation factor (ACF), the RNA-binding subunit of the apolipoprotein-B (apo-B) mRNA-editing enzyme. ACF was purified using synthetic wild-type and mutant apo-B RNAs, which were coupled to cyanogen bromide (CNBr)-activated Sepharose. The methods are plasmid construction for in vitro transcription, affinity chromatography column preparation, protein purification by RNA affinity chromatography, and analysis of the purified protein.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Sela-Brown, A., Silver, J., Brewer, G., and Naveh-Many, T. (2000) Identification of AUF1 as a parathyroid hormone mRNA 3′-untranslated region-binding protein that determines parathyroid hormone mRNA stability. J. Biol. Chem. 275(10), 7424–7429.
Neupert, B., Thompson, N. A., Meyer, C., and Kuhn, L. C. (1990) A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta. Nucleic Acids Res. 18(1), 51–55.
Rouault, T. A., Hentze, M. W., Haile, D. J., Harford, J. B., and Klausner, R. D. (1989) The iron-responsive element binding protein: a method for the affinity purification of a regulatory RNA-binding protein. Proc. Natl. Acad. Sci. USA 86(15), 5768–5772.
Mehta, A. and Driscoll, D. (1998) A sequence-specific RNA-binding protein complements apobec-1 to edit apolipoprotein B mRNA. Mol. Cell. Biol. 18(8), 4426–4432.
Copeland, P. R. and Driscoll, D. M. (1999) Purification, redox sensitivity, and RNA binding properties of SECIS-binding protein 2, a protein involved in selenoprotein biosynthesis. J. Biol. Chem. 274(36), 25,447–25,454.
Copeland, P. R., Fletcher, J. E., Carlson, B. A., Hatfield, D. L., and Driscoll, D. M. (2000) A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs. EMBO J. 19(2), 306–314.
Mehta, A., Kinter, M. T., Sherman, N. E., and Driscoll, D. M. (2000) Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA. Mol. Cell. Biol. 20(5), 1846–1854.
Kaminski, A., Hunt, S. L., Patton, J. G., and Jackson, R. J. (1995) Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA. RNA 1(9), 924–938.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Gerber, C.A., Relich, A., Driscoll, D.M. (2004). Isolation of an mRNA-Binding Protein Involved in C-to-U Editing. In: Gott, J.M. (eds) RNA Interference, Editing, and Modification. Methods in Molecular Biology, vol 265. Humana Press. https://doi.org/10.1385/1-59259-775-0:239
Download citation
DOI: https://doi.org/10.1385/1-59259-775-0:239
Publisher Name: Humana Press
Print ISBN: 978-1-58829-242-1
Online ISBN: 978-1-59259-775-8
eBook Packages: Springer Protocols