Abstract
This chapter explains the use of RNase-assisted RNA chromatography. RNA affinity chromatography is a powerful technique that is used to isolate and identify proteins that bind to a specific RNA ligand. The RNA of interest is attached to beads before protein lysates are passed over the column. In traditional RNA chromatography, bound proteins are eluted with high salt or harsh detergent, which can also release proteins that are nonspecifically bound to the beads. To avoid this, a new method was developed in which RNases are used to cleave RNA from the beads, releasing only RNA binding proteins (RBPs) and leaving behind proteins that are bound to the beads (Michlewski and Caceres, RNA 16(8):1673–1678, 2010). This chapter will describe the isolation of proteins that bind specifically to the distal region of the Selenoprotein S 3′ untranslated region (3′ UTR).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Copeland PR, Driscoll DM (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 PR, Fletcher JE, Carlson BA et al (2000) A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs. EMBO J 19(2):306–314
Budiman M, Bubenik J, Miniard A et al (2009) Eukaryotic initiation factor 4a3 is a selenium-regulated RNA -binding protein that selectively inhibits selenocysteine incorporation. Mol Cell 35:479–489
Miniard A, Middleton L, Budiman NE et al (2010) Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression. Nucleic Acids Res 38:4807–4820
Mellacheruvu D, Wright Z, Couzens A et al (2013) The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods 10:730–736
Michlewski G, Caceres JF (2010) RNase-assisted RNA chromatography. RNA 16(8):1673–1678
Bubenik J, Miniard A, Driscoll D (2013) Alternative transcripts and 3′ UTR elements govern the incorporation of selenocysteine into selenoprotein S. PLoS One 8(4):e62102
Acknowledgments
We wish to thank Alexis Polce and Dr. Vivek Narayan for critical review of the manuscript. This work was supported by NIH grants # R01DK07859 and R01DK107426.
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
Cockman, E.M., Driscoll, D.M. (2018). Identification and Characterization of Proteins that Bind to Selenoprotein 3′ UTRs. In: Chavatte, L. (eds) Selenoproteins. Methods in Molecular Biology, vol 1661. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7258-6_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7258-6_5
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7257-9
Online ISBN: 978-1-4939-7258-6
eBook Packages: Springer Protocols