Identification and Characterization of Proteins that Bind to Selenoprotein 3' UTRs

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).