Using RNA Affinity Purification Followed by Mass Spectrometry to Identify RNA-Binding Proteins (RBPs)

RNA-binding proteins (RBPs) perform key functions in posttranscriptional regulation, adding complexity to the RNA life cycle. RNA interactome capture techniques have been applied to various organisms of interest and detected hundreds of RBPs, some with uncharacterized functions. However, even in many well-studied organisms, the primary sequence motif for most RBPs remains unknown. Here, we describe a 3-day protocol where users couple an RNA sequence of interest that is known to be bound by an RBP(s) with agarose beads, incubate the now tagged RNA sequence with protein lysate, and then pull down the proteins bound to the RNA. Subsequent mass spectrometry allows users to profile the RNA sequence-interacting proteome and pick out any enriched proteins as RBPs of interest. This protocol allows researchers to match sequences to their RBPs and even often identify novel RBPs or new functions for known RBPs.