Protocol for capturing the RNA-binding proteome from plants using orthogonal organic phase separation

RNA-binding proteins (RBPs) regulate many processes related to RNA biogenesis, function, localization, and degradation. Here, we present a protocol for the en masse isolation of RBPs crosslinked to RNA and provide a strategy for subsequent proteomics analysis. We describe the steps for in vivo UV crosslinking of RNA-protein complexes, tissue lysis, fractionation, and purification of crosslinked RNA-protein adducts using organic solvents. Although the protocol was developed for Nicotiana benthamiana leaves, it can be optimized for use in different plants and tissues.