Search

Long non-coding RNAs (lncRNAs) work together with diverse RNA-binding proteins (RBPs) to fulfill key regulations in important cellular functions. Here, we present a protocol to detect lncRNA-RBP interactions in vitrousing a tRNA scaffold containing a streptavidin aptamer pull-down assay. We describe steps for preparing both protein and lncRNA transcripts, lncRNA-protein interaction detection with an in vitrobinding assay, and western blot analysis. This protocol is applicable to screen for RNA-interacting proteins using cell lysates followed by mass spectrometry analysis.

Analyses of long non-coding RNA (lncRNA)-protein interactions provide key clues for understanding the molecular basis of lncRNA-modulated biological processes. Here, we detail a yeast three-hybrid assay to identify the lncRNA-interacting protein. We describe steps for lncRNA bait preparation, screening an RNA-binding proteins (RBPs) cDNA library, and validation of the lncRNA-RBP interaction. The assay can also be further applied to delineate the region of RBP that mediates the RNA-protein interaction.