Analysis of lncRNA-Protein Interactions by RNA-Protein Pull-Down Assays and RNA Immunoprecipitation (RIP)

Long noncoding RNAs (lncRNAs) have important roles in shaping chromatin by targeting chromatin-modifying enzymes to distinct genomic sites. This section covers two methods to analyze lncRNA-protein interactions. The RNA-protein pull-down assays use either bead-bound proteins to capture in vitro transcripts, or immobilized synthetic RNAs to bind proteins from cell lysates. In the RNA immunoprecipitation (RIP) assay, endogenous RNAs are co-immunoprecipitated with a protein of interest. Both the methods can be applied to material from proliferating and quiescent cells, thus providing insights into how lncRNA-protein interactions are altered between these two cellular states.

Schematic overview of the lncRNA-protein interaction assays described in this protocol

Initially, cells expressing the protein of interest are lysed. For RNA-dependent pulldown, the cell lysate is incubated with streptavidin beads coated with an in vitro synthesized, biotinylated lncRNA. For RNA immunoprecipitation (RIP), lncRNA is co-precipitated with the protein of interest from the cell lysate. To pull down lncRNAs in vitro, the immunopurified and immobilized protein is incubated with radiolabeled transcripts