Identification of Intrinsic RNA Binding Specificity of Purified Proteins by in vitro RNA Immunoprecipitation (vitRIP).

RNA-protein interactions are often mediated by dedicated canonical RNA binding domains. However, interactions through non-canonical domains with unknown specificity are increasingly observed, raising the question how RNA targets are recognized. Knowledge of the intrinsic RNA binding specificity contributes to the understanding of target selectivity and function of an individual protein. The presented in vitro RNA immunoprecipitation assay (vitRIP) uncovers intrinsic RNA binding specificities of isolated proteins using the total cellular RNA pool as a library. Total RNA extracted from cells or tissues is incubated with purified recombinant proteins, RNA-protein complexes are immunoprecipitated and bound transcripts are identified by deep sequencing or quantitative RT-PCR. Enriched RNA classes and the nucleotide frequency in these RNAs inform on the intrinsic specificity of the recombinant protein. The simple and versatile protocol can be adapted to other RNA binding proteins and total RNA libraries from any cell type or tissue. Graphic abstract: Figure 1. Schematic of the in vitro RNA immunoprecipitation (vitRIP) protocol.