Immunoprecipation Assay to Quantify the Amount of tRNAs associated with Their Interacting Proteins in Tissue and Cell Culture

Abstract

Transfer RNAs (tRNAs) are highly abundant species and, along their biosynthetic and functional path, they establish interactions with a plethora of proteins. The high number of nucleobase modifications in tRNAs renders conventional RNA quantification approaches unsuitable to study protein-tRNA interactions and their associated functional roles in the cell. We present an immunoprecipitation-based approach to quantify tRNA bound to its interacting protein partner(s). The tRNA-protein complexes are immunoprecipitated from cells or tissues and tRNAs are identified by northern blot and quantified by tRNA-specific fluorescent labeling. The tRNA interacting protein is quantified by an automated western blot and the tRNA amount is presented per unit of the interacting protein. We tested the approach to quantify tRNAGly associated with mutant glycyl-tRNA-synthetase implicated in Charcot-Marie-Tooth disease. This simple and versatile protocol can be easily adapted to any other tRNA binding proteins.Graphic abstract: Figure 1. Schematic of the tRNA-Immunoprecipitation approach.