Detection of Plant RNA–Protein Interactions Using GFP-tag for Immunoprecipitation

The study of RNA metabolism involves understanding how RNA molecules interact with specific RNA-binding proteins (RBPs). In plants, these interactions have traditionally been investigated using a variety of in vivo and in vitro approaches, such as electrophoretic mobility shift assays or the analysis of knockout mutants. More recently, immunoprecipitation-based techniques have been developed. Most of the available protocols rely on crosslinking procedures, magnetic beads, and RNA-seq as the final endpoint analysis. Here, we present a protocol developed to identify specific RNA targets that directly interact with known plant RBPs using GFP-Trap^® agarose (ChromoTek) for immunoprecipitation without the need for crosslinking or RNA-seq. Briefly, a GFP-tagged RNA-binding protein is expressed in plant tissue, protein extracts are incubated with the GFP-Trap^® agarose matrix, and the resulting complexes are isolated. Co-purified RNAs, specifically mRNAs, are then analyzed by RT-PCR to detect bound transcripts. This protocol was first implemented for the study of RNA-protein interaction in <i>Arabidopsis thaliana</i>. This approach presents high potential for analysis in other plant species as well as several advantages, such as its high specificity and low cost. Even though GFP-Trap^® magnetic agarose (ChromoTek) has been used in plant systems to detect RNA-protein interactions, the protocol presented here consists of an alternative that is straightforward to implement when both candidate RNAs and RNA-binding proteins are known, and it can be broadly applied to study RNA-protein interactions in other plant systems. Key features • Can be used to confirm predicted RNA-protein interactions. • Suitable for validating RNA-protein interactions when candidate transcripts and RBPs are already known. • Compatible with downstream analysis by RT-PCR; can be adapted to RNA-seq if high-throughput data is needed. • Does not require crosslinking or specialized equipment beyond standard molecular biology tools for direct and strong RNA-protein interactions.