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 Arabidopsis thaliana. 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.