An advanced protocol for profiling RNA-binding proteins in Arabidopsis using plant phase extraction

RNA-binding proteins (RBPs) are key players in regulating cell fate and essential developmental processes. Systematic profiling of the RNA-binding proteome (RBPome) is thus indispensable for researchers aiming to understand the mechanisms of post-transcriptional gene regulation. RBPome identification methods developed in humans, mice, and bacteria have successfully identified RBPomes in these organisms. However, the biochemical and genetic complexities of plant tissues have greatly hindered the effectiveness of these methods in plants. Moreover, plant RBPs have been predominantly discovered through oligo d(T) based affinity purification (RNA-interactome capture). Since polyadenylated RNA only accounts for less than 5% of the total RNA population in eukaryotic cells, there is a pressing need to develop a comprehensive, yet unbiased, method to capture the full spectrum of RBPs in plants. Here, we describe a detailed protocol of Plant Phase Extraction (PPE), a recently developed method to identify RBPs in Arabidopsis (Zhang Y, Xu Y, Skaggs TH, et al. Plant phase extraction: a method for enhanced discovery of the RNA-binding proteome and its dynamics in plants. Plant Cell 2023; 35: 2750-72.) []. The PPE method enables the efficient enrichment of both poly(A) and non-poly(A) RBPs from various tissues quickly and reproducibly. Most importantly, PPE allows for unveiling dynamic RBP-RNA interactions under various abiotic and biotic stress conditions and during different plant developmental stages. This provides a much broader and more accurate understanding of plant RBPs, marking a significant advancement in plant molecular biology.