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

Authors: ZHANG, YONG - University Of California, Riverside; XU, YE - University Of California, Riverside; Skaggs, Todd; Ferreira, Jorge; CHEN, XUEMEI - Peking University; Sandhu, Devinder

Submitted to: Biology Methods and Protocols
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/6/2023
Publication Date: 8/10/2023
Citation: Zhang, Y., Xu, Y., Skaggs, T.H., Ferreira, J.F., Chen, X., Sandhu, D. 2023. An advanced protocol for profiling RNA-binding proteins in Arabidopsis using plant phase extraction. Biology Methods and Protocols. 8(1). Article bpad016. https://doi.org/10.1093/biomethods/bpad016.
DOI: https://doi.org/10.1093/biomethods/bpad016

Interpretive Summary: RNA-binding proteins, or RBPs, are special kinds of proteins that work together with RNA. They're very important because they help to control what goes on inside cells and how cells grow and change. Until now, scientists had figured out ways to identify these proteins in humans, mice, and bacteria, but it was hard to do the same for plants due to their complex tissues. Recently, we made a big breakthrough. We developed a new method called plant phase extraction (PPE), which lets us isolate and identify these proteins in plants. This method is a big step up from previous methods and performs better in several ways. This is important because now we can study these plant proteins more closely and see what they do under different conditions, such as salinity stress. In this manuscript, we provide a detailed, step-by-step protocol for using PPE to study these proteins in plants so that other scientists can use it in their own research. This information is going to be incredibly valuable to scientists studying plant biology and genetics. It will help them to understand the roles these proteins play in controlling what happens in plant cells and develop varieties that are able to tolerate different types of stresses, such as diseases or harsh weather conditions, which could have major benefits for agriculture and food production.

Technical Abstract: 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 posttranscriptional 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.) [1]. 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.