Plant phase extraction: A method for enhanced discovery of the RNA-binding proteome and its dynamics in plants

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

Submitted to: The Plant Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/13/2023
Publication Date: 5/5/2023
Citation: Zhang, Y., Xu, Y., Skaggs, T.H., Ferreira, J.F., Chen, X., Sandhu, D. 2023. Plant phase extraction: A method for enhanced discovery of the RNA-binding proteome and its dynamics in plants. The Plant Cell. 35(8):2750-2772. https://doi.org/10.1093/plcell/koad124.
DOI: https://doi.org/10.1093/plcell/koad124

Interpretive Summary: RNA-binding proteins (RBPs) interact with RNA and play vital roles in governing cell fate and essential developmental processes. Several methods have been developed to identify RBPs in human cell lines, mouse brains, and bacteria. However, none of these approaches were successful in identifying RBPs in plants. We have developed a novel plant phase extraction (PPE) method for isolating RBPs from plant tissues and showcased the technical advantages of PPE over other methods on multiple levels. Using PPE, we identified 1,169 RBPs, including traditional RBPs participating in various aspects of RNA metabolism. Remarkably, we discovered 496 novel RBPs, many of which are engaged in non-coding RNA binding. Moreover, putative novel RNA binding domains (RBDs) from non-classical RBPs were revealed. We further developed an mRNA pull-down assay, validated some of the moonlighting enzymes as valid RBPs, and, most importantly, demonstrated the active sites of these enzymes with a second role as an RBD. This work has paved the way to characterize novel RBPs and investigate RBP functions under different physiological and stress conditions. Our results will help plant biologists and geneticists decipher mechanisms involved in RBP-mediated regulation of gene expression in plants. This knowledge will be crucial for plant breeders in developing elite genetic material tolerant to various biotic and abiotic stresses.

Technical Abstract: RNA-binding proteins (RBPs) play critical roles in posttranscriptional gene regulation. Current methods of systematically profiling RBPs in plants have been predominantly limited to proteins interacting with polyadenylated (poly(A)) RNAs. We developed a method called plant phase extraction (PPE), which yielded a highly comprehensive RNA-binding proteome(RBPome),uncovering 2,517 RBPs from Arabidopsis (Arabidopsis thaliana) leaf and root samples with a highly diverse array of RNA-binding domains. We identified traditional RBPs that participate in various aspects of RNA metabolism and a plethora of nonclassical proteins moonlighting as RBPs. We uncovered constitutive and tissue-specific RBPs essential for normal development and,more importantly, revealed RBPs crucial for salinity stress responses from a RBP–RNA dynamics perspective. Remarkably, 40% of the RBPs are non-poly(A) RBPs that were not previously annotated as RBPs, signifying the advantage of PPE in unbiasedly retrieving RBPs. We propose that intrinsically disordered regions contribute to their nonclassical binding and provide evidence that enzymatic domains from metabolic enzymes have additional roles in RNA binding. Taken together, our findings demonstrate that PPE is an impactful approach for identifying RBPs from complex plant tissues and pave the way for investigating RBP functions under different physiological and stress conditions at the posttranscriptional level.