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

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.