Plant phase extraction (PPE): A novel method for enhanced discovery of RNA-binding proteome in plants

RNA-binding proteins (RBPs) are versatile effectors in posttranscriptional gene regulation. Systematic profiling of RBPs in plants has been limited to proteins interacting with polyadenylated (poly(A)) RNAs due to the lack of an efficient method of isolating RBPs associated with non-poly(A) RNAs. Here we reported the establishment and application of plant phase extraction (PPE) as a novel method to comprehensively discover the RNA-binding proteome in Arabidopsis leaf tissues, leading to the isolation of 1,169 RBPs, of which 673 corroborate with previously reported RBPs and 496 are novel RBPs. PPE showed unmatched ability in capturing 374 diverse RNA-binding domains (RBDs), while 44% of the RBPs lack recognized RBDs. PPE recovered far more ribosomal and tRNA-related RBPs and metabolic enzymes with moonlighting RBP functions than any other individual report from plants, signifying its advantage in unbiasedly retrieving non-poly(A) and unconventional RBPs. We proposed that intrinsically disordered regions (IDRs) contributed to the non-classical binding of some novel RBPs, and provided the first evidence that enzymatic domains from metabolic enzymes have additional roles in RNA-binding. Taken together, our findings demonstrated that PPE is an effective approach to identifying a wide range of RBPs in complex plant tissues and may have broad biological implications.