Dynamic Mapping of RNA-Binding Proteins During Bacillus subtilis Sporulation Using Orthogonal Organic Phase Separation.

RNA-binding proteins (RBPs) have pleiotropic roles in modulating the physiology of both eukaryotic and prokaryotic cells, enabling them to adapt to environmental variations. The importance of RBPs has led to the development of a variety of methods aiming to identify them. However, most of these approaches have primarily been implemented and optimized in eukaryotic systems. To both uncover novel RBPs involved in Bacillus subtilis sporulation and capture their RNA-binding ability dynamically, we adapted the orthogonal organic phase separation technique (OOPS), which had previously been used in Escherichia coli to reveal its RNA-binding proteome (RBPome). We optimized the UV cross-linking process used to stabilize RNA-protein interactions in vivo and the bacterial lysis process to overcome the robust cell wall of Gram-positive sporulating cells. RNA-protein complexes are then recovered after phase separation steps using guanidinium thiocyanate-phenol-chloroform, and RNA-associated proteins are identified and label-free-quantified by liquid chromatography-mass spectrometry. Collecting samples at various time points during sporulation further enables tracking the dynamics of the RBPome. In addition to being applicable to bacteria and requiring minimal starting material, this method has provided a comprehensive map of the RBPome during sporulation, refining the roles of known factors and revealing new players. Key features • The high-throughput method OOPS, developed in [1], was successfully applied to both sporulating and vegetative cells of a Gram-positive bacterium to specifically purify the RBPome. • OOPS allowed tracking of RBPome remodeling dynamics (both RBP abundance and RNA binding ability) across different stages of B. subtilis sporulation. • OOPS enabled the identification of novel RBPs in the context of sporulation, revealing potential new players in RNA-mediated regulation.