Remodeling of RNA-Binding Proteome and RNA-mediated regulation as a new layer of control of sporulation

Sporulation allows certain bacteria to survive extreme conditions for extended periods posing challenges to public health and food safety. Transcriptional level of regulation relying on σ factors has been well studied in the non-pathogenic model bacterium Bacillus subtilis , while post-transcriptional control remains poorly understood. RNA-binding proteins (RBPs) and small non-coding RNAs (sRNAs) modulate gene expression by affecting mRNA stability or translation. Recent studies suggest that RNA-mediated regulation plays a role in sporulation, but its networks and interactions remain largely uncharacterized, highlighting the need for further investigation. To address this knowledge gap, we adapted OOPS (Orthogonal Organic Phase Separation), a high-throughput method to specifically purify RNA binding proteome (RBPome). By monitoring its dynamics, we observed RBPome is highly remodeled during B. subtilis process. Our approach led to the identification of novel RBPs and potential RNA-mediated post-transcriptional regulators of sporulation. This work provides important insights into the interplay between RNAs and RBPs, advancing the understanding of post-transcriptional regulation in Gram-positive spore-forming bacteria. It also offers new resources for exploring the molecular mechanisms that govern sporulation.