APEX2 proximity labeling of RNA in bacteria

Rapid, spatially controlled methods are needed to investigate RNA localization in bacterial cells. APEX2 proximity labeling was shown to be adaptable to rapid RNA labeling in eukaryotic cells and, through the fusion of APEX2 to different proteins targeted to diverse subcellular locations, has been useful to identify RNA localization in these cells. Therefore, we adapted APEX2 proximity labeling of RNA to bacterial cells by generating an APEX2 fusion to the ribonuclease (RNase) E gene, which is necessary and sufficient for bacterial ribonucleoprotein (BR)-body formation. APEX2 fusion is minimally perturbative, and RNA can be rapidly labeled on the sub-minute timescale with alkyne-phenol, outpacing the rapid speed of mRNA decay in bacteria. Alkyne-phenol provides flexibility in the overall application with copper-catalyzed click chemistry for downstream processes, such as fluorescent dye azides or biotin-azides for purification. Altogether, APEX2 proximity labeling of RNA provides a useful method for studying RNA localization in bacteria.