APEX2 proximity labeling of RNA in bacteria.

Abstract

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 different 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 RNase E gene, which is necessary and sufficient for 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 downstream application with copper catalyzed click-chemistry for downstream applications, such as fluorescent dyeazides or biotin-azides for purification. Altogether, APEX2 proximity labeling of RNA provides a useful method for studying RNA localization in bacteria. Studies over the past several years have shown that distinct RNAs can be targeted to subcellular locations in bacterial cells. The ability to investigate localized RNAs in bacteria is currently limited to imaging-based approaches or to laborious procedures to isolate ribonucleoprotein complexes by grad-seq, HITS-CLIP, or Rloc-seq. However, a major challenge in studying mRNA localization in bacterial cells is that bacterial mRNAs typically last for only a few minutes in the cell, while experiments to investigate their localization or interaction partners can take much longer. Therefore, rapid methods of studying RNA localization are needed to bridge this technical challenge. APEX2 proximity labeling can be applied to RNA in bacteriaAPEX2 RNA labeling reactions occur on the sub-minute timescale.APEX2 workflow requires less material and time than current methods.Alkyne-Phenol APEX2 substrate provides flexibility with click-chemistry.