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APEX2 proximity labeling of RNA in bacteria
Hadi Yassine1, Elizabeta Sirotkin2, Omer Goldberger2
1Department of Biology, Indiana University, Bloomington, IN 47405, USA; Departments of Chemistry and Biological Sciences, Wayne State University, Detroit, MI 48202, USA.
Cell Reports Methods
|October 21, 2025
Summary
Researchers developed a new method for rapid RNA labeling in bacteria using APEX2 proximity labeling. This technique enables precise spatial and temporal investigation of RNA localization, crucial for understanding bacterial gene regulation.
Area of Science:
- Molecular Biology
- Bacterial Cell Biology
- RNA Biology
Background:
- Investigating RNA localization in bacteria requires rapid, spatially controlled methods.
- Existing techniques are often limited by the fast decay rates of bacterial mRNA.
- APEX2 proximity labeling has been successful in eukaryotic cells for RNA localization studies.
Purpose of the Study:
- To adapt APEX2 proximity labeling for rapid RNA labeling in bacterial cells.
- To enable the study of RNA localization with high spatial and temporal resolution.
- To provide a method that can outpace bacterial mRNA decay.
Main Methods:
- Generated an APEX2 fusion to the ribonuclease (RNase) E gene.
- Utilized APEX2 proximity labeling for RNA labeling on a sub-minute timescale.
- Employed alkyne-phenol for labeling and copper-catalyzed click chemistry for downstream applications.
Main Results:
- Successfully adapted APEX2 proximity labeling for bacterial RNA.
- Achieved rapid RNA labeling (sub-minute timescale), outpacing mRNA decay.
- Demonstrated the method's minimal perturbation to cellular processes.
Conclusions:
- APEX2 proximity labeling is a viable and effective method for studying bacterial RNA localization.
- This technique offers flexibility for downstream applications like purification and imaging.
- The method provides a powerful tool for advancing our understanding of bacterial gene expression and regulation.
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