APEX2 RNA Proximity Labeling in Mammalian Cell Lines With Low Biotin Permeability

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Subcellular RNA localization is vital for cellular functions, including development and stress responses.
• Proximity labeling techniques, such as APEX2, offer advantages over traditional methods for detecting localized transcripts and enriched proteins.
• Standard APEX2 proximity labeling protocols face challenges in cell lines with low biotin permeability, such as human induced pluripotent stem cells (hiPSCs).

Purpose of the Study:

• To address the limitations of standard APEX2 proximity labeling in cell lines with poor biotin permeability.
• To develop an optimized protocol for efficient and homogeneous proximity labeling in hiPSCs and MCF10A cells.
• To expand the utility of APEX2 proximity labeling to a broader range of cell types.

Main Methods:

• Adapted the APEX2 proximity labeling protocol by incorporating a mild detergent during biotin incubation to enhance biotin uptake.
• Utilized the rapid hydrogen peroxide-driven APEX2 reaction.
• Included controls for nonspecific molecular localization and employed statistical methods for data analysis.

Main Results:

• The adapted protocol successfully enabled efficient proximity labeling in hiPSCs, overcoming previous limitations.
• Homogeneous biotinylation across single cells was achieved in MCF10A breast epithelial cells.
• The findings identified low biotin permeability as the primary cause of failed or inefficient labeling in standard protocols.

Conclusions:

• The modified APEX2 proximity labeling protocol significantly improves labeling efficiency and homogeneity in cell lines with low biotin permeability.
• This optimized method broadens the application of APEX2 proximity labeling for studying transcript and protein localization in challenging cell models.
• The protocol provides a robust approach for investigating subcellular localization critical for biological processes.