Single-Cell Multimodal Profiling of m⁶A mRNA Modification under Oxidative Stress

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• N6-methyladenosine (m⁶A) RNA modifications are crucial in cellular processes.
• Understanding m⁶A dynamics under stress requires high-resolution, multidimensional analysis.
• Existing methods lack the ability to capture spatial localization and quantitative data simultaneously at the single-cell level.

Purpose of the Study:

• To develop a novel strategy for identifying m⁶A modification with location-, quantification-, and single-cell resolution.
• To investigate the spatial and quantitative changes of m⁶A under oxidative stress conditions.
• To provide a comprehensive analytical framework for studying m⁶A regulatory dynamics.

Main Methods:

• Development of a multisignal integrated nanocluster-driven strategy.
• Application of synchrotron radiation soft X-ray microscopy for 3D nanoscale imaging of m⁶A RNA.
• Analysis of m⁶A RNA aggregation spots (ASs) and their colocalization with stress granules (SGs).

Main Results:

• Achieved the first 3D nanoscale imaging of m⁶A RNA within individual cells.
• Demonstrated that oxidative stress induces significant m⁶A aggregation.
• Observed a 65% colocalization rate between m⁶A ASs and SGs.
• Found that m⁶A AS size positively correlates with oxidative stress intensity.
• Revealed an inverse relationship between m⁶A abundance and oxidative stress intensity.

Conclusions:

• The proposed multimodal strategy enables precise, single-cell resolution analysis of m⁶A modifications.
• Oxidative stress triggers m⁶A RNA aggregation, often associated with stress granules.
• The quantitative and spatial dynamics of m⁶A under stress provide critical insights into cellular regulatory mechanisms.