Surpassing limits of static RNA modification analysis with dynamic NAIL-MS

Area of Science:

• Molecular Biology
• Biochemistry
• Analytical Chemistry

Background:

• Ribonucleic acid (RNA) modifications are crucial for cellular function and are typically quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• LC-MS/MS provides a static snapshot of RNA modification abundance, limiting the understanding of their dynamic processes.

Purpose of the Study:

• To introduce and validate nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) as a method to study the dynamics of RNA modifications.
• To apply NAIL-MS to investigate the demethylation kinetics of specific RNA modifications in response to genotoxic agents.

Main Methods:

• Development and description of labeling techniques for NAIL-MS in prokaryotic (E. coli) and eukaryotic (S. cerevisiae, human cell culture) systems.
• Application of NAIL-MS to quantify the demethylation rates of 1-methyladenosine and 3-methylcytidine in E. coli treated with methyl-methanesulfonate.
• Validation experiments to ensure accurate data interpretation and exclude confounding factors like RNA degradation or turnover.

Main Results:

• NAIL-MS successfully provides access to the dynamic changes in RNA modifications, overcoming the limitations of static LC-MS/MS analysis.
• The study successfully applied NAIL-MS to measure the speed and efficiency of specific RNA demethylation processes.
• NAIL-MS allowed for the exclusion of RNA degradation, turnover, and dilution as concurrent processes affecting methylation levels.

Conclusions:

• NAIL-MS is a powerful tool for elucidating the dynamics of RNA modifications, offering a significant advancement over existing methods.
• The technique enables precise measurement of RNA modification turnover rates and can be used to study cellular responses to damage.
• Future applications of NAIL-MS may include relative quantification of tRNA isoacceptor abundances and other dynamic RNA processes.