GLORI for absolute quantification of transcriptome-wide m⁶A at single-base resolution

Area of Science:

• Molecular Biology
• Epigenetics
• Genomics

Background:

• N⁶-methyladenosine (m⁶A) is the most prevalent epitranscriptomic modification, crucial for regulating mRNA metabolism and cellular processes.
• Existing methods for m⁶A mapping often lack absolute quantification or single-base resolution, limiting comprehensive analysis.

Purpose of the Study:

• To introduce GLORI, a novel sequencing technology for generating a globally absolute-quantitative m⁶A map.
• To provide a detailed protocol and bioinformatics pipeline (GLORI-tools) for m⁶A site identification and analysis.
• To offer a sensitive and robust tool for precise m⁶A biology investigation.

Main Methods:

• Utilized a selective chemical reaction (glyoxal and nitrite) to deaminate unmethylated adenosines, preserving m⁶A.
• Employed modified RNA library construction protocols (eCLIP or commercial kits).
• Developed a tailored bioinformatics pipeline (GLORI-tools) for data analysis.

Main Results:

• GLORI achieves absolute quantification of m⁶A sites at single-base resolution.
• Identified approximately 80,000 m⁶A sites using 50 Gb sequencing data in mammalian cells.
• Demonstrated exceptional sensitivity and robustness compared to existing m⁶A mapping techniques.

Conclusions:

• GLORI provides a precise and unbiased method for global m⁶A profiling.
• The technique facilitates in-depth investigation of m⁶A's role in various biological contexts.
• GLORI is a valuable tool for advancing the field of m⁶A biology.