A metabolic labeling method detects m⁶A transcriptome-wide at single base resolution.

Area of Science:

• Epigenetics
• RNA Biology
• Molecular Biology

Background:

• N6-methyladenosine (m6A) is a crucial epitranscriptomic modification.
• Accurate transcriptome-wide mapping of m6A at base resolution is challenging.
• Understanding m6A's nucleotide-level roles requires precise detection methods.

Purpose of the Study:

• To develop a novel metabolic labeling method for base-resolution m6A mapping.
• To introduce 'm6A-label-seq' for transcriptome-wide m6A detection.
• To overcome limitations of existing m6A sequencing techniques.

Main Methods:

• Metabolic labeling of human and mouse cells with Se-allyl-L-selenohomocysteine, a methionine analog.
• Incorporation of N6-allyladenosine (a6A) into cellular RNAs at potential m6A sites.
• Detection of a6A via iodination-induced misincorporation during complementary DNA synthesis in reverse transcription.

Main Results:

• Identification of thousands of mRNA m6A sites in human and mouse cell lines.
• Demonstration of m6A-label-seq's capability to detect clustered m6A sites.
• Validation of identified sites using an orthogonal method and comparison with existing m6A sequencing techniques.

Conclusions:

• m6A-label-seq provides base-resolution transcriptome-wide m6A mapping.
• The method facilitates the study of m6A roles at the nucleotide level.
• m6A-label-seq shows potential for mapping m6A modifications in nuclear nascent RNA.