Decoding m⁶Am by simultaneous transcription-start mapping and methylation quantification.

Area of Science:

• Molecular Biology
• RNA Biology
• Epigenetics

Background:

• N6,2 -O-dimethyladenosine (m6Am) is a vital epitranscriptomic modification found at the 5 end of mRNA and snRNA.
• Current m6Am detection methods are limited by the assumption of single transcription start sites per gene, failing to account for transcript isoform diversity.

Purpose of the Study:

• To develop and apply a novel method, CROWN-seq, for simultaneous identification of transcription start sites and quantification of m6Am stoichiometry across 5 isoforms.
• To comprehensively map the m6Am landscape in human cells and investigate its relationship with gene expression and transcription initiation.

Main Methods:

• CROWN-seq: A novel sequencing-based method to identify transcription start sites and quantify m6Am modification stoichiometry for individual mRNA 5 isoforms.
• Application of CROWN-seq across nine human cell lines to generate a transcriptome-wide m6Am map.

Main Results:

• m6Am is predominantly a high stoichiometry modification across most cellular mRNAs, with a small fraction exhibiting lower stoichiometry.
• m6Am modification levels positively correlate with transcript expression levels.
• Evidence suggests a link between m6Am modification and specific promoter sequences and transcription initiation mechanisms.

Conclusions:

• CROWN-seq provides a powerful tool to resolve m6Am heterogeneity across transcript isoforms.
• m6Am plays a significant role in regulating gene expression, potentially influencing transcription initiation.
• These findings suggest a novel regulatory function for m6Am in gene expression control.