m⁶Am-seq reveals the dynamic m⁶Am methylation in the human transcriptome

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• N6,2 -O-dimethyladenosine (m6Am) is a recently identified, reversible RNA modification found at the 5 end of mRNA.
• A specific and sensitive method for mapping m6Am across the transcriptome is currently unavailable.
• Distinguishing m6Am from other related RNA modifications like 5 -UTR N6-methyladenosine (m6A) is challenging.

Purpose of the Study:

• To develop and validate a novel technique for transcriptome-wide mapping of m6Am.
• To enable the direct and sensitive detection of m6Am and 5 -UTR m6A modifications.
• To investigate the dynamic regulation and functional significance of m6Am and 5 -UTR m6A during cellular processes.

Main Methods:

• Development of m6Am-seq, a method combining selective in vitro demethylation with RNA immunoprecipitation.
• Application of m6Am-seq for single-base resolution mapping of m6Am in the human transcriptome.
• Analysis of dynamic changes in m6Am and 5 -UTR m6A levels in response to cellular stimuli.

Main Results:

• m6Am-seq successfully distinguishes m6Am from 5 -UTR m6A.
• The method enables high-resolution mapping of both m6Am and 5 -UTR m6A in the human transcriptome.
• m6Am and 5 -UTR m6A were found to respond dynamically to stimuli, with specific sites linked to cellular stress response.

Conclusions:

• m6Am-seq provides a robust and specific tool for studying m6Am and 5 -UTR m6A.
• The study reveals the high-confidence m6Am and 5 -UTR m6A methylome.
• This technique facilitates functional investigations into the roles of these epitranscriptomic marks in cellular regulation and stress response.