Abstract
N6,2′-O-dimethyladenosine (m6Am), a terminal modification adjacent to the mRNA cap, is a newly discovered reversible RNA modification. Yet, a specific and sensitive tool to directly map transcriptome-wide m6Am is lacking. Here, we report m6Am-seq, based on selective in vitro demethylation and RNA immunoprecipitation. m6Am-seq directly distinguishes m6Am and 5′-UTR N6-methyladenosine (m6A) and enables the identification of m6Am at single-base resolution and 5′-UTR m6A in the human transcriptome. Using m6Am-seq, we also find that m6Am and 5′-UTR m6A respond dynamically to stimuli, and identify key functional methylation sites that may facilitate cellular stress response. Collectively, m6Am-seq reveals the high-confidence m6Am and 5′-UTR m6A methylome and provides a robust tool for functional studies of the two epitranscriptomic marks.
Highlights
N6,2′-O-dimethyladenosine (m6Am), a terminal modification adjacent to the mRNA cap, is a newly discovered reversible RNA modification
In addition to m6A, there exists another reversible modification in higher eukaryotes, called N6, 2′-O-dimethyladenosine (m6Am), which is precisely located at the first transcribed nucleotide and adjacent to the cap structure of mRNA5,6
A total of 50–80% of adenosine-starting mammalian mRNAs are believed to be m6Am modified[6]. It is catalyzed by PCIF17–10, a protein that interacts with the phosphorylated CTD of RNAPII11, and could be removed by the m6A demethylase FTO12,13
Summary
N6,2′-O-dimethyladenosine (m6Am), a terminal modification adjacent to the mRNA cap, is a newly discovered reversible RNA modification. M6Am-seq directly distinguishes m6Am and 5′-UTR N6-methyladenosine (m6A) and enables the identification of m6Am at single-base resolution and 5′-UTR m6A in the human transcriptome. Using m6Am-seq, we find that m6Am and 5′-UTR m6A respond dynamically to stimuli, and identify key functional methylation sites that may facilitate cellular stress response. M6Am-seq relies on an in vitro demethylation reaction that selectively removes m6Am while keeps m6A intact, thereby discriminating genuine m6Am at the mRNA cap and 5′-UTR m6A. Using m6Am-seq, we identify 2166 m6Am sites at base resolution from 1652 peaks and 1307 5′-UTR m6A peaks throughout the human transcriptome. We provide a tool to directly and selectively distinguish and profile m6Am and 5′-UTR m6A; we anticipate that m6Am-seq will pave the way for future functional studies of m6Am in various biological systems
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
