N
6
-methyladenosine (m
6
A) is the most abundant internal modification on mammalian messenger RNA (mRNA). It is installed by a writer complex and can be reversed by erasers such as the fat mass and obesity-associated protein (FTO). Despite extensive research, the primary physiological substrates of FTO in mammalian tissues and development remain elusive. Here, we show that FTO mediates m
6
A demethylation of long-interspersed element-1 (LINE1) RNA in mouse embryonic stem cells (mESCs), regulating LINE1 RNA abundance and the local chromatin state, which in turn modulates transcription of LINE1-containing genes. FTO-mediated LINE1 RNA m
6
A demethylation also plays regulatory roles in shaping chromatin state and gene expression during mouse oocyte and embryonic development. Our results suggest broad effects of LINE1 RNA m
6
A demethylation by FTO in mammals.
Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10–20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different ‘writer’ proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.
N
6
–methyladenosine (m
6
A) is the most abundant mRNA modification and plays crucial roles in diverse physiological processes. Utilizing a Massively Parallel Assay for m
6
A (MPm
6
A), we discover that m
6
A specificity is globally regulated by “suppressors” that prevent m
6
A deposition in unmethylated transcriptome regions. We identify Exon Junction Complexes (EJCs) as m
6
A suppressors that protect exon junction-proximal RNA within coding sequences from methylation and regulate mRNA stability through m
6
A suppression. EJC suppression of m
6
A underlies multiple global characteristics of mRNA m
6
A specificity, with the local range of EJC protection sufficient to suppress m
6
A deposition in average-length internal exons, but not in long internal and terminal exons. EJC-suppressed methylation sites co-localize with EJC-suppressed splice sites, suggesting that exon architecture broadly determines local mRNA accessibility to regulatory complexes.
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