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.

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.

Highlights d RNA m 6 A methylation plays essential roles in early B cell development d Loss of RNA m 6 A-writer complex blocks two key transitions in B cell development d RNA m 6 A facilitates IL-7-induced pro-B cell proliferation via its reader YTHDF2 d The large-pre-B-to-small-pre-B transition is independent of YTHDF1/2