N6-methyladenosine (m6A) is the most prevalent internal (non-cap) modification present in the messenger RNA (mRNA) of all higher eukaryotes1,2. Although essential to cell viability and development3–5, the exact role of m6A modification remains to be determined. The recent discovery of two m6A demethylases in mammalian cells highlighted the importance of m6A in basic biological functions and disease6–8. Here we show that m6A is selectively recognized by the human YTH domain family 2 (YTHDF2) protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m6A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies9. The C-terminal domain of YTHDF2 selectively binds to m6A-containing mRNA whereas the N-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m6A modification is recognized by selective-binding proteins to affect the translation status and lifetime of mRNA.

We report here that FTO (fat mass and obesity-associated protein) exhibits efficient oxidative demethylation activity of abundant N6-methyladenosine (m6A) residues in RNA in vitro. FTO knockdown with siRNA led to an increased level of m6A in mRNA, whereas overexpression of FTO resulted in a decreased level of m6A in human cells. We further show that FTO partially colocalizes with nuclear speckles, supporting m6A in nuclear RNA as a physiological substrate of FTO.

SUMMARY N6-methyladenosine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m6A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m6A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m6A modification has fundamental and broad functions in mammalian cells.

N6-methyladenosine (m6A) is the most prevalent and reversible internal modification in mammalian messenger and non-coding RNAs. We report here that human METTL14 catalyzes m6A RNA methylation. Together with METTL3, the only previously known m6A methyltransferase, these two proteins form a stable heterodimer core complex of METTL3-14 that functions in cellular m6A deposition on mammalian nuclear RNAs. WTAP, a mammalian splicing factor, can interact with this complex and affect this methylation.

Cellular RNAs carry diverse chemical modifications that used to be regarded as static and having minor roles in 'fine-tuning' structural and functional properties of RNAs. In this Review, we focus on reversible methylation through the most prevalent mammalian mRNA internal modification, N(6)-methyladenosine (m(6)A). Recent studies have discovered protein 'writers', 'erasers' and 'readers' of this RNA chemical mark, as well as its dynamic deposition on mRNA and other types of nuclear RNA. These findings strongly indicate dynamic regulatory roles that are analogous to the well-known reversible epigenetic modifications of DNA and histone proteins. This reversible RNA methylation adds a new dimension to the developing picture of post-transcriptional regulation of gene expression.