SUMMARY Methylation of the N6 position of adenosine (m6A) is a post-transcriptional modification of RNA whose prevalence and physiological relevance is poorly understood. The recent discovery that FTO, an obesity risk gene, encodes an m6A demethylase implicates m6A as an important regulator of physiological processes. Here we present a method for transcriptome-wide m6A localization, which combines m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq). We use this method to identify mRNAs of 7,676 mammalian genes that contain m6A, indicating that m6A is a common base modification of mRNA. The m6A modification exhibits tissue-specific regulation and is markedly increased throughout brain development. We find that m6A sites are enriched near stop codons and in 3' UTRs, and we uncover an association between m6A residues and microRNA binding sites within 3' UTRs. These findings provide a resource for identifying transcripts that are substrates for adenosine methylation and reveal insights into the epigenetic regulation of the mammalian transcriptome.

SUMMARY Protein translation typically begins with the recruitment of the 43S ribosomal complex to the 5′ cap of mRNAs by a cap-binding complex. However, some transcripts are translated in a cap-independent manner through poorly understood mechanisms. Here, we show that mRNAs containing N6-methyladenosine (m6A) in their 5′ UTR can be translated in a cap-independent manner. A single 5′ UTR m6A directly binds eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex to initiate translation in the absence of the cap-binding factor eIF4E. Inhibition of adenosine methylation selectively reduces translation of mRNAs containing 5′UTR m6A. Additionally, increased m6A levels in the Hsp70 mRNA regulate its cap-independent translation following heat shock. Notably, we find that diverse cellular stresses induce a transcriptome-wide redistribution of m6A, resulting in increased numbers of mRNAs with 5′ UTR m6A. These data show that 5′ UTR m6A bypasses 5′ cap-binding proteins to promote translation under stresses.

N6-methyladenosine (m6A) is a modified base that has long been known to be present in noncoding RNAs, ribosomal RNA, polyadenylated RNA and at least one mammalian mRNA. However, our understanding of the prevalence of this modification has been fundamentally redefined by transcriptome-wide m6A mapping studies, which have shown that m6A is present in a large subset of the transcriptome in specific regions of mRNA. This suggests that mRNA may undergo post-transcriptional methylation to regulate its fate and function, analogous to methyl modifications in DNA. Thus, the pattern of methylation constitutes an mRNA ‘epitranscriptome’. The identification of adenosine methyltransferases (‘writers’), m6A demethylating enzymes (‘erasers’) and m6A binding proteins (‘readers’) is helping to define cellular pathways for the post-transcriptional regulation of mRNAs.

In recent years, m6A has emerged as an abundant and dynamically regulated modification throughout the transcriptome. Recent technological advances have enabled the transcriptome-wide identification of m6A residues, which in turn has provided important insights into the biology and regulation of this pervasive regulatory mark. Also central to our current understanding of m6A are the discovery and characterization of m6A readers, writers, and erasers. Over the last few years, studies into the function of these proteins have led to important discoveries about the regulation and function of m6A. However, during this time our understanding of these proteins has also evolved considerably, sometimes leading to the reversal of early conceptions regarding the reading, writing and erasing of m6A. In this review, we summarize recent advances in m6A research, and we highlight how these new findings have reshaped our understanding of how m6A is regulated in the transcriptome.

Dopaminergic (DA) signaling governs the control of complex behaviors, and its deregulation has been implicated in a wide range of diseases. Here we demonstrate that inactivation of the Fto gene, encoding a nucleic acid demethylase, impairs dopamine receptor type 2 (D2R) and type 3 (D3R) (collectively, 'D2-like receptor')-dependent control of neuronal activity and behavioral responses. Conventional and DA neuron-specific Fto knockout mice show attenuated activation of G protein-coupled inwardly-rectifying potassium (GIRK) channel conductance by cocaine and quinpirole. Impaired D2-like receptor-mediated autoinhibition results in attenuated quinpirole-mediated reduction of locomotion and an enhanced sensitivity to the locomotor- and reward-stimulatory actions of cocaine. Analysis of global N(6)-methyladenosine (m(6)A) modification of mRNAs using methylated RNA immunoprecipitation coupled with next-generation sequencing in the midbrain and striatum of Fto-deficient mice revealed increased adenosine methylation in a subset of mRNAs important for neuronal signaling, including many in the DA signaling pathway. Several proteins encoded by these mRNAs had altered expression levels. Collectively, FTO regulates the demethylation of specific mRNAs in vivo, and this activity relates to the control of DA transmission.