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.

N6-methyladenosine (m6A) is an abundant nucleotide modification in mRNA that is required for the differentiation of mouse embryonic stem cells. However, it remains unknown whether m6A controls differentiation of normal and/or malignant myeloid hematopoietic cells. Here we show that shRNA-mediated depletion of the m6A-forming enzyme METTL3 in human hematopoietic stem/progenitor cells promotes differentiation coupled with reduced proliferation. Conversely, overexpression of wild-type METTL3, but not the catalytic-dead form of METTL3, inhibits differentiation and increases cell growth. METTL3 mRNA and protein is expressed more abundantly in acute myeloid leukemia (AML) cells compared to healthy hematopoietic stem/progenitor cells and other types of tumors. Furthermore, METTL3 depletion in humanmyeloid leukemia cell lines induces differentiation and apoptosis and delays leukemia in recipient mice in vivo. Single-nucleotide resolution mapping of m6A coupled with ribosome profiling reveals that m6A promotes the translation of c-MYC, BCL2 and PTEN mRNAs in human myeloid leukemia MOLM13 cells. Moreover, loss of METTL3 leads to increased levels of pAKT, which contributes to the differentiation effects of METTL3 depletion. Overall these results provide a rationale for therapeutic targeting of METTL3 in myeloid leukemia.

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA. Very little is known of the function of m6A in the immune system or its role in host–pathogen interactions. Here we investigated the topology, dynamics, and bidirectional influences of the viral–host RNA methylomes during HIV-1 infection of human CD4 T cells. We show that viral infection triggers a massive increase in m6A in both host and viral mRNAs. In HIV-1 mRNA, we identified 14 methylation peaks in coding and noncoding regions, splicing junctions, and splicing regulatory sequences. We also identified a set of 56 human gene transcripts that were uniquely methylated in HIV-1-infected T cells and were enriched for functions in viral gene expression. The functional relevance of m6A for viral replication was demonstrated by silencing of the m6A writer or the eraser enzymes, which decreased or increased HIV-1 replication, respectively. Furthermore, methylation of two conserved adenosines in the stem loop II region of HIV-1 Rev Response Element (RRE) RNA enhanced binding of HIV-1 Rev protein to the RRE in vivo and influenced nuclear export of RNA. Our results identify a new mechanism for the control of HIV-1 replication and its interaction with the host immune system.

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.

Recent studies have found methyl-6-adenosine in thousands of mammalian genes, and this modification is most pronounced near the beginning of the 3' UTR. We present a perspective on current work and new single-molecule sequencing methods for detecting RNA base modifications.

SUMMARY The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station’s history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.

Figure 3B has been corrected to show the general coverage of the Yersinia pestis pMT1 plasmid, but not the murine toxin gene (yMT). The initial claim of ''.consistent 203 coverage across the murine toxin gene.'' was erroneously based on looking at gene annotation coordinates from different reference sequences. No reads mapped to the yMT gene when updated annotations were used. The Summary, Results, and Discussion sections have been revised to remove and clarify misleading and speculative text about pathogenic organisms. We now state that although all our metagenomic analysis tools identified reads with similarity to B. anthracis and Y. pestis sequences, there is minimal coverage to the backbone genome of these organisms, and there is no strong evidence to suggest these organisms are in fact present, and no evidence of pathogenicity. The figure and the text have been corrected online and in the print version.

Methyl-6-adenosine (m6A) has been hypothesized to exist since the 1970s,1 but little has been known about the specific RNAs, or sites within them, that are affected by this RNA modification. Here, we report that recent work has shown RNA modifications like m6A, collectively called the “epitranscriptome,” are a pervasive feature of mammalian cells and likely play a role in development and disease. An enrichment of m6A near the last CDS of thousands of genes has implicated m6A in transcript processing, translational regulation and potentially a mechanism for regulating miRNA maturation. Also, because the sites of m6A show strong evolutionary conservation and have been replicated in nearly identical sites between mouse and human, strong evolutionary pressures are likely being maintained for this mark.2,3 Finally, we note that m6A is one of over 100 modifications of RNA that have been reported,4 and with the combination of high-throughput, next-generation sequencing (NGS) techniques, immunoprecipitation with appropriate antibodies and splicing-aware peak-finding, the dynamics of the epitranscriptome can now be mapped and characterized to discern their specific cellular roles.