Methylation of the N6 position of adenosine (m6A) is a posttranscriptional modification of RNA with poorly understood prevalence and physiological relevance. 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.
Display omitted
► m6A is a widespread RNA modification in many tissues with high levels in the brain ► MeRIP-Seq identifies m6A in 7,913 genes encoding both coding and noncoding RNAs ► m6A is enriched near stop codons and within 3′ UTRs in both mouse and human mRNAs ► The transcriptome-wide landscape of m6A provides important insights into m6A function
Adenosine methylation (m6A) is a widespread RNA modification found in >7,000 mammalian genes, encoding both mRNAs and noncoding RNAs, with an especially high prevalence in the developing brain. In both mouse and human mRNAs, m6A is enriched within 3′ UTRs that also contain miRNA-binding sites.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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.
N
-methyladenosine (m
A) is an abundant nucleotide modification in mRNA that is required for the differentiation of mouse embryonic stem cells. However, it remains unknown whether the m
A ...modification controls the differentiation of normal and/or malignant myeloid hematopoietic cells. Here we show that shRNA-mediated depletion of the m
A-forming enzyme METTL3 in human hematopoietic stem/progenitor cells (HSPCs) promotes cell differentiation, coupled with reduced cell proliferation. Conversely, overexpression of wild-type METTL3, but not of a catalytically inactive form of METTL3, inhibits cell differentiation and increases cell growth. METTL3 mRNA and protein are expressed more abundantly in acute myeloid leukemia (AML) cells than in healthy HSPCs or other types of tumor cells. Furthermore, METTL3 depletion in human myeloid leukemia cell lines induces cell differentiation and apoptosis and delays leukemia progression in recipient mice in vivo. Single-nucleotide-resolution mapping of m
A coupled with ribosome profiling reveals that m
A promotes the translation of c-MYC, BCL2 and PTEN mRNAs in the human acute myeloid leukemia MOLM-13 cell line. Moreover, loss of METTL3 leads to increased levels of phosphorylated AKT, which contributes to the differentiation-promoting effects of METTL3 depletion. Overall, these results provide a rationale for the therapeutic targeting of METTL3 in myeloid leukemia.
N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of eukaryotic mRNA. Very little is known of the function of m(6)A in the immune system or its role in host-pathogen ...interactions. Here, we investigate 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 m(6)A 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 m(6)A for viral replication was demonstrated by silencing of the m(6)A 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.
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 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 match a station’s history, such as marine-associated bacteria in a hurricane-flooded station. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of cities.
Display omitted
•Almost half of all DNA present on the subway’s surfaces matches no known organism.•Hundreds of species of bacteria are in the subway, mostly harmless. More riders bring more diversity.•One station flooded during Hurricane Sandy still resembles a marine environment.•Human allele frequencies in DNA on surfaces can mirror US Census data.
Afshinnekoo et al. describe a city-scale molecular profile of DNA collected from a city's subway system, public surfaces, and one waterway. These data enable a baseline analysis of bacterial, eukaryotic, and aracheal organisms in the built environment of mass transit and urban life.
Methyl-6-adenosine (m
6
A) 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 m
6
A, collectively called the "epitranscriptome," are a pervasive feature of mammalian cells and likely play a role in development and disease. An enrichment of m
6
A near the last CDS of thousands of genes has implicated m
6
A in transcript processing, translational regulation and potentially a mechanism for regulating miRNA maturation. Also, because the sites of m
6
A 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 m
6
A 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.
PDF
