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Key Points N 6 -methyladenosine (m 6 A) is a prevalent post-transcriptional modification in mammalian mRNA. m 6 A is enriched in consensus sequences within long exons, near stop codons and at the 3′ untranslated regions (3′ UTRs). m 6 A is the first confirmed reversible mRNA modification with dedicated methyltransferases, demethylases and binding (effector) proteins. To date, four components of the m 6 A methyltransferase complex, two m 6 A demethylases and several m 6 A-binding proteins have been identified in mammals. m 6 A exerts its effects by directly recruiting effector proteins or by modulating RNA secondary structures, which modulate mRNA metabolism, including maturation, translation and decay. There is evidence to indicate that methylated transcripts can be sorted to synchronously fast track their metabolism. m 6 A has regulatory roles in many cellular processes, including circadian rhythm maintenance, stem cell differentiation and stress responses. m 6 A may facilitate cell-state transitions by regulating the metabolism of transcripts of key transcription factors. In addition to m 6 A, other modifications exist in mammalian mRNA, including N 1 -methyladenosine (m 1 A), 5-methylcytosine (m 5 C), pseudouridine and 2′- O -methylation (2′OMe). This collection of chemical modifications modulates nearly all aspects of RNA metabolism and related physiological processes, adding another layer to the already complex gene expression regulation pathways in eukaryotes, particularly in mammals. Reversible mRNA methylation is an emerging mode of eukaryotic post-transcriptional gene regulation. N 6 -methyladenosine (m 6 A) affects mRNA processing, translation and decay during cell differentiation, embryonic development and stress responses. Other mRNA modifications — N 1 -methyladenosine (m 1 A), 5-methylcytosine (m 5 C) and pseudouridine — together with m 6 A code a new layer of information that controls protein synthesis. The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N 6 -methyladenosine (m 6 A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N 6 -adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N 1 -methyladenosine (m 1 A), 5-methylcytosine (m 5 C) and pseudouridine, together with m 6 A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis.

Dear Editor, The TET family of dioxygenases can oxidize 5-meth- ylcytosine （5mC） to 5-hydroxymethylcytosine （5hmC）, 5-formylcytosine （5fC）, and 5-carboxylcytosine （5caC） in mammalian genomic DNA via a stepwise manner [ 1- 5]. 5fC and 5caC are selectively recognized and excised by mammalian thymine DNA glycosylase （TDG）, and restored to normal cytosine through base excision repair [3, 6-9]. Once converted to 5fC and 5caC, the modified cytosine base is presumably committed to demethylation through the TDG-dependent pathway or other potential mechanisms. Thus 5fC and 5caC specifically mark active demethylation in the mammalian genome.

RNA–protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA–protein interactions in living cells in an unbiased manner. We used two approaches to target the engineered peroxidase APEX2 to specific cellular RNAs for RNA-centered proximity biotinylation of protein interaction partners. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured candidate binding partners for hTR, including more than a dozen proteins not previously linked to hTR. We validated the interaction between hTR and the N⁶-methyladenosine (m⁶A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m⁶A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA–protein interactions in living cells.

DNA and histone modifications have notable effects on gene expression 1 . Being the most prevalent internal modification in mRNA, the N 6 -methyladenosine (m 6 A) mRNA modification is as an important post-transcriptional mechanism of gene regulation 2 – 4 and has crucial roles in various normal and pathological processes 5 – 12 . However, it is unclear how m 6 A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m 6 A deposition globally. We show that m 6 A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m 6 A methyltransferase complex (MTC), which in turn facilitates the binding of the m 6 A MTC to adjacent RNA polymerase II, thereby delivering the m 6 A MTC to actively transcribed nascent RNAs to deposit m 6 A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m 6 A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m 6 A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation. METTL14 recognizes the trimethyl mark on lysine 36 of histone H3 that directs m 6 A modifications co-transcriptionally.

Two articles recently published in Nature and Cell report the first transcriptome-wide maps of pseudouridine （ψ） at single-base reso- lution through selective chemical labeling, suggesting new mechanisms and functions of ψ in mRNA and noncoding RNA molecules.

N 6 -methyladenosine (m 6 A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m 6 A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m 6 A methyltransferases decreases RSV replication and gene expression whereas knockdown of m 6 A demethylases has the opposite effect. The G gene transcript contains the most m 6 A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m 6 A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m 6 A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m 6 A methylation upregulates RSV replication and pathogenesis and identify viral m 6 A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses. Here, Xue et al. identify N 6 -methyladenosine (m 6 A) modification sites in RNAs of respiratory syncytial virus (RSV) and show that these sites, particularly sites in the transcript encoding for the viral glycoprotein, affect virus replication in primary human cells and cotton rats.

N 6 -methyladenosine (m 6 A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m 6 A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m 6 A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC ) in an m 6 A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m 6 A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m 6 A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m 6 A readers in post-transcriptional gene regulation and cancer biology. Huang et al. identify IGF2BPs as an additional class of N 6 -methyladenosine (m 6 A) reader proteins. They find that IGF2BPs selectively bind to m 6 A-containing mRNAs and promote their stability.

Drugs and antibiotics induce oxidation and mobilization of membrane-bound copper( I ) ions to copper( II ) species within the E. coli cytosol, causing oxidation of a single cysteine residue of the multiple antibiotic-resistance regulator MarR, that leads to formation of disulfide-bonded MarR tetramers and release of dimers from sites of transcriptional activity. The widely conserved multiple antibiotic resistance regulator (MarR) family of transcription factors modulates bacterial detoxification in response to diverse antibiotics, toxic chemicals or both. The natural inducer for Escherichia coli MarR, the prototypical transcription repressor within this family, remains unknown. Here we show that copper signaling potentiates MarR derepression in E. coli . Copper( II ) oxidizes a cysteine residue (Cys80) on MarR to generate disulfide bonds between two MarR dimers, thereby inducing tetramer formation and the dissociation of MarR from its cognate promoter DNA. We further discovered that salicylate, a putative MarR inducer, and the clinically important bactericidal antibiotics norfloxacin and ampicillin all stimulate intracellular copper elevation, most likely through oxidative impairment of copper-dependent envelope proteins, including NADH dehydrogenase-2. This membrane-associated copper oxidation and liberation process derepresses MarR, causing increased bacterial antibiotic resistance. Our study reveals that this bacterial transcription regulator senses copper( II ) as a natural signal to cope with stress caused by antibiotics or the environment.