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N 6 -methyladenosine (m 6 A) is the most abundant chemical modification in mRNA and plays important roles in human and mouse embryonic stem cell pluripotency, maintenance, and differentiation. We have recently reported that m 6 A is involved in the postnatal control of β-cell function in physiological states and in type 1 and 2 diabetes. However, the precise mechanisms by which m 6 A acts to regulate the development of human and mouse pancreas are unexplored. Here, we show that the m 6 A landscape is dynamic during human pancreas development, and that METTL14, one of the m 6 A writer complex proteins, is essential for the early differentiation of both human and mouse pancreatic cells., Competing Interests: Disclosure and competing interests statement SK is an employee of Boehringer Ingelheim Pharmaceuticals, Inc. RNK is on the Scientific Advisory Board of Novo Nordisk, Biomea and Inversago Therapeutics. CH is a scientific founder, a member of the scientific advisory board and equity holder of Aferna Bio, Inc. and AccuaDX Inc., a scientific co-founder and equity holder of Accent Therapeutics, Inc., and a member of the scientific advisory board of Rona Therapeutics. RIG is a co-founder, scientific advisory board member, and equity holder of Redona Therapeutics. The Gregory lab receives or has received research funding from Sanofi, Astellas, and Ono. The authors declare no competing interests., (© 2024. The Author(s).)

Brown adipose tissue (BAT) regulates systemic metabolism by releasing signaling lipids. N 6 -methyladenosine (m 6 A) is the most prevalent and abundant post-transcriptional mRNA modification and has been reported to regulate BAT adipogenesis and energy expenditure. Here, we demonstrate that the absence of m 6 A methyltransferase-like 14 (METTL14) modifies the BAT secretome to improve systemic insulin sensitivity independent of UCP1. Using lipidomics, we identify prostaglandin E2 (PGE2) and prostaglandin F2a (PGF2a) as BAT-secreted insulin sensitizers. PGE2 and PGF2a inversely correlate with insulin sensitivity in humans and protect mice from high-fat-diet-induced insulin resistance by suppressing specific AKT phosphatases. Mechanistically, METTL14-mediated m 6 A promotes the decay of PTGES2 and CBR1, the genes encoding PGE2 and PGF2a biosynthesis enzymes, in brown adipocytes via YTHDF2/3. Consistently, BAT-specific knockdown of Ptges2 or Cbr1 reverses the insulin-sensitizing effects in M14 KO mice. Overall, these findings reveal a novel biological mechanism through which m 6 A-dependent regulation of the BAT secretome regulates systemic insulin sensitivity., Competing Interests: Declaration of interests R.N.K. is on the scientific advisory boards of Novo Nordisk, Biomea, Inversago, and REDD. C.H. is a scientific founder and a member of the scientific advisory board of Accent Therapeutics. M.B. received honoraria as a consultant and speaker from Amgen, AstraZeneca, Bayer, Boehringer Ingelheim, Lilly, Novo Nordisk, Novartis, Pfizer, and Sanofi., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Recent studies have suggested that mRNA internal m 7 G and its writer protein METTL1 are closely related to cell metabolism and cancer regulation. Here, we identify that IGF2BP family proteins IGF2BP1-3 can preferentially bind internal mRNA m 7 G. Such interactions, especially IGF2BP3 with m 7 G, could promote the degradation of m 7 G target transcripts in cancer cells. IGF2BP3 is more responsive to changes of m 7 G modification, while IGF2BP1 prefers m 6 A to stabilize the bound transcripts. We also demonstrate that p53 transcript, TP53, is m 7 G-modified at its 3'UTR in cancer cells. In glioblastoma, the methylation level and the half lifetime of the modified transcript could be modulated by tuning IGF2BP3, or by site-specific targeting of m 7 G through a dCas13b-guided system, resulting in modulation of cancer progression and chemosensitivity., (© 2024. The Author(s).)

Plants employ distinct mechanisms to respond to environmental changes. Modification of mRNA by N  6 -methyladenosine (m 6 A), known to affect the fate of mRNA, may be one such mechanism to reprogram mRNA processing and translatability upon stress. However, it is difficult to distinguish a direct role from a pleiotropic effect for this modification due to its prevalence in RNA. Through characterization of the transient knockdown-mutants of m 6 A writer components and mutants of specific m 6 A readers, we demonstrate the essential role that m 6 A plays in basal resistance and pattern-triggered immunity (PTI). A global m 6 A profiling of mock and PTI-induced Arabidopsis plants as well as formaldehyde fixation and cross-linking immunoprecipitation-sequencing of the m 6 A reader, EVOLUTIONARILY CONSERVED C-TERMINAL REGION2 (ECT2) showed that while dynamic changes in m 6 A modification and binding by ECT2 were detected upon PTI induction, most of the m 6 A sites and their association with ECT2 remained static. Interestingly, RNA degradation assay identified a dual role of m 6 A in stabilizing the overall transcriptome while facilitating rapid turnover of immune-induced mRNAs during PTI. Moreover, polysome profiling showed that m 6 A enhances immune-associated translation by binding to the ECT2/3/4 readers. We propose that m 6 A plays a positive role in plant immunity by destabilizing defense mRNAs while enhancing their translation efficiency to create a transient surge in the production of defense proteins., Competing Interests: Competing interests statement:X.D. is a founder of Upstream Biotechnology Inc. and a member of its scientific advisory board, as well as a scientific advisory board member of Inari Agriculture Inc. and Aferna Bio, Inc. G.H.G. is a founder of Upstream Biotechnology Inc. C.H. is a scientific founder, a member of the scientific advisory board and equity holder of Aferna Bio, Inc. and Ellis Bio Inc., a scientific cofounder and equity holder of Accent Therapeutics, Inc., and a member of the scientific advisory board of Rona Therapeutics and Element Biosciences.

N 6 -methyladenosine (m 6 A) plays critical roles in regulating mRNA metabolism. However, comprehensive m 6 A methylomes in different plant tissues with single-base precision have yet to be reported. Here, we present transcriptome-wide m 6 A maps at single-base resolution in different tissues of rice and Arabidopsis using m 6 A-SAC-seq. Our analysis uncovers a total of 205,691 m 6 A sites distributed across 22,574 genes in rice, and 188,282 m 6 A sites across 19,984 genes in Arabidopsis. The evolutionarily conserved m 6 A sites in rice and Arabidopsis ortholog gene pairs are involved in controlling tissue development, photosynthesis and stress response. We observe an overall mRNA stabilization effect by 3' UTR m 6 A sites in certain plant tissues. Like in mammals, a positive correlation between the m 6 A level and the length of internal exons is also observed in plant mRNA, except for the last exon. Our data suggest an active m 6 A deposition process occurring near the stop codon in plant mRNA. In addition, the MTA-installed plant mRNA m 6 A sites correlate with both translation promotion and translation suppression, depicting a more complicated regulatory picture. Our results therefore provide in-depth resources for relating single-base resolution m 6 A sites with functions in plants and uncover a suppression-activation model controlling m 6 A biogenesis across species., (© 2024. The Author(s).)

Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes., (© 2022. The Author(s).)

N 6 -methyladenosine (m 6 A) is the most abundant messenger RNA (mRNA) modification and plays crucial roles in diverse physiological processes. Using a massively parallel assay for m 6 A (MPm 6 A), we discover that m 6 A specificity is globally regulated by suppressors that prevent m 6 A deposition in unmethylated transcriptome regions. We identify exon junction complexes (EJCs) as m 6 A suppressors that protect exon junction-proximal RNA within coding sequences from methylation and regulate mRNA stability through m 6 A suppression. EJC suppression of m 6 A underlies multiple global characteristics of mRNA m 6 A specificity, with the local range of EJC protection sufficient to suppress m 6 A deposition in average-length internal exons but not in long internal and terminal exons. EJC-suppressed methylation sites colocalize with EJC-suppressed splice sites, which suggests that exon architecture broadly determines local mRNA accessibility to regulatory complexes.

N 6 -methyladenosine (m 6 A) is the most abundant internal modification on mammalian messenger RNA. It is installed by a writer complex and can be reversed by erasers such as the fat mass and obesity-associated protein FTO. Despite extensive research, the primary physiological substrates of FTO in mammalian tissues and development remain elusive. Here, we show that FTO mediates m 6 A demethylation of long-interspersed element-1 (LINE1) RNA in mouse embryonic stem cells (mESCs), regulating LINE1 RNA abundance and the local chromatin state, which in turn modulates the transcription of LINE1-containing genes. FTO-mediated LINE1 RNA m 6 A demethylation also plays regulatory roles in shaping chromatin state and gene expression during mouse oocyte and embryonic development. Our results suggest broad effects of LINE1 RNA m 6 A demethylation by FTO in mammals.

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2'O-methylation, pseudouridylation, N 6 -methyladenosine (m 6 A), and N 6,6- dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m 6 A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m 6 A modification, the methyltransferase responsible for the 18S rRNA m 6 A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m 6 A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m 6 A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m 6 A in noncoding RNAs., Competing Interests: Conflict of interest C. H. is a scientific founder and a member of the scientific advisory board of Accent Therapeutics, Inc., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

METTL16 has recently been identified as an RNA methyltransferase responsible for the deposition of N 6 -methyladenosine (m 6 A) in a few transcripts. Whether METTL16 methylates a large set of transcripts, similar to METTL3 and METTL14, remains unclear. Here we show that METTL16 exerts both methyltransferase activity-dependent and -independent functions in gene regulation. In the cell nucleus, METTL16 functions as an m 6 A writer to deposit m 6 A into hundreds of its specific messenger RNA targets. In the cytosol, METTL16 promotes translation in an m 6 A-independent manner. More specifically, METTL16 directly interacts with the eukaryotic initiation factors 3a and -b as well as ribosomal RNA through its Mtase domain, thereby facilitating the assembly of the translation-initiation complex and promoting the translation of over 4,000 mRNA transcripts. Moreover, we demonstrate that METTL16 is critical for the tumorigenesis of hepatocellular carcinoma. Collectively, our studies reveal previously unappreciated dual functions of METTL16 as an m 6 A writer and a translation-initiation facilitator, which together contribute to its essential function in tumorigenesis., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

RNA carries a diverse array of chemical modifications that play important roles in the regulation of gene expression. N 6 -methyladenosine (m 6 A), installed onto mRNA by the METTL3/METTL14 methyltransferase complex, is the most prevalent mRNA modification. m 6 A methylation regulates gene expression by influencing numerous aspects of mRNA metabolism, including pre-mRNA processing, nuclear export, decay, and translation. The importance of m 6 A methylation as a mode of post-transcriptional gene expression regulation is evident in the crucial roles m 6 A-mediated gene regulation plays in numerous physiological and pathophysiological processes. Here, we review current knowledge on the mechanisms by which m 6 A exerts its functions and discuss recent advances that underscore the multifaceted role of m 6 A in the regulation of gene expression. We highlight advances in our understanding of the regulation of m 6 A deposition on mRNA and its context-dependent effects on mRNA decay and translation, the role of m 6 A methylation of non-coding chromosomal-associated RNA species in regulating transcription, and the activities of the RNA demethylase FTO on diverse substrates. We also discuss emerging evidence for the therapeutic potential of targeting m 6 A regulators in disease., (© 2021 The Authors.)

Bacterial infection triggers a cytokine storm that needs to be resolved to maintain the host's wellbeing. Here, we report that ablation of m 6 A methyltransferase subunit METTL14 in myeloid cells exacerbates macrophage responses to acute bacterial infection in mice, leading to high mortality due to sustained production of pro-inflammatory cytokines. METTL14 depletion blunts Socs1 m 6 A methylation and reduces YTHDF1 binding to the m 6 A sites, which diminishes SOCS1 induction leading to the overactivation of TLR4/NF-κB signaling. Forced expression of SOCS1 in macrophages depleted of METTL14 or YTHDF1 rescues the hyper-responsive phenotype of these macrophages in vitro and in vivo. We further show that LPS treatment induces Socs1 m 6 A methylation and sustains SOCS1 induction by promoting Fto mRNA degradation, and forced FTO expression in macrophages mimics the phenotype of METTL14-depleted macrophages. We conclude that m 6 A methylation-mediated SOCS1 induction is required to maintain the negative feedback control of macrophage activation in response to bacterial infection., Competing Interests: Declaration of Interests C.H. is a scientific founder and a member of the scientific advisory board of Accent Therapeutics, Inc., (Copyright © 2020 Elsevier Inc. All rights reserved.)

N 6 -methyladenosine (m 6 A) plays important roles in regulating messenger RNA processing. Despite rapid progress in this field, little is known about the genetic determinants of m 6 A modification and their role in common diseases. In this study, we mapped the quantitative trait loci (QTLs) of m 6 A peaks in 60 Yoruba (YRI) lymphoblastoid cell lines. We found that m 6 A QTLs are largely independent of expression and splicing QTLs and are enriched with binding sites of RNA-binding proteins, RNA structure-changing variants and transcriptional features. Joint analysis of the QTLs of m 6 A and related molecular traits suggests that the downstream effects of m 6 A are heterogeneous and context dependent. We identified proteins that mediate m 6 A effects on translation. Through integration with data from genome-wide association studies, we show that m 6 A QTLs contribute to the heritability of various immune and blood-related traits at levels comparable to splicing QTLs and roughly half of expression QTLs. By leveraging m 6 A QTLs in a transcriptome-wide association study framework, we identified putative risk genes of these traits.

Background: Pancreatic cancer is one of the most lethal human cancers. N 6 -methyladenosine (m 6 A), a common eukaryotic mRNA modification, plays critical roles in both physiological and pathological processes. However, its role in pancreatic cancer remains elusive., Methods: LC/MS was used to profile m 6 A levels in pancreatic cancer and normal tissues. Bioinformatics analysis, real-time PCR, immunohistochemistry, and western blotting were used to identify the role of m 6 A regulators in pancreatic cancer. The biological effects of methyltransferase-like 14 (METTL14), an mRNA methylase, were investigated using in vitro and in vivo models. MeRIP-Seq and RNA-Seq were used to assess the downstream targets of METTL14., Results: We found that the m 6 A levels were elevated in approximately 70% of the pancreatic cancer samples. Furthermore, we demonstrated that METTL14 is the major enzyme that modulates m 6 A methylation (frequency and site of methylation). METTL14 overexpression markedly promoted pancreatic cancer cell proliferation and migration both in vitro and in vivo, via direct targeting of the downstream PERP mRNA (p53 effector related to PMP-22) in an m 6 A-dependent manner. Methylation of the target adenosine lead to increased PERP mRNA turnover, thus decreasing PERP (mRNA and protein) levels in pancreatic cancer cells., Conclusions: Our data suggest that the upregulation of METTL14 leads to the decrease of PERP levels via m 6 A modification, promoting the growth and metastasis of pancreatic cancer; therefore METTL14 is a potential therapeutic target for its treatment.

The molecular mechanisms that govern the maturation of oligodendrocyte lineage cells remain unclear. Emerging studies have shown that N 6 -methyladenosine (m 6 A), the most common internal RNA modification of mammalian mRNA, plays a critical role in various developmental processes. Here, we demonstrate that oligodendrocyte lineage progression is accompanied by dynamic changes in m 6 A modification on numerous transcripts. In vivo conditional inactivation of an essential m 6 A writer component, METTL14, results in decreased oligodendrocyte numbers and CNS hypomyelination, although oligodendrocyte precursor cell (OPC) numbers are normal. In vitro Mettl14 ablation disrupts postmitotic oligodendrocyte maturation and has distinct effects on OPC and oligodendrocyte transcriptomes. Moreover, the loss of Mettl14 in oligodendrocyte lineage cells causes aberrant splicing of myriad RNA transcripts, including those that encode the essential paranodal component neurofascin 155 (NF155). Together, our findings indicate that dynamic RNA methylation plays an important regulatory role in oligodendrocyte development and CNS myelination., (Copyright © 2019 Elsevier Inc. All rights reserved.)

N 6 -Methyladenosine (m 6 A) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to m 6 A-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans. Using HEK293T cells, siRNA-mediated gene knockdown, cytoplasmic and nuclear fractions, RNA-Seq, and LC-MS/MS analyses, we demonstrate here that FMRP binds directly to a collection of m 6 A sites on mRNAs. FMRP depletion increased mRNA m 6 A levels in the nucleus. Moreover, the abundance of FMRP targets in the cytoplasm relative to the nucleus was decreased in Fmr1 -KO mice, an effect also observed in highly methylated genes. We conclude that FMRP may affect the nuclear export of m 6 A-modified RNA targets., (© 2019 Hsu et al.)

Chemical modifications to messenger RNA are increasingly recognized as a critical regulatory layer in the flow of genetic information, but quantitative tools to monitor RNA modifications in a whole-transcriptome and site-specific manner are lacking. Here we describe a versatile platform for directed evolution that rapidly selects for reverse transcriptases that install mutations at sites of a given type of RNA modification during reverse transcription, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N 1 -methyladenosine (m 1 A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m 1 A sites. The optimal evolved reverse transcriptase enabled detection of well-characterized m 1 A sites and revealed hundreds of m 1 A sites in human mRNA. This work develops and validates the reverse transcriptase evolution platform, and provides new tools, analysis methods and datasets to study m 1 A biology.

N 6 -methyladenosine (m 6 A) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional m 6 A-dependent alternative splicing regulation is still poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an m 6 A reader protein that binds RNA through RRM and Arg-Gly-Gly (RGG) motifs. Here, we show that hnRNPG directly binds to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) using RGG motifs in its low-complexity region. Through interactions with the phosphorylated CTD and nascent RNA, hnRNPG associates co-transcriptionally with RNAPII and regulates alternative splicing transcriptome-wide. m 6 A near splice sites in nascent pre-mRNA modulates hnRNPG binding, which influences RNAPII occupancy patterns and promotes exon inclusion. Our results reveal an integrated mechanism of co-transcriptional m 6 A-mediated splicing regulation, in which an m 6 A reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and m 6 A-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

N 6 -methyladenosine (m 6 A) is one of the most abundant messenger RNA modifications in eukaryotes involved in various pivotal processes of RNA metabolism. The most popular high-throughput m 6 A identification method depends on the anti-m 6 A antibody but suffers from poor reproducibility and limited resolution. Exact location information is of great value for understanding the dynamics, machinery, and functions of m 6 A. Here, we developed a precise and high-throughput antibody-independent m 6 A identification method based on the m 6 A-sensitive RNA endoribonuclease recognizing ACA motif (m 6 A-sensitive RNA-Endoribonuclease-Facilitated sequencing or m 6 A-REF-seq). Whole-transcriptomic, single-base m 6 A maps generated by m 6 A-REF-seq quantitatively displayed an explicit distribution pattern with enrichment near stop codons. We used independent methods to validate methylation status and abundance of individual m 6 A sites, confirming the high reliability and accuracy of m 6 A-REF-seq. We applied this method on five tissues from human, mouse, and rat, showing that m 6 A sites are conserved with single-nucleotide specificity and tend to cluster among species.

N 7 -methylguanosine (m 7 G) is a positively charged, essential modification at the 5' cap of eukaryotic mRNA, regulating mRNA export, translation, and splicing. m 7 G also occurs internally within tRNA and rRNA, but its existence and distribution within eukaryotic mRNA remain to be investigated. Here, we show the presence of internal m 7 G sites within mammalian mRNA. We then performed transcriptome-wide profiling of internal m 7 G methylome using m 7 G-MeRIP sequencing (MeRIP-seq). To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively converts internal m 7 G sites into abasic sites, inducing misincorporation at these sites during reverse transcription. This base-resolution m 7 G-seq enabled transcriptome-wide mapping of m 7 G in human tRNA and mRNA, revealing distribution features of the internal m 7 G methylome in human cells. We also identified METTL1 as a methyltransferase that installs a subset of m 7 G within mRNA and showed that internal m 7 G methylation could affect mRNA translation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cellular RNAs are naturally decorated with a variety of chemical modifications. The structural diversity of the modified nucleosides provides regulatory potential to sort groups of RNAs for organized metabolism and functions, thus affecting gene expression. Recent years have witnessed a burst of interest in and understanding of RNA modification biology, thanks to the emerging transcriptome-wide sequencing methods for mapping modified sites, highly sensitive mass spectrometry for precise modification detection and quantification, and extensive characterization of the modification "effectors," including enzymes ("writers" and "erasers") that alter the modification level and binding proteins ("readers") that recognize the chemical marks. However, challenges remain due to the vast heterogeneity in expression abundance of different RNA species, further complicated by divergent cell-type-specific and tissue-specific expression and localization of the effectors as well as modifications. In this review, we highlight recent progress in understanding the function of N 6 -methyladenosine (m 6 A), the most abundant internal mark on eukaryotic mRNA, in light of the specific biological contexts of m 6 A effectors. We emphasize the importance of context for RNA modification regulation and function., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

The post-transcriptional modification 2'-O-Methyl (2'OMe) could be present on the ribose of all four ribonucleosides, and is highly prevalent in a wide variety of RNA species, including the 5' RNA cap of viruses and higher eukaryotes, as well as internally in transfer RNA and ribosomal RNA. Recent studies have suggested that 2'OMe is also located internally in low-abundance RNA species such as viral RNA and mRNA. To profile 2'OMe on different RNA species, we have developed Nm-seq, which could identify 2'OMe sites at single base resolution. Nm-seq is particularly useful for identifying 2'OMe sites located at the 3' terminal ends of small RNAs. Here, we present an optimized protocol for Nm-seq and a protocol for applying Nm-seq to identify 2'OMe sites on small RNA 3' terminal ends., (Copyright © 2018 Elsevier Inc. All rights reserved.)

There is growing evidence that tumour neoantigens have important roles in generating spontaneous antitumour immune responses and predicting clinical responses to immunotherapies 1,2 . Despite the presence of numerous neoantigens in patients, complete tumour elimination is rare, owing to failures in mounting a sufficient and lasting antitumour immune response 3,4 . Here we show that durable neoantigen-specific immunity is regulated by mRNA N 6 -methyadenosine (m 6 A) methylation through the m 6 A-binding protein YTHDF1 5 . In contrast to wild-type mice, Ythdf1-deficient mice show an elevated antigen-specific CD8 + T cell antitumour response. Loss of YTHDF1 in classical dendritic cells enhanced the cross-presentation of tumour antigens and the cross-priming of CD8 + T cells in vivo. Mechanistically, transcripts encoding lysosomal proteases are marked by m 6 A and recognized by YTHDF1. Binding of YTHDF1 to these transcripts increases the translation of lysosomal cathepsins in dendritic cells, and inhibition of cathepsins markedly enhances cross-presentation of wild-type dendritic cells. Furthermore, the therapeutic efficacy of PD-L1 checkpoint blockade is enhanced in Ythdf1 -/- mice, implicating YTHDF1 as a potential therapeutic target in anticancer immunotherapy.

Investigations over the past eight years of chemical modifications on messenger RNA (mRNA) have revealed a new level of posttranscriptional gene regulation in eukaryotes. Rapid progress in our understanding of these modifications, particularly, N 6 -methyladenosine (m 6 A), has revealed their roles throughout the life cycle of an mRNA transcript. m 6 A methylation provides a rapid mechanism for coordinated transcriptome processing and turnover that is important in embryonic development and cell differentiation. In response to cellular signals, m 6 A can also regulate the translation of specific pools of transcripts. These mechanisms can be hijacked in human diseases, including numerous cancers and viral infection. Beyond m 6 A, many other mRNA modifications have been mapped in the transcriptome, but much less is known about their biological functions. As methods continue to be developed, we will be able to study these modifications both more broadly and in greater depth, which will likely reveal a wealth of new RNA biology.

RNA modifications have recently emerged as critical posttranscriptional regulators of gene expression programs. They affect diverse eukaryotic biological processes, and the correct deposition of many of these modifications is required for normal development. Messenger RNA (mRNA) modifications regulate various aspects of mRNA metabolism. For example, N 6 -methyladenosine (m 6 A) affects the translation and stability of the modified transcripts, thus providing a mechanism to coordinate the regulation of groups of transcripts during cell state maintenance and transition. Similarly, some modifications in transfer RNAs are essential for RNA structure and function. Others are deposited in response to external cues and adapt global protein synthesis and gene-specific translational accordingly and thereby facilitate proper development., (Copyright © 2018, American Association for the Advancement of Science.)

N 6 -methyladenosine (m 6 A) messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation in development and cancer. To study the roles of m 6 A mRNA methylation in cell proliferation and tumorigenicity, we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltransferase complex (METTL14). We found that about 70% of endometrial tumours exhibit reductions in m 6 A methylation that are probably due to either this METTL14 mutation or reduced expression of METTL3, another component of the methyltransferase complex. These changes lead to increased proliferation and tumorigenicity of endometrial cancer cells, likely through activation of the AKT pathway. Reductions in m 6 A methylation lead to decreased expression of the negative AKT regulator PHLPP2 and increased expression of the positive AKT regulator mTORC2. Together, these results reveal reduced m 6 A mRNA methylation as an oncogenic mechanism in endometrial cancer and identify m 6 A methylation as a regulator of AKT signalling.

Background: N 6 -methyladenosine (m 6 A) modification in mRNAs was recently shown to be dynamically regulated, indicating a pivotal role in multiple developmental processes. Most recently, it was shown that the Mettl3-Mettl14 writer complex of this mark is required for the temporal control of cortical neurogenesis. The m 6 A reader protein Ythdf2 promotes mRNA degradation by recognizing m 6 A and recruiting the mRNA decay machinery., Results: We show that the conditional depletion of the m 6 A reader protein Ythdf2 in mice causes lethality at late embryonic developmental stages, with embryos characterized by compromised neural development. We demonstrate that neural stem/progenitor cell (NSPC) self-renewal and spatiotemporal generation of neurons and other cell types are severely impacted by the loss of Ythdf2 in embryonic neocortex. Combining in vivo and in vitro assays, we show that the proliferation and differentiation capabilities of NSPCs decrease significantly in Ythdf2 -/- embryos. The Ythdf2 -/- neurons are unable to produce normally functioning neurites, leading to failure in recovery upon reactive oxygen species stimulation. Consistently, expression of genes enriched in neural development pathways is significantly disturbed. Detailed analysis of the m 6 A-methylomes of Ythdf2 -/- NSPCs identifies that the JAK-STAT cascade inhibitory genes contribute to neuroprotection and neurite outgrowths show increased expression and m 6 A enrichment. In agreement with the function of Ythdf2, delayed degradation of neuron differentiation-related m 6 A-containing mRNAs is seen in Ythdf2 -/- NSPCs., Conclusions: We show that the m 6 A reader protein Ythdf2 modulates neural development by promoting m 6 A-dependent degradation of neural development-related mRNA targets.

N 6 -methyladenosine (m 6 A) is an abundant modification in eukaryotic mRNA, regulating mRNA dynamics by influencing mRNA stability, splicing, export, and translation. However, the precise m 6 A regulating machinery still remains incompletely understood. Here we demonstrate that ZC3H13, a zinc-finger protein, plays an important role in modulating RNA m 6 A methylation in the nucleus. We show that knockdown of Zc3h13 in mouse embryonic stem cell significantly decreases global m 6 A level on mRNA. Upon Zc3h13 knockdown, a great majority of WTAP, Virilizer, and Hakai translocate to the cytoplasm, suggesting that Zc3h13 is required for nuclear localization of the Zc3h13-WTAP-Virilizer-Hakai complex, which is important for RNA m 6 A methylation. Finally, Zc3h13 depletion, as does WTAP, Virilizer, or Hakai, impairs self-renewal and triggers mESC differentiation. Taken together, our findings demonstrate that Zc3h13 plays a critical role in anchoring WTAP, Virilizer, and Hakai in the nucleus to facilitate m 6 A methylation and to regulate mESC self-renewal., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

N 6 -methyladenosine (m 6 A), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether m 6 A regulates mammalian brain development is unknown. Here, we show that m 6 A depletion by Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages. m 6 A depletion by Mettl3 knockdown also leads to a prolonged cell cycle and maintenance of radial glia cells. m 6 A sequencing of embryonic mouse cortex reveals enrichment of mRNAs related to transcription factors, neurogenesis, the cell cycle, and neuronal differentiation, and m 6 A tagging promotes their decay. Further analysis uncovers previously unappreciated transcriptional prepatterning in cortical neural stem cells. m 6 A signaling also regulates human cortical neurogenesis in forebrain organoids. Comparison of m 6 A-mRNA landscapes between mouse and human cortical neurogenesis reveals enrichment of human-specific m 6 A tagging of transcripts related to brain-disorder risk genes. Our study identifies an epitranscriptomic mechanism in heightened transcriptional coordination during mammalian cortical neurogenesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

RNA contains over 150 types of chemical modifications. Although many of these chemical modifications were discovered several decades ago, their functions were not immediately apparent. Discoveries of RNA demethylases, along with advances in mass spectrometry and high-throughput sequencing techniques, have caused research into RNA modifications to progress at an accelerated rate. Post-transcriptional RNA modifications make up an epitranscriptome that extensively regulates gene expression and biological processes. Here, we present an overview of recent advances in the field that are shaping our understanding of chemical modifications, their impact on development and disease, and the dynamic mechanisms through which they regulate gene expression.

N 6 -methyladenosine (m 6 A) is the most abundant internal modification of eukaryotic messenger RNA (mRNA) and plays critical roles in RNA biology. The function of this modification is mediated by m 6 A-selective 'reader' proteins of the YTH family, which incorporate m 6 A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m 6 A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m 6 A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m 6 A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.

RNA modifications are integral to the regulation of RNA metabolism. One abundant mRNA modification is N 6 -methyladenosine (m 6 A), which affects various aspects of RNA metabolism, including splicing, translation and degradation. Current knowledge about the proteins recruited to m 6 A to carry out these molecular processes is still limited. Here we describe comprehensive and systematic mass-spectrometry-based screening of m 6 A interactors in various cell types and sequence contexts. Among the main findings, we identified G3BP1 as a protein that is repelled by m 6 A and positively regulates mRNA stability in an m 6 A-regulated manner. Furthermore, we identified FMR1 as a sequence-context-dependent m 6 A reader, thus revealing a connection between an mRNA modification and an autism spectrum disorder. Collectively, our data represent a rich resource and shed further light on the complex interplay among m 6 A, m 6 A interactors and mRNA homeostasis.

The discovery of multiple RNA modifications in the past few years has broadened our views of the structures and potential functions of RNA species, but deciphering which modifications are made where and how remains a challenge. A new study by Xu et al. applies a combination of mass spectrometry, biochemistry, genetics, and cellular biology tools to reveal the two mammalian methyltransferases that are responsible for m 3 C installation in tRNA and a third that mediates the previously unknown installation of m 3 C in mammalian mRNA., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The ribose of RNA nucleotides can be 2'-O-methylated (Nm). Despite advances in high-throughput detection, the inert chemical nature of Nm still limits sensitivity and precludes mapping in mRNA. We leveraged the differential reactivity of 2'-O-methylated and 2'-hydroxylated nucleosides to periodate oxidation to develop Nm-seq, a sensitive method for transcriptome-wide mapping of Nm with base precision. Nm-seq uncovered thousands of Nm sites in human mRNA with features suggesting functional roles.

Phenotypic differences within populations and between closely related species are often driven by variation and evolution of gene expression. However, most analyses have focused on the effects of genomic variation at cis- regulatory elements such as promoters and enhancers that control transcriptional activity, and little is understood about the influence of post-transcriptional processes on transcript evolution. Post-transcriptional modification of RNA by N 6 -methyladenosine (m 6 A) has been shown to be widespread throughout the transcriptome, and this reversible mark can affect transcript stability and translation dynamics. Here we analyze m 6 A mRNA modifications in lymphoblastoid cell lines (LCLs) from human, chimpanzee and rhesus, and we identify patterns of m 6 A evolution among species. We find that m 6 A evolution occurs in parallel with evolution of consensus RNA sequence motifs known to be associated with the enzymatic complexes that regulate m 6 A dynamics, and expression evolution of m 6 A-modified genes occurs in parallel with m 6 A evolution., (© 2017 Ma et al.; Published by Cold Spring Harbor Laboratory Press.)

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., Competing Interests: statement The authors declare no competing interests.

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.

Chemical modifications in cellular RNA are diverse and abundant. Commonly found in ribosomal RNA (rRNA), transfer RNA (tRNA), long-noncoding RNA (lncRNA), and small nuclear (snRNA), these components play various structural and functional roles. Until recently, the roles of chemical modifications within messenger RNA (mRNA) have been understudied. Recent maps of several mRNA modifications have suggested regulatory functions for these marks. This review summarizes recent advances in identifying and understanding biological roles of posttranscriptional mRNA modification, or 'RNA epigenetics', with an emphasis on the most common internal modification of eukaryotic mRNA, N(6)-methyladenosine (m(6)A). We also discuss YTH proteins as direct mediators of m(6)A function and the emerging role of this mark in a new layer of gene expression regulation., (Published by Elsevier Ltd.)

N(6)-methyladenosine (m(6)A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA). Recent discoveries of demethylases and specific binding proteins of m(6)A as well as m(6)A methylomes obtained in mammals, yeast and plants have revealed regulatory functions of this RNA modification. Although m(6)A is present in the ribosomal RNA of bacteria, its occurrence in mRNA still remains elusive. Here, we have employed ultra-high pressure liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) to calculate the m(6)A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m(6)A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m(6)A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved m(6)A pattern that is distinct from those in eukaryotes. Most m(6)A peaks are located inside open reading frames and carry a unique consensus motif of GCCAU. Functional enrichment analysis of bacterial m(6)A peaks indicates that the majority of m(6)A-modified genes are associated with respiration, amino acids metabolism, stress response and small RNAs, suggesting potential functional roles of m(6)A in these pathways., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

N(6)-methyladenosine (m(6)A) is the most prevalent and internal modification that occurs in the messenger RNAs (mRNA) of most eukaryotes, although its functional relevance remained a mystery for decades. This modification is installed by the m(6)A methylation "writers" and can be reversed by demethylases that serve as "erasers." In this review, we mainly summarize recent progress in the study of the m(6)A mRNA methylation machineries across eukaryotes and discuss their newly uncovered biological functions. The broad roles of m(6)A in regulating cell fates and embryonic development highlight the existence of another layer of epigenetic regulation at the RNA level, where mRNA is subjected to chemical modifications that affect protein expression., (© 2015 Yue et al.; Published by Cold Spring Harbor Laboratory Press.)

RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs). However, RBMs can be buried within their local RNA structures, thus inhibiting RNA-protein interactions. N(6)-methyladenosine (m(6)A), the most abundant and dynamic internal modification in eukaryotic messenger RNA, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs, but how m(6)A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m(6)A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism 'the m(6)A-switch'. We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m(6)A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m(6)A-switches among HNRNPC-binding sites; and global m(6)A reduction decreased HNRNPC binding at 2,798 high-confidence m(6)A-switches. We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology.

The N 6-methyladenosine (m6A) modification of mRNA has a crucial function in regulating pluripotency in murine stem cells: it facilitates resolution of naïve pluripotency towards differentiation.

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