N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency.

N(6)-methyladenosine (m(6)A) is the most abundant internal modification in mammalian mRNA. This modification is reversible and non-stoichiometric and adds another layer to the dynamic control of mRNA metabolism. The stability of m(6)A-modified mRNA is regulated by an m(6)A reader protein, human YTHDF2, which recognizes m(6)A and reduces the stability of target transcripts. Looking at additional functional roles for the modification, we find that another m(6)A reader protein, human YTHDF1, actively promotes protein synthesis by interacting with translation machinery. In a unified mechanism of m(6)A-based regulation in the cytoplasm, YTHDF2-mediated degradation controls the lifetime of target transcripts, whereas YTHDF1-mediated translation promotion increases translation efficiency, ensuring effective protein production from dynamic transcripts that are marked by m(6)A. Therefore, the m(6)A modification in mRNA endows gene expression with fast responses and controllable protein production through these mechanisms.

Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification.

We report the identification of 67 previously undescribed histone modifications, increasing the current number of known histone marks by about 70%. We further investigated one of the marks, lysine crotonylation (Kcr), confirming that it represents an evolutionarily-conserved histone posttranslational modification. The unique structure and genomic localization of histone Kcr suggest that it is mechanistically and functionally different from histone lysine acetylation (Kac). Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in postmeiotic male germ cells.

Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in the Cell Nucleus and Cytoplasm.

FTO, the first RNA demethylase discovered, mediates the demethylation of internal N6-methyladenosine (m6A) and N6, 2-O-dimethyladenosine (m6Am) at the +1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal m6A and cap m6Am in mRNA, internal m6A in U6 RNA, internal and cap m6Am in snRNAs, and N1-methyladenosine (m1A) in tRNA. FTO-mediated demethylation has a greater effect on the transcript levels of mRNAs possessing internal m6A than the ones with cap m6Am in the tested cells. We also show that FTO can directly repress translation by catalyzing m1A tRNA demethylation. Collectively, FTO-mediated RNA demethylation occurs to m6A and m6Am in mRNA and snRNA as well as m1A in tRNA.

Histone H3 trimethylation at lysine 36 guides m6A RNA modification co-transcriptionally.

DNA and histone modifications have notable effects on gene expression1. Being the most prevalent internal modification in mRNA, the N6-methyladenosine (m6A) mRNA modification is as an important post-transcriptional mechanism of gene regulation2-4 and has crucial roles in various normal and pathological processes5-12. However, it is unclear how m6A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. We show that m6A 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 m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m6A 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 m6A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation.

m6A facilitates hippocampus-dependent learning and memory through YTHDF1.

N6-methyladenosine (m6A), the most prevalent internal RNA modification on mammalian messenger RNAs, regulates the fates and functions of modified transcripts through m6A-specific binding proteins1-5. In the nervous system, m6A is abundant and modulates various neural functions6-11. Whereas m6A marks groups of mRNAs for coordinated degradation in various physiological processes12-15, the relevance of m6A for mRNA translation in vivo remains largely unknown. Here we show that, through its binding protein YTHDF1, m6A promotes protein translation of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory. Mice with genetic deletion of Ythdf1 show learning and memory defects as well as impaired hippocampal synaptic transmission and long-term potentiation. Re-expression of YTHDF1 in the hippocampus of adult Ythdf1-knockout mice rescues the behavioural and synaptic defects, whereas hippocampus-specific acute knockdown of Ythdf1 or Mettl3, which encodes the catalytic component of the m6A methyltransferase complex, recapitulates the hippocampal deficiency. Transcriptome-wide mapping of YTHDF1-binding sites and m6A sites on hippocampal mRNAs identified key neuronal genes. Nascent protein labelling and tether reporter assays in hippocampal neurons showed that YTHDF1 enhances protein synthesis in a neuronal-stimulus-dependent manner. In summary, YTHDF1 facilitates translation of m6A-methylated neuronal mRNAs in response to neuronal stimulation, and this process contributes to learning and memory.

An in-library ligation strategy and its application in CRISPR/Cas9 screening of high-order gRNA combinations.

Simultaneous targeting multiple genes is a big advantage of CRISPR (clustered regularly interspaced short palindromic repeats) genome editing but challenging to achieve in CRISPR screening. The crosstalk among genes or gene products is a common and fundamental mechanism to ensure cellular stability and functional diversity. However, the screening approach to map high-order gene combinations to the interesting phenotype is still lacking. Here, we developed a universal in-library ligation strategy and applied it to generate multiplexed CRISPR library, which could perturb four pre-designed targets in a cell. We conducted in vivo CRISPR screening for potential guide RNA (gRNA) combinations inducing anti-tumor immune responses. Simultaneously disturbing a combination of three checkpoints in CD8+ T cells was demonstrated to be more effective than disturbing Pdcd1 only for T cell activation in the tumor environment. This study developed a novel in-library ligation strategy to facilitate the multiplexed CRISPR screening, which could extend our ability to explore the combinatorial outcomes from coordinated gene behaviors.

m6A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition.

The maternal-to-zygotic transition (MZT) is one of the most profound and tightly orchestrated processes during the early life of embryos, yet factors that shape the temporal pattern of vertebrate MZT are largely unknown. Here we show that over one-third of zebrafish maternal messenger RNAs (mRNAs) can be N6-methyladenosine (m6A) modified, and the clearance of these maternal mRNAs is facilitated by an m6A-binding protein, Ythdf2. Removal of Ythdf2 in zebrafish embryos decelerates the decay of m6A-modified maternal mRNAs and impedes zygotic genome activation. These embryos fail to initiate timely MZT, undergo cell-cycle pause, and remain developmentally delayed throughout larval life. Our study reveals m6A-dependent RNA decay as a previously unidentified maternally driven mechanism that regulates maternal mRNA clearance during zebrafish MZT, highlighting the critical role of m6A mRNA methylation in transcriptome switching and animal development.

RNA m6A methylation regulates the ultraviolet-induced DNA damage response.

Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein). In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of m6A in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol κ overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA m6A modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.

Corrigendum: RNA m6A methylation regulates the ultraviolet-induced DNA damage response.

This corrects the article DOI: 10.1038/nature21671.

A metabolic labeling method detects m6A transcriptome-wide at single base resolution.

Transcriptome-wide mapping of N6-methyladenosine (m6A) at base resolution remains an issue, impeding our understanding of m6A roles at the nucleotide level. Here, we report a metabolic labeling method to detect mRNA m6A transcriptome-wide at base resolution, called 'm6A-label-seq'. Human and mouse cells could be fed with a methionine analog, Se-allyl-L-selenohomocysteine, which substitutes the methyl group on the enzyme cofactor SAM with the allyl. Cellular RNAs could therefore be metabolically modified with N6-allyladenosine (a6A) at supposed m6A-generating adenosine sites. We pinpointed the mRNA a6A locations based on iodination-induced misincorporation at the opposite site in complementary DNA during reverse transcription. We identified a few thousand mRNA m6A sites in human HeLa, HEK293T and mouse H2.35 cells, carried out a parallel comparison of m6A-label-seq with available m6A sequencing methods, and validated selected sites by an orthogonal method. This method offers advantages in detecting clustered m6A sites and holds promise to locate nuclear nascent RNA m6A modifications.

The m6A Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in Arabidopsis.

The epitranscriptomic mark N6-methyladenosine (m6A) can be written, read, and erased via the action of a complex network of proteins. m6A binding proteins read m6A marks and transduce their downstream regulatory effects by altering RNA metabolic processes. The characterization of m6A readers is an essential prerequisite for understanding the roles of m6A in plants, but the identities of m6A readers have been unclear. Here, we characterized the YTH-domain family protein ECT2 as an Arabidopsis thaliana m6A reader whose m6A binding function is required for normal trichome morphology. We developed the formaldehyde cross-linking and immunoprecipitation method to identify ECT2-RNA interaction sites at the transcriptome-wide level. This analysis demonstrated that ECT2 binding sites are strongly enriched in the 3' untranslated regions (3' UTRs) of target genes and led to the identification of a plant-specific m6A motif. Sequencing analysis suggested that ECT2 plays dual roles in regulating 3' UTR processing in the nucleus and facilitating mRNA stability in the cytoplasm. Disruption of ECT2 accelerated the degradation of three ECT2 binding transcripts related to trichome morphogenesis, thereby affecting trichome branching. The results shed light on the underlying mechanisms of the roles of m6A in RNA metabolism, as well as plant development and physiology.

Publisher Correction: N6-Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation.

In the version of this article originally published, the references were incorrectly re-ordered during production. The hyphen in "N6-methyladenosine" in the title was also superscript. The errors have been corrected in the HTML and PDF versions of the paper.

N6-Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation.

N6-Methyladenosine (m6A) RNA modification is present in messenger RNAs (mRNA), ribosomal RNAs (rRNA), and spliceosomal RNAs (snRNA) in humans. Although mRNA m6A modifications have been extensively studied and shown to play critical roles in many cellular processes, the identity of m6A methyltransferases for rRNAs and the function of rRNA m6A modifications are unknown. Here we report a new m6A methyltransferase, ZCCHC4, which primarily methylates human 28S rRNA and also interacts with a subset of mRNAs. ZCCHC4 knockout eliminates m6A4220 modification in 28S rRNA, reduces global translation, and inhibits cell proliferation. We also find that ZCCHC4 protein is overexpressed in hepatocellular carcinoma tumors, and ZCCHC4 knockout significantly reduces tumor size in a xenograft mouse model. Our results highlight the functional significance of an rRNA m6A modification in translation and in tumor biology.

ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.

Transfer RNA demethylase ALKBH3 promotes cancer progression via induction of tRNA-derived small RNAs.

Transfer RNA is heavily modified and plays a central role in protein synthesis and cellular functions. Here we demonstrate that ALKBH3 is a 1-methyladenosine (m1A) and 3-methylcytidine (m3C) demethylase of tRNA. ALKBH3 can promote cancer cell proliferation, migration and invasion. In vivo study confirms the regulation effects of ALKBH3 on growth of tumor xenograft. The m1A demethylated tRNA is more sensitive to angiogenin (ANG) cleavage, followed by generating tRNA-derived small RNAs (tDRs) around the anticodon regions. tDRs are conserved among species, which strengthen the ribosome assembly and prevent apoptosis triggered by cytochrome c (Cyt c). Our discovery opens a potential and novel paradigm of tRNA demethylase, which regulates biological functions via generation of tDRs.

The RNA-binding protein FMRP facilitates the nuclear export of N6-methyladenosine-containing mRNAs.

N6-Methyladenosine (m6A) 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 m6A-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 m6A sites on mRNAs. FMRP depletion increased mRNA m6A 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 m6A-modified RNA targets.

Correction to: YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma.

An amendment to this paper has been published and can be accessed via the original article.

ALKBH10B Is an RNA N6-Methyladenosine Demethylase Affecting Arabidopsis Floral Transition.

N6-methyladenosine (m6A) is the most abundant, internal, posttranscriptional modification in mRNA among all higher eukaryotes. In mammals, this modification is reversible and plays broad roles in the regulation of mRNA metabolism and processing. Despite its importance, previous studies on the role and mechanism of m6A methylation in Arabidopsis thaliana have been limited. Here, we report that ALKBH10B is a demethylase that oxidatively reverses m6A methylation in mRNA in vitro and in vivo. Depletion of ALKBH10B in the alkbh10b mutant delays flowering and represses vegetative growth. Complementation with wild-type ALKBH10B, but not a catalytically inactive mutant (ALKBH10B H366A/E368A), rescues these effects in alkbh10b-1 mutant plants, suggesting the observed phenotypes are controlled by the catalytic action of ALKBH10B We show that ALKBH10B-mediated mRNA demethylation affects the stability of target transcripts, thereby influencing floral transition. We identified 1190 m6A hypermethylated transcripts in the alkbh10b-1 mutant involved in plant development. The discovery and characterization of the archetypical RNA demethylase in Arabidopsis sheds light on the occurrence and functional role(s) of reversible mRNA methylation in plants and defines the role of m6A RNA modification in Arabidopsis floral transition.

Genome-wide mapping reveals that deoxyuridine is enriched in the human centromeric DNA.

Uracil in DNA can be generated by cytosine deamination or dUMP misincorporation; however, its distribution in the human genome is poorly understood. Here we present a selective labeling and pull-down technology for genome-wide uracil profiling and identify thousands of uracil peaks in three different human cell lines. Surprisingly, uracil is highly enriched at the centromere of the human genome. Using mass spectrometry, we demonstrate that human centromeric DNA contains a higher level of uracil. We also directly verify the presence of uracil within two centromeric uracil peaks on chromosomes 6 and 11. Moreover, centromeric uracil is preferentially localized within the binding regions of the centromere-specific histone CENP-A and can be excised by human uracil-DNA glycosylase UNG. Collectively, our approaches allow comprehensive analysis of uracil in the human genome and provide robust tools for mapping and future functional studies of uracil in DNA.

N6-methyladenosine-dependent regulation of messenger RNA stability.

N(6)-methyladenosine (m(6)A) is the most prevalent internal (non-cap) modification present in the messenger RNA of all higher eukaryotes. Although essential to cell viability and development, the exact role of m(6)A modification remains to be determined. The recent discovery of two m(6)A demethylases in mammalian cells highlighted the importance of m(6)A in basic biological functions and disease. Here we show that m(6)A is selectively recognized by the human YTH domain family 2 (YTHDF2) 'reader' protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m(6)A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies. The carboxy-terminal domain of YTHDF2 selectively binds to m(6)A-containing mRNA, whereas the amino-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m(6)A modification is recognized by selectively binding proteins to affect the translation status and lifetime of mRNA.