A Unified Model for the Function of YTHDF Proteins in Regulating m6A-Modified mRNA.

N6-methyladenosine (m6A) is the most abundant mRNA nucleotide modification and regulates critical aspects of cellular physiology and differentiation. m6A is thought to mediate its effects through a complex network of interactions between different m6A sites and three functionally distinct cytoplasmic YTHDF m6A-binding proteins (DF1, DF2, and DF3). In contrast to the prevailing model, we show that DF proteins bind the same m6A-modified mRNAs rather than different mRNAs. Furthermore, we find that DF proteins do not induce translation in HeLa cells. Instead, the DF paralogs act redundantly to mediate mRNA degradation and cellular differentiation. The ability of DF proteins to regulate stability and differentiation becomes evident only when all three DF paralogs are depleted simultaneously. Our study reveals a unified model of m6A function in which all m6A-modified mRNAs are subjected to the combined action of YTHDF proteins in proportion to the number of m6A sites.

DDX21 mediates co-transcriptional RNA m6A modification to promote transcription termination and genome stability.

N6-methyladenosine (m6A) is a crucial RNA modification that regulates diverse biological processes in human cells, but its co-transcriptional deposition and functions remain poorly understood. Here, we identified the RNA helicase DDX21 with a previously unrecognized role in directing m6A modification on nascent RNA for co-transcriptional regulation. DDX21 interacts with METTL3 for co-recruitment to chromatin through its recognition of R-loops, which can be formed co-transcriptionally as nascent transcripts hybridize onto the template DNA strand. Moreover, DDX21's helicase activity is needed for METTL3-mediated m6A deposition onto nascent RNA following recruitment. At transcription termination regions, this nexus of actions promotes XRN2-mediated termination of RNAPII transcription. Disruption of any of these steps, including the loss of DDX21, METTL3, or their enzymatic activities, leads to defective termination that can induce DNA damage. Therefore, we propose that the R-loop-DDX21-METTL3 nexus forges the missing link for co-transcriptional modification of m6A, coordinating transcription termination and genome stability.

A lncRNA from the FTO locus acts as a suppressor of the m6A writer complex and p53 tumor suppression signaling.

N6-methyladenosine (m6A) of mRNAs modulated by the METTL3-METTL14-WTAP-RBM15 methyltransferase complex and m6A demethylases such as FTO play important roles in regulating mRNA stability, splicing, and translation. Here, we demonstrate that FTO-IT1 long noncoding RNA (lncRNA) was upregulated and positively correlated with poor survival of patients with wild-type p53-expressing prostate cancer (PCa). m6A RIP-seq analysis revealed that FTO-IT1 knockout increased mRNA m6A methylation of a subset of p53 transcriptional target genes (e.g., FAS, TP53INP1, and SESN2) and induced PCa cell cycle arrest and apoptosis. We further showed that FTO-IT1 directly binds RBM15 and inhibits RBM15 binding, m6A methylation, and stability of p53 target mRNAs. Therapeutic depletion of FTO-IT1 restored mRNA m6A level and expression of p53 target genes and inhibited PCa growth in mice. Our study identifies FTO-IT1 lncRNA as a bona fide suppressor of the m6A methyltransferase complex and p53 tumor suppression signaling and nominates FTO-IT1 as a potential therapeutic target of cancer.

RNA m6A methylation across the transcriptome.

Since the early days of foundational studies of nucleic acids, many chemical moieties have been discovered to decorate RNA and DNA in diverse organisms. In mammalian cells, one of these chemical modifications, N6-methyl adenosine (m6A), is unique in a way that it is highly abundant not only on RNA polymerase II (RNAPII) transcribed, protein-coding transcripts but also on non-coding RNAs, such as ribosomal RNAs and snRNAs, mediated by distinct, evolutionarily conserved enzymes. Here, we review RNA m6A modification in the light of the recent appreciation of nuclear roles for m6A in regulating chromatin states and gene expression, as well as the recent discoveries of the evolutionarily conserved methyltransferases, which catalyze methylation of adenosine on diverse sets of RNAs. Considering that the substrates of these enzymes are involved in many important biological processes, this modification warrants further research to understand the molecular mechanisms and functions of m6A in health and disease.

Lactylation-driven METTL3-mediated RNA m6A modification promotes immunosuppression of tumor-infiltrating myeloid cells.

Tumor-infiltrating myeloid cells (TIMs) are crucial cell populations involved in tumor immune escape, and their functions are regulated by multiple epigenetic mechanisms. The precise regulation mode of RNA N6-methyladenosine (m6A) modification in controlling TIM function is still poorly understood. Our study revealed that the increased expression of methyltransferase-like 3 (METTL3) in TIMs was correlated with the poor prognosis of colon cancer patients, and myeloid deficiency of METTL3 attenuated tumor growth in mice. METTL3 mediated m6A modification on Jak1 mRNA in TIMs, the m6A-YTHDF1 axis enhanced JAK1 protein translation efficiency and subsequent phosphorylation of STAT3. Lactate accumulated in tumor microenvironment potently induced METTL3 upregulation in TIMs via H3K18 lactylation. Interestingly, we identified two lactylation modification sites in the zinc-finger domain of METTL3, which was essential for METTL3 to capture target RNA. Our results emphasize the importance of lactylation-driven METTL3-mediated RNA m6A modification for promoting the immunosuppressive capacity of TIMs.

Dynamic control of chromatin-associated m6A methylation regulates nascent RNA synthesis.

N6-methyladenosine (m6A) methylation is co-transcriptionally deposited on mRNA, but a possible role of m6A on transcription remains poorly understood. Here, we demonstrate that the METTL3/METTL14/WTAP m6A methyltransferase complex (MTC) is localized to many promoters and enhancers and deposits the m6A modification on nascent transcripts, including pre-mRNAs, promoter upstream transcripts (PROMPTs), and enhancer RNAs. PRO-seq analyses demonstrate that nascent RNAs originating from both promoters and enhancers are significantly decreased in the METTL3-depleted cells. Furthermore, genes targeted by the Integrator complex for premature termination are depleted of METTL3, suggesting a potential antagonistic relationship between METTL3 and Integrator. Consistently, we found the Integrator complex component INTS11 elevated at promoters and enhancers upon loss of MTC or nuclear m6A binders. Taken together, our findings suggest that MTC-mediated m6A modification protects nascent RNAs from Integrator-mediated termination and promotes productive transcription, thus unraveling an unexpected layer of gene regulation imposed by RNA m6A modification.

The Mettl3 epitranscriptomic writer amplifies p53 stress responses.

The p53 transcription factor drives anti-proliferative gene expression programs in response to diverse stressors, including DNA damage and oncogenic signaling. Here, we seek to uncover new mechanisms through which p53 regulates gene expression using tandem affinity purification/mass spectrometry to identify p53-interacting proteins. This approach identified METTL3, an m6A RNA-methyltransferase complex (MTC) constituent, as a p53 interactor. We find that METTL3 promotes p53 protein stabilization and target gene expression in response to DNA damage and oncogenic signals, by both catalytic activity-dependent and independent mechanisms. METTL3 also enhances p53 tumor suppressor activity in in vivo mouse cancer models and human cancer cells. Notably, METTL3 only promotes tumor suppression in the context of intact p53. Analysis of human cancer genome data further supports the notion that the MTC reinforces p53 function in human cancer. Together, these studies reveal a fundamental role for METTL3 in amplifying p53 signaling in response to cellular stress.

Hidden codes in mRNA: Control of gene expression by m6A.

Information in mRNA has largely been thought to be confined to its nucleotide sequence. However, the advent of mapping techniques to detect modified nucleotides has revealed that mRNA contains additional information in the form of chemical modifications. The most abundant modified nucleotide is N6-methyladenosine (m6A), a methyl modification of adenosine. Although early studies viewed m6A as a dynamic and tissue-specific modification, it is now clear that the mRNAs that contain m6A and the location of m6A in those transcripts are largely universal and are influenced by gene architecture, i.e., the size and location of exons and introns. m6A can affect nuclear processes such as splicing and epigenetic regulation, but the major effect of m6A on mRNAs is to promote degradation in the cytoplasm. m6A marks a functionally related cohort of mRNAs linked to certain biological processes, including cell differentiation and cell fate determination. m6A is also enriched in other cohorts of mRNAs and can therefore affect their respective cellular processes and pathways. Future work will focus on understanding how the m6A pathway is regulated to achieve control of m6A-containing mRNAs.

m6A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development.

N6-methyladenosine (m6A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m6A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m6A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m6A modified in their native state, characterized by low folding energies, and predominantly protein coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mavs or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m6A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.

Mechanism of noncoding RNA-associated N6-methyladenosine recognition by an RNA processing complex during IgH DNA recombination.

Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (SµGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N6-methyladenosine (m6A) RNA modification drives recognition and 3' end processing of SµGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m6A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe SµGLT. Direct suppression of m6A modification of SµGLT or of m6A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgH-associated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m6A modification, and m6A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells.

Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy.

The microtubule-associated protein tau oligomerizes, but the actions of oligomeric tau (oTau) are unknown. We have used Cry2-based optogenetics to induce tau oligomers (oTau-c). Optical induction of oTau-c elicits tau phosphorylation, aggregation, and a translational stress response that includes stress granules and reduced protein synthesis. Proteomic analysis identifies HNRNPA2B1 as a principle target of oTau-c. The association of HNRNPA2B1 with endogenous oTau was verified in neurons, animal models, and human Alzheimer brain tissues. Mechanistic studies demonstrate that HNRNPA2B1 functions as a linker, connecting oTau with N6-methyladenosine (m6A) modified RNA transcripts. Knockdown of HNRNPA2B1 prevents oTau or oTau-c from associating with m6A or from reducing protein synthesis and reduces oTau-induced neurodegeneration. Levels of m6A and the m6A-oTau-HNRNPA2B1 complex are increased up to 5-fold in the brains of Alzheimer subjects and P301S tau mice. These results reveal a complex containing oTau, HNRNPA2B1, and m6A that contributes to the integrated stress response of oTau.

Stabilization of ERK-Phosphorylated METTL3 by USP5 Increases m6A Methylation.

N6-methyladenosine (m6A) is the most abundant mRNA modification and is installed by the METTL3-METTL14-WTAP methyltransferase complex. Although the importance of m6A methylation in mRNA metabolism has been well documented recently, regulation of the m6A machinery remains obscure. Through a genome-wide CRISPR screen, we identify the ERK pathway and USP5 as positive regulators of the m6A deposition. We find that ERK phosphorylates METTL3 at S43/S50/S525 and WTAP at S306/S341, followed by deubiquitination by USP5, resulting in stabilization of the m6A methyltransferase complex. Lack of METTL3/WTAP phosphorylation reduces decay of m6A-labeled pluripotent factor transcripts and traps mouse embryonic stem cells in the pluripotent state. The same phosphorylation can also be found in ERK-activated human cancer cells and contribute to tumorigenesis. Our study reveals an unrecognized function of ERK in regulating m6A methylation.

METTL3 and N6-Methyladenosine Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation.

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.

Endoribonucleolytic Cleavage of m6A-Containing RNAs by RNase P/MRP Complex.

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.

Where, When, and How: Context-Dependent Functions of RNA Methylation Writers, Readers, and Erasers.

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 N6-methyladenosine (m6A), the most abundant internal mark on eukaryotic mRNA, in light of the specific biological contexts of m6A effectors. We emphasize the importance of context for RNA modification regulation and function.

Structural Basis for Regulation of METTL16, an S-Adenosylmethionine Homeostasis Factor.

S-adenosylmethionine (SAM) is an essential metabolite that acts as a cofactor for most methylation events in the cell. The N6-methyladenosine (m6A) methyltransferase METTL16 controls SAM homeostasis by regulating the abundance of SAM synthetase MAT2A mRNA in response to changing intracellular SAM levels. Here we present crystal structures of METTL16 in complex with MAT2A RNA hairpins to uncover critical molecular mechanisms underlying the regulated activity of METTL16. The METTL16-RNA complex structures reveal atomic details of RNA substrates that drive productive methylation by METTL16. In addition, we identify a polypeptide loop in METTL16 near the SAM binding site with an autoregulatory role. We show that mutations that enhance or repress METTL16 activity in vitro correlate with changes in MAT2A mRNA levels in cells. Thus, we demonstrate the structural basis for the specific activity of METTL16 and further suggest the molecular mechanisms by which METTL16 efficiency is tuned to regulate SAM homeostasis.

METTL3 is essential for normal progesterone signaling during embryo implantation via m6A-mediated translation control of progesterone receptor.

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P4) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P4 signaling. Conditional deletion of methyltransferase-like 3 (Mettl3), encoding the m6A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter (Pgr-Cre) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m6A modification. A luciferase assay revealed that the m6A modification in the 5' untranslated region (5'-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization in vitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P4 signaling during embryo implantation via m6A-mediated translation control of Pgr mRNA.

A Role for N6-Methyladenine in DNA Damage Repair.

The leading cause of mutation due to oxidative damage is 8-oxo-2'-deoxyguanosine (8-oxoG) mispairing with adenine (Ade), which can occur in two ways. First, guanine of a G:C DNA base pair can be oxidized. If not repaired in time, DNA polymerases can mispair Ade with 8-oxoG in the template. This 8-oxoG:A can be repaired by enzymes that remove Ade opposite to template 8-oxoG, or 8-oxoG opposite to Cyt. Second, free 8-oxo-dGTP can be misincorporated by DNA polymerases into DNA opposite template Ade. However, there is no known repair activity that removes 8-oxoG opposite to template Ade. We suggest that a major role of N6-methyladenine in mammalian DNA is minimizing incorporation of 8-oxoG opposite to Ade by DNA polymerases following adduct formation.

N6-methyladenosine triggers renal fibrosis via enhancing translation and stability of ZEB2 mRNA.

In recent years, a surge in studies investigating N6-methyladenosine (m6A) modification in human diseases has occurred. However, the specific roles and mechanisms of m6A in kidney disease remain incompletely understood. This study revealed that m6A plays a positive role in regulating renal fibrosis (RF) by inducing epithelial-to-mesenchymal phenotypic transition (EMT) in renal tubular cells. Through comprehensive analyses, including m6A sequencing, RNA sequencing, and functional studies, we confirmed the pivotal involvement of zinc finger E-box binding homeobox 2 (ZEB2) in m6A-mediated RF and EMT. Notably, the m6A-modified coding sequence (CDS) of ZEB2 mRNA significantly enhances its translational elongation and mRNA stability by interacting with the YTHDF1/eEF-2 complex and IGF2BP3, respectively. Moreover, targeted demethylation of ZEB2 mRNA using the dm6ACRISPR system substantially decreases ZEB2 expression and disrupts the EMT process in renal tubular epithelial cells. In vivo and clinical data further support the positive influence of m6A/ZEB2 on RF progression. Our findings highlight the m6A-mediated regulation of RF through ZEB2, revealing a novel therapeutic target for RF treatment and enhancing our understanding of the impact of mRNA methylation on kidney disease.

Epstein-Barr virus suppresses N6-methyladenosine modification of TLR9 to promote immune evasion.

Epstein-Barr virus (EBV) is a human tumor virus associated with a variety of malignancies, including nasopharyngeal carcinoma, gastric cancers, and B-cell lymphomas. N6-methyladenosine (m6A) modifications modulate a wide range of cellular processes and participate in the regulation of virus-host cell interactions. Here, we discovered that EBV infection downregulates toll-like receptor 9 (TLR9) m6A modification levels and thus inhibits TLR9 expression. TLR9 has multiple m6A modification sites. Knockdown of METTL3, an m6A "writer", decreases TLR9 protein expression by inhibiting its mRNA stability. Mechanistically, Epstein-Barr nuclear antigen 1 increases METTL3 protein degradation via K48-linked ubiquitin-proteasome pathway. Additionally, YTHDF1 was identified as an m6A "reader" of TLR9, enhancing TLR9 expression by promoting mRNA translation in an m6A -dependent manner, which suggests that EBV inhibits TLR9 translation by "hijacking" host m6A modification mechanism. Using the METTL3 inhibitor STM2457 inhibits TLR9-induced B cell proliferation and immunoglobulin secretion, and opposes TLR9-induced immune responses to assist tumor cell immune escape. In clinical lymphoma samples, the expression of METTL3, YTHDF1, and TLR9 was highly correlated with immune cells infiltration. This study reveals a novel mechanism that EBV represses the important innate immunity molecule TLR9 through modulating the host m6A modification system.