Hypoxia blocks ferroptosis of hepatocellular carcinoma via suppression of METTL14 triggered YTHDF2-dependent silencing of SLC7A11

Residue hepatocellular carcinoma (HCC) cells enduring hypoxic environment triggered by interventional embolization obtain more malignant potential with little clarified mechanism. The N⁶-methyladenosine (m⁶A) biological activity plays essential roles in diverse physiological processes. However, its role under hypoxic condition remains largely unexplored. RT-qPCR and Western blot were used to evaluate METTL14 expression in hypoxic HCC cells. MDA assay and electronic microscopy photography were used to evaluate ferroptosis. The correlation between SLC7A11 and METTL14 was conducted by bioinformatical analysis. Flow cytometry was used to verify the effect of SLC7A11 on ROS production. Cell counting kit-8 assay was performed to detect cells proliferation ability. Hypoxia triggered suppression of METTL14 in a HIF-1α–dependent manner potently abrogated ferroptosis of HCC cells. Mechanistic investigation identified SLC7A11 was a direct target of METTL14. Both in vitro and in vivo assay demonstrated that METTL14 induced m⁶A modification at 5’UTR of SLC7A11 mRNA, which in turn underwent degradation relied on the YTHDF2-dependent pathway. Importantly, ectopic expression of SLC7A11 strongly blocked METTL14-induced tumour-suppressive effect in hypoxic HCC. Our investigations lay the emphasis on the hypoxia-regulated ferroptosis in HCC cells and identify the HIF-1α /METTL14/YTHDF2/SLC7A11 axis as a potential therapeutic target for the HCC interventional embolization treatment.     

Dynamic assembly of the mRNA m6A methyltransferase complex is regulated by METTL3 phase separation

m⁶A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m⁶A-modified base. The recruitment of the mRNA m⁶A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of two gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.

Approximately one-fourth of eukaryotic mRNAs harbor at least one m6A-modified base, but how is this regulated? This study shows that cells can use liquid-liquid phase separation to regulate dynamic assembly of the mRNA m6A methyltransferase complex (METTL3/METTL14/WTAP), with stoichiometries that depend on condensate partitioning in a substrate binding-dependent manner.     

WTAP facilitates progression of endometrial cancer via CAV-1/NF-κB axis

The N⁶-methyladenosine (m⁶A) modification is one of the most prevalent methylations in eukaryotic messenger RNA (mRNA), and it is essential for the development of many important biological processes such as multiple types of tumors. One of the most important enzymes catalyzing generation of m⁶A on mRNA is Wilms' tumor 1-associating protein (WTAP); however, the potential role of WTAP in endometrial cancer (EC) still remains unknown. Here, we investigated WTAP expression level in cancer tissue and paracancerous tissue from an EC patient. Subsequently, WTAP was knocked down by small interfering RNA in EC cell line of Ishikawa and HEC-1A, respectively. Cell proliferation, migration, and invasion were studied. The expression of caveolin-1 (CAV-1) was detected by quantitative polymerase chain reaction (qPCR). The enrichments of m⁶A and METTL3 on CAV-1 were detected using RNA immunoprecipitation-qPCR. The activity of nuclear factor-κB (NF-κB) was studied using Western blot. We observed that WTAP was dramatically upregulated in the cancer tissue, and there was an enhancement in cell proliferation, migration, and invasion and a decrease in EC apoptosis in vivo and in vitro, which indicated higher tumor malignancy and worse survival outcome. After WTAP was knocked down in EC cells, CAV-1 was significantly upregulated and the enrichments of m⁶A and METTL3 at 3′-untranslated region (UTR) region of CAV-1 were decreased. Moreover, the activity of NF-κB signaling pathway was inhibited by its regulator CAV-1. Taken together, we concluded that WTAP could methylate 3′-UTR of CAV-1 and downregulate CAV-1 expression to activate NF-κB signaling pathway in EC, which promoted EC progression.     

m6A甲基转移酶家族Fiona/METT-10/METTL16的功能

RNA碱基上的化学修饰在其功能的精准调节中发挥关键作用,其中m⁶A是自然界中最普遍的RNA修饰之一,且该修饰在调控RNA稳定性、pre-mRNA剪接、翻译等方面具有重要功能。在真核生物中,m⁶A修饰主要由两种甲基转移酶完成,其在哺乳动物中分别命名为METTL3和METTL16。与METTL3相似,METTL16的底物多种多样,包括pre-mRNA、rRNA、snRNA和lncRNA等,因此似乎难以用一种分子机理解释METTL16对不同RNA底物进行m⁶A修饰的功能。此外,METTL16还在翻译调控中发挥重要作用,但此过程不依赖其甲基转移酶活性,这进一步增加了高度保守的METTL16的功能复杂性。本综述总结了METTL16及其同源蛋白质的结构域、甲基化底物以及它们的潜在功能,着重阐述了在不同物种中关于METTL16研究结果的矛盾之处,并推测METTL16调控S-腺苷基甲硫氨酸(SAM)代谢的功能是趋同进化的一个潜在案例。     

Molecular basis for human mitochondrial tRNA m3C modification by alternatively spliced METTL8

METTL8 has recently been identified as the methyltransferase catalyzing 3-methylcytidine biogenesis at position 32 (m³C32) of mitochondrial tRNAs. METTL8 also potentially participates in mRNA methylation and R-loop biogenesis. How METTL8 plays multiple roles in distinct cell compartments and catalyzes mitochondrial tRNA m³C formation remain unclear. Here, we discovered that alternative mRNA splicing generated several isoforms of METTL8. One isoform (METTL8-Iso1) was targeted to mitochondria via an N-terminal pre-sequence, while another one (METTL8-Iso4) mainly localized to the nucleolus. METTL8-Iso1-mediated m³C32 modification of human mitochondrial tRNA^Thr (hmtRNA^Thr) was not reliant on t⁶A modification at A37 (t⁶A37), while that of hmtRNA^Ser(UCN) critically depended on i⁶A modification at A37 (i⁶A37). We clarified the hmtRNA^Thr substrate recognition mechanism, which was obviously different from that of hmtRNA^Ser(UCN), in terms of requiring a G35 determinant. Moreover, SARS2 (mitochondrial seryl-tRNA synthetase) interacted with METTL8-Iso1 in an RNA-independent manner and modestly accelerated m³C modification activity. We further elucidated how nonsubstrate tRNAs in human mitochondria were efficiently discriminated by METTL8-Iso1. In summary, our results established the expression pattern of METTL8, clarified the molecular basis for m³C32 modification by METTL8-Iso1 and provided the rationale for the involvement of METTL8 in tRNA modification, mRNA methylation or R-loop biogenesis.     

N~6 -methyl-adenosine (m ~6A) in RNA: An Old Modification with A Novel Epigenetic Function

N6 -methyl-adenosine (m6A) is one of the most common and abundant modifications on RNA molecules present in eukaryotes. However, the biological significance of m6A methylation remains largely unknown. Several independent lines of evidence suggest that the dynamic regulation of m6A may have a profound impact on gene expression regulation. The m6A modification is catalyzed by an unidentified methyltransferase complex containing at least one subunit methyltransferase like 3 (METTL3). m6A modification on messenger RNAs (mRNAs) mainly occurs in the exonic regions and 3’-untranslated region (3’-UTR) as revealed by high-throughput m6A-seq. One significant advance in m6A research is the recent discovery of the first two m6A RNA demethylases fat mass and obesity-associated (FTO) gene and ALKBH5, which catalyze m6A demethylation in an a-ketoglutarate (a-KG)-and Fe2+-dependent manner. Recent studies in model organisms demonstrate that METTL3, FTO and ALKBH5 play important roles in many biological processes, ranging from development and metabolism to fertility. Moreover, perturbation of activities of these enzymes leads to the disturbed expression of thousands of genes at the cellular level, implicating a regulatory role of m6A in RNA metabolism. Given the vital roles of DNA and histone methylations in epigenetic regulation of basic life processes in mammals, the dynamic and reversible chemical m6A modification on RNA may also serve as a novel epigenetic marker of profound biological significances.     

METTL3 inhibition ameliorates liver damage in mouse with hepatitis B virus-associated acute-on-chronic liver failure by regulating miR-146a-5p maturation

Hepatitis B virus (HBV)-associated acute-on-chronic liver failure (ACLF) is a clinical syndrome of severe liver damage. HBV infection is affected by N6-methyladenosine (m⁶A) RNA modification. Here, we investigated whether methyltransferase-like 3 (METTL3)-mediated m⁶A methylation can affect ACLF. Human hepatic cells (THLE-2) were treated with lipopolysaccharide (LPS) to induce cell damage. Proliferation, apoptosis and m⁶A modification were measured by MTT assay, flow cytometry and Dot blot assay. Our results showed that HBV infection significantly enhanced the levels of m⁶A modification and elevated the expression of METTL3 and mature-miR-146a-5p in THLE-2 cells, which was repressed by cycloleucine (m⁶A inhibitor). METTL3 overexpression enhanced m⁶A modification and promoted mature-miR-146a-5p expression. METTL3 overexpression promoted HBV replication and apoptosis, enhanced the levels of pro-inflammatory cytokines, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg), and repressed cell proliferation in THLE-2 cells, which attributed to repress miR-146a-5p maturation. Moreover, a severe liver failure mouse model was established by HBV infection to verify the impact of METTL3 knockdown on liver damage in vivo. HBV-infection led to a severe liver damage and increase of apoptosis in hepatic tissues of mice, which was abolished by METTL3 knockdown. METTL3 knockdown reduced METTL3 expression and impeded miR-146a-5p maturation in HBV-infected mice. In conclusion, this work demonstrates that METTL3 inhibition ameliorates liver damage in mouse with HBV-associated ACLF, which contributes to repress miR-146a-5p maturation. Thus, this article suggests a novel therapeutic avenue to prevent and treat HBV-associated ACLF.     

Integrative Analyses of m6A Regulators Identify that METTL3 is Associated with HPV Status and Immunosuppressive Microenvironment in HPV-related Cancers

Although m6A modifications are associated with tumor progression, and anti-tumor immune responses, the role of m6A regulators in HPV-related carcinogenesis has not been well resolved. To provide evidence for the role of m6A regulators in HPV-related carcinogenesis and identify potential therapeutic targets for HPV-related cancers, integrative analyses of m6A regulators in 1,485 head and neck squamous cell carcinoma (HNSC) patients and 507 cervical squamous cell carcinoma (CESC) patients was performed and identified that an m6A regulator, METTL3, was highly expressed in tumors and was related to the poor prognosis in HNSC and CESC. In HPV-positive tumors, METTL3 was positively associated with tumor HPV status, such as HPV integration status, E6 and unspliced-E6 expression, and p16 expression. Further analysis demonstrated that METTL3 ^high status was negatively correlated with tumor immune cell infiltrations and facilitated the expression of immunosuppressive immune checkpoint molecules (i.e., PD-L1). Cell-derived xenograft models demonstrated that METTL3 inhibitor combined with anti-PD1 therapy promoted immunotherapy of CESC in vivo. Overall, this study identified that METTL3 ^high status, is associated with poor prognosis and HPV status, and serves as a mediator of the immunosuppressive tumor microenvironment in HPV-associated cancer, which provides a promising therapeutic target for anti-cancer immunotherapy.     

METTL3 promotes prostatic hyperplasia by regulating PTEN expression in an m6A-YTHDF2-dependent manner

Uncontrolled epithelial cell proliferation in the prostate transition zone and the hyper-accumulation of mesenchymal-like cells derived from the epithelial-mesenchymal transition (EMT) of prostatic epithelium are two key processes in benign prostatic hyperplasia (BPH). m⁶A RNA modification affects multiple cellular processes, including cell proliferation, apoptosis, and differentiation. In this study, the aberrant up-regulation of methylase METTL3 in BPH samples suggests its potential role in BPH development. Elevated m⁶A modification in the prostate of the BPH rat was partially reduced by METTL3 knockdown. METTL3 knockdown also partially reduced the prostatic epithelial thickness and prostate weight, significantly improved the histological features of the prostate, inhibited epithelial proliferation and EMT, and promoted apoptosis. In vitro, METTL3 knockdown decreased TGF-β-stimulated BPH-1 cell proliferation, m⁶A modification, and EMT, whereas promoted cell apoptosis. METTL3 increased the m⁶A modification of PTEN and inhibited its expression through the reading protein YTHDF2. PTEN knockdown aggravated the molecular, cellular, and pathological alterations in the prostate of BPH rats and amplified TGF-β-induced changes in BPH-1 cells. More importantly, PTEN knockdown partially abolished the improving effects of METTL3 knockdown both in vivo and in vitro. In conclusion, the level of m⁶A modification is elevated in BPH; the METTL3/YTHDF2/PTEN axis disturbs the balance between epithelial proliferation and apoptosis, promotes EMT, and accelerates BPH development in an m⁶A modification-related manner.Subject terms: Cell biology, Molecular biology     

The m6A methyltransferase METTL3 modifies PGC-1α mRNA promoting mitochondrial dysfunction and oxLDL-induced inflammation in monocytes

Mitochondrial biogenesis and energy metabolism are essential for regulating the inflammatory state of monocytes. This state is partially controlled by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a coactivator that regulates mitochondrial biogenesis and energy metabolism. Disruption of these processes can also contribute to the initiation of chronic inflammatory diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis. Methyltransferase-like 3 (METTL3)-dependent N⁶-methyladenosine (m⁶A) methylation has recently been shown to regulate a variety of inflammatory processes. However, the role of m⁶A mRNA methylation in affecting mitochondrial metabolism in monocytes under inflammation is unclear, nor is there an established relationship between m⁶A methylation and PGC-1α. In this study, we identified a novel mechanism by which METTL3 acts during oxidized low-density lipoprotein (oxLDL)-induced monocyte inflammation, where METTL3 and YTH N⁶-methyladenosine RNA binding protein 2 (YTHDF2) cooperatively modify PGC-1α mRNA, mediating its degradation, decreasing PGC-1α protein levels, and thereby enhancing the inflammatory response. METTL3 coordinated with YTHDF2 to suppress the expression of PGC-1α, as well as that of cytochrome c (CYCS) and NADH:ubiquinone oxidoreductase subunit C2 (NDUFC2) and reduced ATP production and oxygen consumption rate (OCR). This subsequently increased the accumulation of cellular and mitochondrial reactive oxygen species (ROS) and the levels of proinflammatory cytokines in inflammatory monocytes. These data may provide new insights into the role of METTL3-dependent m⁶A modification of PGC-1α mRNA in the monocyte inflammation response. These data also contribute to a more comprehensive understanding of the pathogenesis of monocyte-macrophage inflammation-associated diseases, such as pulmonary fibrosis, atherosclerosis, and rheumatoid arthritis.     

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2′O-methylation, pseudouridylation, N⁶-methyladenosine (m⁶A), 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 m⁶A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m⁶A modification, the methyltransferase responsible for the 18S rRNA m⁶A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m⁶A 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 m⁶A 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 m⁶A in noncoding RNAs.     

Human METTL16 is a N6‐methyladenosine (m6A) methyltransferase that targets pre‐mRNAs and various non‐coding RNAs

N⁶‐methyladenosine (m⁶A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m⁶A modifications are installed by the METTL3–METTL14 complex, others appear to be introduced independently, implying that additional human m⁶A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m⁶A “writer” protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre‐mRNAs. We demonstrate that METTL16 is responsible for N⁶‐methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5′ splice sites of pre‐mRNAs during splicing, suggesting that METTL16‐mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m⁶A methyltransferase in human cells expands our understanding of the mechanisms by which the m⁶A landscape is installed on cellular RNAs.     

Dysregulation of LINC00470 and METTL3 promotes chemoresistance and suppresses autophagy of chronic myelocytic leukaemia cells

Cytoplasmic lncRNAs have been found to directly interact with target mRNAs and regulate their stability. In this study, we aimed to study the molecular mechanism underlying the function of m⁶A as a central regulator in chemoresistance and CML proliferation. In this study, we established three mice groups (control group, ADR-R group and ADR-R + shLINC00470 group). We detected PTEN mRNA expression in the presence of LINC00470 in the mice models, as well as in the KCL22 and K562 cells. LINC00470 was significantly enriched for PTEN mRNA to exhibit a negative regulatory relationship between LINC00470 and PTEN mRNA. However, the alteration of LINC00470 had no effect on the luciferase activity of PTEN promoter, while the half-life of PTEN mRNA was affected. It was further validated that LINC00470 down-regulated PTEN expression by positively regulating the m6A modification of PTEN mRNA via RNA methyltransferase METTL3. Moreover, the relative expression of LC3II, Beclin-1, ATG7 and ATG5 was all decreased in cells treated with LINC00470, and down-regulated PTEN expression was observed in chemo-resistant cells, while the expression of PTEN was rescued by the transfection of shMETTL3 into chemo-resistant cells. Moreover, the knockdown of METTL3 also restored the normal level of PTEN m⁶A modification and LINC00470 expression in chemo-resistant cells. In conclusion, our results demonstrated the molecular mechanism underlying the effect of LINC00470 on CML by reducing the PTEN stability via RNA methyltransferase METTL3, thus leading to the inhibition of cell autophagy while promoting chemoresistance in CML.     

Methyltransferase METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs

A subset of eukaryotic tRNAs is methylated in the anticodon loop, forming 3-methylcytosine (m³C) modifications. In mammals, the number of tRNAs containing m³C modifications has been expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. However, whereas the enzymes catalyzing m³C formation in nuclear-encoded tRNAs have been identified, the proteins responsible for m³C modification in mt-tRNAs are unknown. Here, we show that m³C formation in human mt-tRNAs is dependent upon the methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase, as well as with mt-tRNAs containing m³C. We demonstrate that human cells deficient in METTL8 exhibit loss of m³C modification in mt-tRNAs, but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m³C in METTL8-deficient cells could be rescued by re-expression of WT METTL8, but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, we found METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA, suggesting a role for m³C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m³C formation in mt-tRNAs and uncover a potential function for m³C modification in mitochondrial tRNA structure.