METTL3 promotes chemoresistance in small cell lung cancer by inducing mitophagy.

BACKGROUND: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Although most patients are initially sensitive to first-line combination chemotherapy with cisplatin and etoposide, chemotherapy drug resistance easily develops and quickly leads to tumour progression. Therefore, understanding the mechanisms of chemotherapy drug resistance and how to reverse it is key to improving the prognosis of patients with SCLC. Moreover, N6-methyladenosine (m6A) is the most abundant mRNA modification and is catalysed by the methyltransferase complex, in which methyltransferase-like 3 (METTL3) is the sole catalytic subunit. METHODS: The effects of METTL3 on chemoresistance in SCLC cells were determined using qRT-PCR, Western blotting, immunohistochemistry, cell counting kit (CCK-8) assays, flow cytometry, and tumorigenicity experiments. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), MeRIP qPCR, immunofluorescence, and drug inhibitor experiments were performed to confirm the molecular mechanism of Decapping Protein 2 (DCP2), which is involved in the chemoresistance of SCLC. RESULTS: In the present study, we found that METTL3 is a marker for poor SCLC prognosis, and it is highly expressed in chemoresistant SCLC cells. METTL3 promotes SCLC chemoresistance by positively regulating mitophagy. METTL3 induces m6A methylation of DCP2 and causes the degradation of DCP2, which promotes mitochondrial autophagy through the Pink1-Parkin pathway, leading to chemotherapy resistance. We also found that STM2457, a novel METTL3 inhibitor, can reverse SCLC chemoresistance. CONCLUSIONS: The m6A methyltransferase METTL3 regulates Pink1-Parkin pathway-mediated mitophagy and mitochondrial damage in SCLC cells by targeting DCP2, thereby promoting chemotherapy resistance in patients with SCLC.

OGT Binding Peptide-Tagged Strategy Increases Protein O-GlcNAcylation Level in E. coli.

O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in E. coli. OBP (P1, P2, or P3) was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in E. coli. Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research.

N6-Methyladenosine Modification of ANLN Enhances Hepatocellular Carcinoma Bone Metastasis.

Bones are categorized as the second most prevalent location of extra-hepatic metastasis in Hepatocellular Carcinoma (HCC), which is linked to an extremely poor prognosis due to limited therapeutic options. N6-methyladenosine (m6A) is a prominent modification involved in HCC, but the exact mechanisms on how m6A modifications induce HCC bone metastases (BM) remain unclear. The key modulators responsible for the abundant m6A RNA modification-induced HCC BM was found to be the METTL3 and YTHDF1. The expression of Anillin actin-binding protein (ANLN) was dramatically higher in HCC with BM tissues, and its messenger RNA (mRNA) stability was enhanced via m6A epitranscriptomic regulation by METTL3 and YTHDF1. High METTL3 and YTHDF1 expression along with nuclear ANLN protein was clinically correlated with BM in HCC patients. Furthermore, HCC BM was attributed to over-expression of nuclear ANLN forming a transcriptional complex with SP1 which enhanced KIF2C transcriptional activity to activate the mTORC1 pathway, therefore increased the expression of RANKL and disproportionated RANKL-OPG expression in bone microenvironment leading to malignant neoplasms invade bone tissue. In addition, inhibition of ANLN m6A modification by DZNeP attenuated HCC BM. This data provides meaningful understanding of the modulation and association of m6A epitranscriptomic-regulated BM in HCC, and moreover, defines potentially valuable therapeutic targets.

METTL3 enhances the effect of YTHDF1 on NEDD1 mRNA stability by m6A modification in diffuse large B-cell lymphoma cells.

AIM: Diffuse large B-cell lymphoma (DLBCL) remains the most frequent subpopulation of lymphoma, and N6-methyladenosine (m6A) was implicated in the DLBCL progression. Herein, we sought to decipher the m6A-asociated mechanism of NEDD1 in DLBCL development. METHODS: The NEDD1 expression profile in DLBCL was assessed by quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot. NEDD1 was artificially downregulated or upregulated in DLBCL cells, followed by EdU, Transwell assays and flow cytometry. The Hedgehog pathway activity was assayed by a dual-luciferase assay. The m6A methylation of NEDD1 in DLBCL was assessed by meRIP-qPCR, and the regulatory mechanism of METTL3 on NEDD1 was validated. The LDH assay was conducted to examine the impact of CD8+ T cells on DLBCL cells. The DLBCL cells were administrated into mice to evaluate the tumorigenic activity and ki-67 activity in tumor tissues. RESULTS: NEDD1 was overexpressed in DLBCL. Depletion of NEDD1 inhibited the aggressiveness of SU-DHL-8 and OCI-LY1 cells, whereas overexpression of NEDD1 expedited the aggressiveness of SU-DHL-8 and OCI-LY1 cells. METTL3 promoted NEDD1 translation in an m6A-dependent manner via YTHDF1. Depletion of METTL3 inhibited SU-DHL-8 and OCI-LY1 cell activity through regulation of NEDD1. NEDD1 reversed the repressive effect of METTL3 loss on the aggressiveness of SU-DHL-8 and OCI-LY1 cells. NEDD1 activated the Hedgehog signaling to promote immune escape of DLBCL. CONCLUSIONS: METTL3 promotes translation of NEDD1 via YTHDF1-depedndent m6A modification, thereby activating the Hedgehog signaling pathway to promote immune escape of DLBCL cells.

Methyltransferase-like 3 aggravates endoplasmic reticulum stress in preeclampsia by targeting TMBIM6 in YTHDF2-dependent manner.

BACKGROUND: With the increasing morbidity and mortality of preeclampsia (PE), it has posed a huge challenge to public health. Previous studies have reported endoplasmic reticulum (ER) stress could contribute to trophoblastic dysfunction which was associated with the N6-methyladenosine (m6A) modification by methyltransferase-like 3 (METTL3), resulting in PE. However, little was known about the relationship between METTL3 and ER stress in PE. Thus, in vitro and in vivo studies were performed to clarify the mechanism about how METTL3 affects the trophoblasts under ER stress in PE and to explore a therapeutic approach for PE. METHODS: An ER stress model in HTR-8/SVneo cells and a preeclamptic rat model were used to study the mechanism and explore a therapeutic approach for PE. Western blot, immunohistochemistry, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and methylated RNA immunoprecipitation (MeRIP)-qPCR were performed to detect the protein, RNA, and methylated transmembrane BAX inhibitor motif containing 6 (TMBIM6) expression levels. The m6A colorimetric and mRNA stability assays were used to measure the m6A levels and TMBIM6 stability, respectively. Short hairpin RNAs (shRNAs) were used to knockdown METTL3 and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). Flow cytometry and Transwell assays were performed to evaluate the apoptosis and invasion abilities of trophoblasts. RESULTS: Upregulated METTL3 and m6A levels and downregulated TMBIM6 levels were observed in preeclamptic placentas under ER stress. The ER stress model was successfully constructed, and knockdown of METTL3 had a beneficial effect on HTR-8/SVneo cells under ER stress as it decreased the levels of methylated TMBIM6 mRNA. Moreover, overexpression of TMBIM6 was beneficial to HTR-8/SVneo cells under ER stress as it could neutralize the harmful effects of METTL3 overexpression. Similar to the knockdown of METTL3, downregulation of YTHDF2 expression resulted in the increased expression and mRNA stability of TMBIM6. Finally, improved systemic symptoms as well as protected placentas and fetuses were demonstrated in vivo. CONCLUSIONS: METTL3/YTHDF2/TMBIM6 axis exerts a significant role in trophoblast dysfunction resulting in PE while inhibiting METTL3 may provide a novel therapeutic approach for PE.

METTL3 depletion contributes to tumour progression and drug resistance via N6 methyladenosine-dependent mechanism in HR+HER2-breast cancer.

BACKGROUND: Chemotherapy is an important strategy for the treatment of hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+HER2-) breast cancer (BC), but this subtype has a low response rate to chemotherapy. Growing evidence indicates that N6-methyladenosine (m6A) is the most common RNA modification in eukaryotic cells and that methyltransferase-like 3 (METTL3) participates in tumour progression in several cancer types. Therefore, exploring the function of METTL3 in HR+HER2- BC initiation and development is still important. METHODS: mRNA and protein expression levels were analysed by quantitative real-time polymerase chain reaction and western blotting, respectively. Cell proliferation was detected by CCK-8 and colony formation assays. Cell cycle progression was assessed by flow cytometry. Cell migration and invasion were analysed by wound healing assays and transwell assays, respectively, and apoptosis was analysed by TUNEL assays. Finally, m6A modification was analysed by methylated RNA immunoprecipitation. RESULTS: Chemotherapy-induced downregulation of the m6A modification is regulated by METTL3 depletion in HR+HER2- BC. METTL3 knockdown in MCF-7/T47D cells decreased the drug sensitivity of HR+HER2- BC cells by promoting tumour proliferation and migration and inhibiting apoptosis. Mechanistically, CDKN1A is a downstream target of METTL3 that activates the AKT pathway and promotes epithelial-mesenchymal transformation (EMT). Moreover, a decrease in BAX expression was observed when m6A modification was inhibited with METTL3 knockdown, and apoptosis was inhibited by the reduction of caspase-3/-9/-8. CONCLUSION: METTL3 depletion promotes the proliferation and migration and decreases the drug sensitivity of HR+HER2- BC via regulation of the CDKN1A/EMT and m6A-BAX/caspase-9/-3/-8 signalling pathways, which suggests METTL3 played a tumour-suppressor role and it could be a potential biomarker for predicting the prognosis of patients with HR+HER2- BC.

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.

METTL14-dependent maturation of pri-miR-17 regulates mitochondrial homeostasis and induces chemoresistance in colorectal cancer.

miR-17-5p has been found to be involved in the proliferation and metastasis of colorectal cancer (CRC), and N6-methyladenosine (m6A) modification is the most common RNA modification in eukaryotes. However, whether miR-17-5p contributes to chemotherapy sensitivity in CRC via m6A modification is unclear. In this study, we found that overexpression of miR-17-5p led to less apoptosis and lower drug sensitivity in vitro and in vivo under the 5-fluorouracil (5-FU) treatment, which indicated miR-17-5p led to 5-FU chemotherapy resistance. Bioinformatic analysis suggested that miR-17-5p-mediated chemoresistance was associated with mitochondrial homeostasis. miR-17-5p directly bound to the 3' untranslated region of Mitofusin 2 (MFN2), leading to decreased mitochondrial fusion and enhanced mitochondrial fission and mitophagy. Meanwhile, methyltransferase-like protein 14 (METTL14) was downregulated in CRC, resulting in lower m6A level. Moreover, the low level of METTL14 promoted the expression of pri-miR-17 and miR-17-5p. Further experiments suggested that m6A mRNA methylation initiated by METTL14 inhibits pri-miR-17 mRNA decay via reducing the recognition of YTHDC2 to the "GGACC" binding site. The METTL14/miR-17-5p/MFN2 signaling axis may play a critical role in 5-FU chemoresistance in CRC.

METTL3 regulates breast cancer-associated alternative splicing switches.

Alternative splicing (AS) enables differential inclusion of exons from a given transcript, thereby contributing to the transcriptome and proteome diversity. Aberrant AS patterns play major roles in the development of different pathologies, including breast cancer. N6-methyladenosine (m6A), the most abundant internal modification of eukaryotic mRNA, influences tumor progression and metastasis of breast cancer, and it has been recently linked to AS regulation. Here, we identify a specific AS signature associated with breast tumorigenesis in vitro. We characterize for the first time the role of METTL3 in modulating breast cancer-associated AS programs, expanding the role of the m6A-methyltransferase in tumorigenesis. Specifically, we find that both m6A deposition in splice site boundaries and in splicing and transcription factor transcripts, such as MYC, direct AS switches of specific breast cancer-associated transcripts. Finally, we show that five of the AS events validated in vitro are associated with a poor overall survival rate for patients with breast cancer, suggesting the use of these AS events as a novel potential prognostic biomarker.

Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis.

In Caenorhabditis elegans, the N6-methyladenosine (m6A) modification by METT10, at the 3'-splice sites in S-adenosyl-l-methionine (SAM) synthetase (sams) precursor mRNA (pre-mRNA), inhibits sams pre-mRNA splicing, promotes alternative splicing coupled with nonsense-mediated decay of the pre-mRNAs, and thereby maintains the cellular SAM level. Here, we present structural and functional analyses of C. elegans METT10. The structure of the N-terminal methyltransferase domain of METT10 is homologous to that of human METTL16, which installs the m6A modification in the 3'-UTR hairpins of methionine adenosyltransferase (MAT2A) pre-mRNA and regulates the MAT2A pre-mRNA splicing/stability and SAM homeostasis. Our biochemical analysis suggested that C. elegans METT10 recognizes the specific structural features of RNA surrounding the 3'-splice sites of sams pre-mRNAs, and shares a similar substrate RNA recognition mechanism with human METTL16. C. elegans METT10 also possesses a previously unrecognized functional C-terminal RNA-binding domain, kinase associated 1 (KA-1), which corresponds to the vertebrate-conserved region (VCR) of human METTL16. As in human METTL16, the KA-1 domain of C. elegans METT10 facilitates the m6A modification of the 3'-splice sites of sams pre-mRNAs. These results suggest the well-conserved mechanisms for the m6A modification of substrate RNAs between Homo sapiens and C. elegans, despite their different regulation mechanisms for SAM homeostasis.

RBM15 suppresses hepatic insulin sensitivity of offspring of gestational diabetes mellitus mice via m6A-mediated regulation of CLDN4.

BACKGROUND: Gestational diabetes Mellitus (GDM) is a common pregnancy-specific disease with high morbidity, which is linked to a high risk of obesity and diabetes in offspring. N6-methyladenosine modification of RNA is emerging as an important epigenetic mechanism that is widely manifested in many diseases. This study aimed to investigate the mechanism of m6A methylation in metabolic syndrome in offspring result from intrauterine hyperglycemia. METHODS: GDM mice were established by feeding a high-fat diet 1 weeks before pregnancy. The m6A RNA methylation quantification kit was used to detect liver tissue methylation levels. PCR array was used to determine the expression of the m6A methylation modification enzyme. Immunohistochemistry, qRT-PCR, and western blot were used to examine the expression of RBM15, METTL13, IGF2BP1, and IGF2BP2. Subsequently, methylated RNA immunoprecipitation sequencing combined with mRNA sequencing, followed by dot blot and glucose uptake tests, were performed. RESULTS: In this study, we found that offspring from a GDM mother were more vulnerable to glucose intolerance and insulin resistance. GC-MS revealed significant metabolic changes including saturated fatty acids and unsaturated fatty acids in liver of GDM offspring. We also demonstrated that global mRNA m6A methylation level was significantly increased in the fetal liver of GDM mice, indicating epigenetic change may have a strong relationship with the mechanism of metabolism syndrome. Concordantly, RBM15, the RNA binding methyltransferase, was upregulated in the liver. In vitro, RBM15 suppressed insulin sensitivity and increased insulin resistance through m6A-regulated epigenetic inhabitation of CLDN4. Moreover, MeRIP-sequencing and mRNA-sequencing revealed that differently regulated genes with differential m6A peaks were enriched in metabolic pathways. CONCLUSION: Our study revealed the essential role of RBM15 in insulin resistance and the effect of RBM15-regulated m6A modification in the metabolic syndrome of offspring of GDM mice.

SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

METTL3 inhibits autoreactive Th17 cell responses in experimental autoimmune uveitis via stabilizing ASH1L mRNA.

Methyltransferase like 3 (METTL3), a primary N6-methyladenosine (m6A) methyltransferase, has been implicated in various biological and pathological processes including immune responses. However, the functions and mechanisms of METTL3 in pathogenic T helper (Th)17 cells are poorly understood. Here we found significantly decreased METTL3 expression along with reduced m6A levels in eyeballs and T cells of experimental autoimmune uveitis (EAU). Overexpression of METTL3 ameliorated the development of EAU and suppressed pathogenic Th17 cell responses in vivo and in vitro. Mechanistically, METTL3 promoted the expression of absent, small, or homeotic-like 1 (ASH1L) via enhancing its stability in a YT521-B homology domain containing 2 (YTHDC2)-dependent manner, which further decreased the expression of IL-17 and IL-23 receptor (IL-23R), resulting in reduced pathogenic Th17 responses. Together, our data reveal a pivotal role of METTL3 in regulating pathogenic Th17 responses, which may contribute to human uveitis therapy.

MetBil as a novel molecular regulator in ischemia-induced cardiac fibrosis via METTL3-mediated m6A modification.

Cardiac fibrosis is a common pathological manifestation in multiple cardiovascular diseases and often results in myocardial stiffness and cardiac dysfunctions. LncRNA (long noncoding RNA) participates in a number of pathophysiological processes. However, its role in cardiac fibrosis remains unclear. The purpose of this study was to investigate the role and molecular mechanism of MetBil in regulating cardiac fibrosis. Our data showed that METTL3 binding lncRNA (MetBil) was significantly increased both in fibrotic tissue following myocardial infarction (MI) in mice and in cardiac fibroblasts (CFs) exposed to TGF-ß1 (20 ng/mL) or 20% FBS. Overexpression of MetBil augmented collagen deposition, CF proliferation and activation while silencing MetBil exhibited the opposite effects. Importantly, heterozygous knockout of MetBil alleviated cardiac fibrosis and improved cardiac function after MI. RNA pull-down and RNA-binding protein immunoprecipitation assay showed that METTL3 is a direct downstream target of MetBil; consistently, MetBil and METTL3 were co-localized in both the nucleus and cytoplasm of CFs. Interestingly, MetBil regulated METTL3 expression at protein level, but not mRNA level, in ubiquitin-proteasome pathway. Enforced expression of METTL3 canceled the antifibrotic effects of silencing MetBil reflected by increased collagen production, CF proliferation and activation. Most notably, the m6A-modified fibrosis-regulated genes mediated by METTL3 are profoundly involved in the regulation of MetBil in the cardiac fibrosis following MI. Our study reveals that MetBil as a novel regulator of fibrosis promotes cardiac fibrosis via interacting with METTL3 and regulating the expression of the methylated fibrosis-associated genes, providing a new intervening target for fibrosis-associated cardiac diseases.

m6A-modified circRNA MYO1C participates in the tumor immune surveillance of pancreatic ductal adenocarcinoma through m6A/PD-L1 manner.

Emerging evidence indicates the critical roles of N6-methyladenosine (m6A) modification in human cancers. Herein, our work reported that a novel m6A-modified circRNA from the MYO1C gene, circMYO1C, upregulated in the pancreatic ductal adenocarcinoma (PDAC). Our findings demonstrated that circMYO1C is highly expressed in PDAC tissues. Functionally, circMYO1C promoted the proliferation and migration of PDAC cells in vitro and its silencing reduced the tumor growth in vivo. Mechanistically, circMYO1C cyclization was mediated by m6A methyltransferase METTL3. Moreover, methylated RNA immunoprecipitation sequencing (MeRIP-seq) unveiled the remarkable m6A modification on PD-L1 mRNA. Moreover, circMYO1C targeted the m6A site of PD-L1 mRNA to enhance its stability by cooperating with IGF2BP2, thereby accelerating PDAC immune escape. In conclusion, these findings highlight the oncogenic role of METTL3-induced circMYO1C in PDAC tumorigenesis via an m6A-dependent manner, inspiring a novel strategy to explore PDAC epigenetic therapy.

N6-methyladenosine modification in 18S rRNA promotes tumorigenesis and chemoresistance via HSF4b/HSP90B1/mutant p53 axis.

Aberrant N6-methyladenosine (m6A) modification on mRNA is correlated with cancer progression. However, the role of m6A on ribosomal RNA (rRNA) in cancer remains poorly understood. Our current study reveals that METTL5/TRMT112 and their mediated m6A modification at the 18S rRNA 1832 site (m6A1832) are elevated in nasopharyngeal carcinoma (NPC) and promote oncogenic transformation in vitro and in vivo. Moreover, loss of catalytic activity of METTL5 abolishes its oncogenic functions. Mechanistically, m6A1832 18S rRNA modification facilitates the assembly of 80S ribosome via bridging the RPL24-18S rRNA interaction, therefore promoting the translation of mRNAs with 5' terminal oligopyrimidine (5' TOP) motifs. Further mechanistic analysis reveals that METTL5 enhances HSF4b translation to activate the transcription of HSP90B1, which binds with oncogenic mutant p53 (mutp53) protein and prevents it from undergoing ubiquitination-dependent degradation, therefore facilitating NPC tumorigenesis and chemoresistance. Overall, our findings uncover an innovative mechanism underlying rRNA epigenetic modification in regulating mRNA translation and the mutp53 pathway in cancer.

The m6A methyltransferase METTL3 affects cell proliferation and migration by regulating YAP expression in Hirschsprung disease.

BACKGROUND: METTL3, an mRNA m6A methyltransferase, has been implicated in various steps of mRNA metabolism, such as stabilization, splicing, nuclear transportation, translation, and degradation. However, whether METTL3 dysregulation is involved in Hirschsprung disease (HSCR) development remains unclear. In this study, we preliminarily elucidated the role of METTL3 in HSCR and sought to identify the associated molecular mechanism. METHODS: The gene expression levels of YAP and several methyltransferases, demethylases, and effectors were evaluated by RT-qPCR. Protein levels were evaluated by western blot and immunohistochemistry. Cell proliferation and migration were detected by CCK-8 and Transwell assays, respectively. The overall levels of m6A modification were determined by colorimetry. RESULTS: We found that m6A levels were reduced in the stenotic intestinal tissue of patients with HSCR. When METTL3 was knocked down in SH-SY5Y and HEK-293T cells, the proliferative and migratory abilities of the cells were inhibited, m6A modification levels were reduced, and YAP expression was increased. Importantly, YAP and METTL3 expression displayed a negative correlation in both cell lines as well as in HSCR tissue. CONCLUSIONS: Our results provide evidence for an interaction between METTL3 and YAP in HSCR, and further suggest that METTL3 is involved in the pathogenesis of HSCR by regulating neural crest cell proliferation and migration upstream of YAP.

NSUN2 promotes osteosarcoma progression by enhancing the stability of FABP5 mRNA via m5C methylation.

5-methylcytosine (m5C) modification, which is mainly induced by the RNA methyltransferase NSUN2 (NOP2/Sun domain family, member 2), is an important chemical posttranscriptional modification in mRNA and has been proven to play important roles in the progression of many cancers. However, the functions and underlying molecular mechanisms of NSUN2-mediated m5C in osteosarcoma (OS) remain unclear. In this study, we found NSUN2 was highly expressed in OS tissues and cells. We also discovered that higher expression of NSUN2 predicted poorer prognosis of OS patients. Our study showed that NSUN2 could promote the progression of OS cells. Moreover, we employed RNA sequencing, RNA immunoprecipitation (RIP), and methylated RIP to screen and validate the candidate targets of NSUN2 and identified FABP5 as the target. We observed that NSUN2 stabilized FABP5 mRNA by inducing m5C modification and further promoted fatty acid metabolism in OS cells. Moreover, both knocking down the expression of FABP5 and adding fatty acid oxidation inhibitor could counterbalance the promoting effect of NSUN2 on the progression of OS. Our study confirms that NSUN2 can up-regulate the expression of FABP5 by improving the stability of FABP5 mRNA via m5C, so as to promote fatty acid metabolism in OS cells, and finally plays the role in promoting the progression of OS. Our findings suggest that NSUN2 is a promising prognostic marker for OS patients and may serve as a potential therapeutic target for OS treatment. A schematic illustration was proposed to summarize our findings.

Simultaneous Measurement of DNA Methylation and Nucleosome Occupancy in Single Cells Using scNOMe-Seq.

Single-cell Nucleosome Occupancy and Methylome sequencing (scNOMe-seq) is a multimodal assay that simultaneously measures endogenous DNA methylation and nucleosome occupancy (i.e., chromatin accessibility) in single cells. scNOMe-seq combines the activity of a GpC Methyltransferase, an enzyme which methylates cytosines in GpC dinucleotides, with bisulfite conversion, whereby unmethylated cytosines are converted into thymines. Because GpC Methyltransferase acts only on cytosines present in non-nucleosomal regions of the genome, the subsequent bisulfite conversion step not only detects the endogenous DNA methylation, but also reveals the genome-wide pattern of chromatin accessibility. Implementing this technology at the single-cell level helps to capture the dynamics governing methylation and accessibility vary across individual cells and cell types. Here, we provide a scalable plate-based protocol for preparing scNOMe-seq libraries from single nucleus suspensions.

N6-methyadenosine modified SUV39H2 regulates homologous recombination through epigenetic repression of DUSP6 in gastric cancer.

Despite many advances in treatment over the past few years, the poor 5-year survival rate and high recurrence rate of gastric cancer (GC) remain unsatisfactory. As the most abundant epigenetic modification in the eukaryotic mRNA, N6-methyladenosine (m6A) methylation participates in tumor progression and tissue development. During tumor progression, DNA damage repair mechanisms can be reprogrammed to give new growth advantages on tumor clones whose genomic integrity is disturbed. Here we detected the elevated SUV39H2 expression in GC tissues and cell lines. Functionally, SUV39H2 promoted GC proliferation and inhibited apoptosis in vitro and in vivo. Mechanistically, METTL3-mediated m6A modification promotes mRNA stability of SUV39H2 in an IGF2BP2 dependent manner, resulting in upregulated mRNA expression of SUV39H2. As a histone methyltransferase, SUV39H2 was verified to increase the phosphorylation level of ATM through transcriptional repression of DUSP6, thereby promoting HRR and ultimately inhibiting GC chemosensitivity to cisplatin. Collectively, these results indicate the specific mechanism of m6A-modified SUV39H2 as a histone methyltransferase promoting HRR to inhibit the chemosensitivity of GC. SUV39H2 is expected to become a key target in the precision targeted therapy of GC.