bims-rimeca 2023-02-19 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2023–02–19
27 papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

m6A-modified circRNA MYO1C participates in the tumor immune surveillance of pancreatic ductal adenocarcinoma through m6A/PD-L1 manner.
The m6A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment.
Thyroid cancer risk prediction model using m6A RNA methylation regulators: integrated bioinformatics analysis and histological validation.
N-methyladenosine reader YTHDF2-mediated long noncoding RNA FENDRR degradation promotes cell proliferation in endometrioid endometrial carcinoma.
The N6-methyladenosine writer WTAP contributes to the induction of immune tolerance post kidney transplantation by targeting regulatory T cells.
N6-Methyladenosine RNA Modification in Host Cells Regulates Peste des Petits Ruminants Virus Replication.
Clinical and biological significance of RNA N6-methyladenosine regulators in Alzheimer disease.
METTL3 promotes SMSCs chondrogenic differentiation by targeting the MMP3, MMP13, and GATA3.
Target-promoted specific activation of m6A-DNAzyme for SPEXPAR-amplified and highly sensitive non-label electrochemical assay of FTO demethylase.
Structure of the Caenorhabditis elegans m6A methyltransferase METT10 that regulates SAM homeostasis.
Expression and significance of m6A-RNA-methylation in oral cancer and precancerous lesion.
NSUN2 promotes osteosarcoma progression by enhancing the stability of FABP5 mRNA via m5C methylation.
ALKBH5-mediated m6 A demethylation of TIRAP mRNA promotes radiation-induced liver fibrosis and decreases radiosensitivity of hepatocellular carcinoma.
YTHDC1 maintains trophoblasts function by promoting degradation of mA6-modified circMPP1.
m 6 A RNA methylation orchestrates transcriptional dormancy during developmental pausing.
Decreased ubiquitin modifying enzyme A20 associated with hyper-responsiveness to ovalbumin challenge following intrauterine growth restriction.
N6-methyladenine regulator-mediated RNA methylation modification patterns in immune microenvironment regulation of osteoarthritis.
Identifying RNA Modifications by Direct RNA Sequencing Reveals Complexity of Epitranscriptomic Dynamics in Rice.
Evaluation of N6-methyldeoxyadenosine antibody-based genomic profiling in eukaryotes.
DOT1L promotes cell proliferation and invasion by epigenetically regulating STAT5B in renal cell carcinoma.
The m6A methyltransferase METTL3 affects cell proliferation and migration by regulating YAP expression in Hirschsprung disease.
Methyltransferase-like 3 modulates osteogenic differentiation of adipose-derived stem cells in osteoporotic rats.
RNA Methylome Reveals the m6A-mediated Regulation of Flavor Metabolites in Tea Leaves under Solar-withering.
Nsun2 coupling with RoRγt shapes the fate of Th17 cells and promotes colitis.
Transcriptome-wide high-throughput m6 A sequencing of differential m6 A methylation patterns in the decidual tissues from RSA patients.
METTL14 modulates glycolysis to inhibit colorectal tumorigenesis in p53-wild-type cells.
m6A modifications of circular RNAs in ischemia-induced retinal neovascularization.

Cell Death Dis. 2023 Feb 14. 14(2): 120

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

Hua Guan, Kun Tian, Wei Luo, Mingfei Li.

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.

DOI: https://doi.org/10.1038/s41419-023-05570-0
JCI Insight. 2023 Feb 16. pii: e162436. [Epub ahead of print]

The m6A methyltransferase METTL16 negatively regulates MCP1 expression in mesenchymal stem cells during monocyte recruitment.

Zhaoqiang Zhang, Zhongyu Xie, Jiajie Lin, Zehang Sun, Zhikun Li, Wenhui Yu, Yipeng Zeng, Guiwen Ye, Jinteng Li, Feng Ye, Zepeng Su, Yunshu Che, Peitao Xu, Chenying Zeng, Peng Wang, Yanfeng Wu, Huiyong Shen.

  Mesenchymal stem cells (MSCs) possess strong immunoregulatory functions, one aspect of which is recruiting monocytes from peripheral vessels to local tissue by secreting MCP1. However, the regulatory mechanisms of MCP1 secretion in MSCs are still unclear. Recently, N6-methyladenosine (m6A) modification was reported to be involved in the functional regulation of MSCs. In this study, we demonstrated that methyltransferase-like 16 (METTL16) negatively regulated MCP1 expression in MSCs through m6A modification. Specifically, the expression of METTL16 in MSCs decreased gradually and was negatively correlated with the expression of MCP1 after coculture with monocytes. Knocking down METTL16 markedly enhanced MCP1 expression and the ability to recruit monocytes. Mechanistically, knocking down METTL16 decreased MCP1 mRNA degradation, which was mediated by the m6A reader YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). We further revealed that YTHDF2 specifically recognized m6A sites on MCP1 mRNA in the CDS region and thus negatively regulated MCP1 expression. Moreover, an in vivo assay showed that MSCs transfected with METTL16 siRNA showed a stronger ability to recruit monocytes. These findings reveal a potential mechanism by which the m6A methylase METTL16 regulates MCP1 expression through YTHDF2-mediated mRNA degradation and suggest a potential strategy to manipulate MCP1 expression in MSCs.

Keywords:  Adult stem cells; Chemokines; Epigenetics; Immunology; Stem cells

DOI:  https://doi.org/10.1172/jci.insight.162436
Aging (Albany NY). 2023 Feb 15. 15

Thyroid cancer risk prediction model using m6A RNA methylation regulators: integrated bioinformatics analysis and histological validation.

Wei Zhou, Junchao Lin, Jinqiang Liu, Rui Zhang, Aqiang Fan, Qibin Xie, Liu Hong, Daiming Fan.

   BACKGROUND: Epigenetic reprogramming has been reported to play a critical role in the progression of thyroid cancer. RNA methylation accounts for more than 60% of all RNA modifications, and N6-methyladenosine (m6A) is the most common modification of RNAs in higher organisms. The purpose of this study was to explore the related modification mode of m6A regulators construction and its evaluation on the clinical prognosis and therapeutic effect of thyroid cancer.
METHODS: The levels of 23 m6A regulators in The Cancer Genome Atlas (TCGA) were analyzed. Differentially expressed genes (DEGs) and survival analysis were performed based on TCGA-THCA clinicopathological and follow-up information, and the mRNA levels of representative genes were verified using clinical thyroid cancer data. In order to detect the effects of m6A regulators and their DEGs, consensus cluster analysis was carried out, and the expression of different m6A scores in Tumor Mutation Burden (TMB) and immune double antibodies (PD-1 antibody and CTLA4 antibody) were evaluated to predict the correlation between m6A score and thyroid cancer tumor immunotherapy response.
RESULTS: Different expression patterns of m6A regulatory factors were detected in thyroid cancer tumors and normal tissues, and several prognoses related m6A genes were obtained. Two different m6A modification patterns were determined by consensus cluster analysis. Two different subgroups were established by screening overlapping DEGs between two m6A clusters, with cluster A having the best prognosis. According to the m6A score extracted from DEGs, thyroid cancer patients can be divided into high and low score subgroups. Patients with lower m6A score have longer survival time and better clinical features. The relationship between m6A score and Tumor Mutation Burden (TMB) and its correlation with the expression of PD-1 antibody and CTLA4 antibody proved that m6A score could be used as a potential predictor of the efficacy of immunotherapy in thyroid cancer patients.
CONCLUSIONS: We screened DEGs from cluster m6A and constructed a highly predictive model with prognostic value by dividing TCGA-THCA into two different clusters and performing m6A score analysis. This study will help clarify the overall impact of m6A modification patterns on thyroid cancer progression and formulate more effective immunotherapy strategies.

Keywords:  N6-methyladenosine (m6A); The Cancer Genome Atlas (TCGA); geneCluster; m6Acluster; m6Ascore; thyroid cancer

DOI:  https://doi.org/10.18632/aging.204525
Lab Invest. 2021 Jun;pii: S0023-6837(22)00629-8. [Epub ahead of print]101(6): 775-784

N-methyladenosine reader YTHDF2-mediated long noncoding RNA FENDRR degradation promotes cell proliferation in endometrioid endometrial carcinoma.

Jie Shen, Xiao-Ping Feng, Ru-Bing Hu, Hao Wang, Yan-Li Wang, Jian-Hua Qian, Yun-Xiao Zhou.

Dysregulation of long noncoding RNA (LncRNA) FENDDR has been shown to be closely related to the progression of several cancers. However, its role and upstream regulatory mechanism in endometrioid endometrial carcinoma (EEC) remains unclear. This study was conducted using the cancerous tissues of EEC patients (n = 60), EEC cell lines, and a xenograft mouse model. The expression level of LncRNA FENDRR was decreased and the N-methyladenosine (m6A) methylation levels of LncRNA FENDRR was elevated in cancerous tissues of EEC patients. In vitro experiments demonstrated that YTH domain-containing 2 (YTHDF2), an m6A reader, recognized the abundance of m6A-modified LncRNA FENDRR in EEC cells and promoted its degradation. LncRNA FENDRR overexpression suppressed cell proliferation and facilitated cell apoptosis in the EEC cell line HEC-1B by reducing the protein level of SRY-related HMG box transcription factor 4 (SOX4). Interference of LncRNA FENDRR reversed the inhibitory effect of sh-YTHDF2 on cell proliferation and the promoting effect of sh-YTHDF2 on cell apoptosis in HEC-1B cells by silencing FENDRR. Finally, in vivo experiments confirmed that overexpression of LncRNA FENDRR retarded the growth of EEC cells. In conclusion, YTHDF2-mediated LncRNA FENDRR degradation promotes cell proliferation by elevating SOX4 expression in EEC.

DOI: https://doi.org/10.1038/s41374-021-00543-3
Lab Invest. 2022 11;pii: S0023-6837(22)00264-1. [Epub ahead of print]102(11): 1268-1279

The N6-methyladenosine writer WTAP contributes to the induction of immune tolerance post kidney transplantation by targeting regulatory T cells.

Zhigang Wang, Yuanbo Qi, Yonghua Feng, Hongen Xu, Junxiang Wang, Luyu Zhang, Jie Zhang, Xinyue Hou, Guiwen Feng, Wenjun Shang.

N6-methyladenosine (m6A) modification is involved in diverse immunoregulation, while the relationship between m6A modification and immune tolerance post kidney transplantation remains unclear. Expression of Wilms tumor 1-associating protein (WTAP), an m6A writer, was firstly detected in tolerant kidney transplant recipients (TOL). Then the role of WTAP on regulatory T (Treg) cell differentiation and function in CD4+ T cells from kidney transplant recipients with immune rejection (IR) was investigated. The potential target of WTAP and effect of WTAP on immune tolerance in vivo were subsequently verified. WTAP was upregulated in CD4+ T cells of TOL and positively correlated with Treg cell proportion. In vitro, WTAP overexpression promoted Treg cell differentiation and enhanced Treg cell-mediated suppression toward naïve T cells. Forkhead box other 1 (Foxo1) was identified as a target of WTAP. WTAP enhanced m6A modification of Foxo1 mRNA in coding sequence (CDS) region, leading to up-regulation of Foxo1. Overexpression of m6A demethylase removed the effect of WTAP overexpression, while Foxo1 overexpression reversed these effects. WTAP overexpression alleviated allograft rejection in model mice, as evidenced by reduced inflammatory response and increased Treg population. Our study suggests that WTAP plays a positive role in induction of immune tolerance post kidney transplant by promoting Treg cell differentiation and function.

DOI: https://doi.org/10.1038/s41374-022-00811-w
Microbiol Spectr. 2023 Feb 14. e0266622

N6-Methyladenosine RNA Modification in Host Cells Regulates Peste des Petits Ruminants Virus Replication.

Owais Khan, Gunturu Narasimha Tanuj, Divyaprakash R Choravada, Kaushal Kishore Rajak, S Chandra Sekar, Madhu Cholenahalli Lingaraju, Sujoy K Dhara, Praveen K Gupta, Bishnu Prasad Mishra, Triveni Dutt, Ravi Kumar Gandham, Basavaraj Sajjanar.

  N6-methyladenosine (m6A) modification is a major RNA epigenetic regulatory mechanism. The dynamics of m6A levels in viral genomic RNA and their mRNAs have been shown to have either pro- or antiviral functions, and therefore, m6A modifications influence virus-host interactions. Currently, no reports are available on the effect of m6A modifications in the genome of Peste des petits ruminants virus (PPRV). In the present study, we took PPRV as a model for nonsegmented negative-sense single-stranded RNA viruses and elucidate the role of m6A modification on viral replication. We detected m6A-modified sites in the mRNA of the virus and host cells, as well as the PPRV RNA genome. Further, it was found that the level of m6A modification in host cells alters the viral gene expression. Knockdown of the METTL3 and FTO genes (encoding the m6A RNA modification writer and eraser proteins, respectively) results in alterations of the levels of m6A RNA modifications in the host cells. Experiments using these genetically modified clones of host cells infected with PPRV revealed that both higher and lower m6A RNA modification in the host cells negatively affect PPRV replication. We found that m6A-modified viral transcripts had better stability and translation efficiency compared to the unmodified mRNA. Altogether, from these data, we conclude that the m6A modification of RNA regulates PPRV replication. These findings contribute toward a way forward for developing novel antiviral strategies against PPRV by modulating the dynamics of host m6A RNA modification. IMPORTANCE Peste des petits ruminants virus (PPRV) causes a severe disease in sheep and goats. PPRV infection is a major problem, causing significant economic losses to small ruminant farmers in regions of endemicity. N6-methyladenosine (m6A) is an important RNA modification involved in various functions, including virus-host interactions. In the present study, we used stable clones of Vero cells, having knocked down the genes encoding proteins involved in dynamic changes of the levels of m6A modification. We also used small-molecule compounds that interfere with m6A methylation. This resulted in a platform of host cells with various degrees of m6A RNA modification. The host cells with these different microenvironments were useful for studying the effect of m6A RNA modification on the expression of viral genes and viral replication. The results pinpoint the level of m6A modifications that facilitate the maximum replication of PPRV. These findings will be useful in increasing the virus titers in cultured cells needed for the economical development of the vaccine. Furthermore, the findings have guiding significance for the development of novel antiviral strategies for limiting PPRV replication in infected animals.

Keywords:  N6-methyladenosine (m6A); RNA modifications; small ruminant morbillivirus; virus replication; virus-host interactions

DOI:  https://doi.org/10.1128/spectrum.02666-22
Medicine (Baltimore). 2023 Feb 17. 102(7): e32945

Clinical and biological significance of RNA N6-methyladenosine regulators in Alzheimer disease.

Zhiqiang Qiu, Xuanyang Bai, Xinye Han, Peishen Wang, Xiang Wang, Yuxia Lv, Yihua An.

RNA N6-methyladenosine (m6A) regulators are essential for a variety of biological functions, such as early development, viral infections, and cancer. However, their roles in Alzheimer disease (AD) are still not very clear. Here, 16 significant m6A regulators were identified using difference analysis between AD patients and non-demented controls based on the GSE132903 dataset from the Gene Expression Omnibus database. Using these 16 m6A regulators, a nomogram model was established to predict the prevalence of AD. We found that patients could obtain a good clinical benefit based on this model. In addition, we revealed 2 distinct m6A patterns and 2 distinct m6A gene patterns in AD and demonstrated their prognostic and risk assessment significance. This present work comprehensively evaluated the functions of m6A regulators in the diagnosis and subtype classification of AD. These results suggested they have potential prognostic and risk assessment significance in AD.

DOI: https://doi.org/10.1097/MD.0000000000032945
Regen Ther. 2023 Mar;22 148-159

METTL3 promotes SMSCs chondrogenic differentiation by targeting the MMP3, MMP13, and GATA3.

Bin Hu, Xiangjie Zou, Yaohui Yu, Yiqiu Jiang, Hongyao Xu.

   Objective: Synovium-derived mesenchymal stem cells (SMSCs) are multipotential non-hematopoietic progenitor cells that can differentiate into various mesenchymal lineages in adipose and bone tissue, especially in chondrogenesis. Post-transcriptional methylation modifications are relative to the various biological development procedures. N6-methyladenosine (m6A) methylation has been identified as one of the abundant widespread post-transcriptional modifications. However, the connection between the SMSCs differentiation and m6A methylation remains unknown and needs further exploration.
Methods: SMSCs were derived from synovial tissues of the knee joint of male Sprague-Dawley (SD) rats. In the chondrogenesis of SMSCs, m6A regulators were detected by quantitative real-time PCR (RT-PCR) and Western blot (WB). We observed the situation that the knockdown of m6A "writer" protein methyltransferase-like (METTL)3 in the chondrogenesis of SMSCs. We also mapped the transcript-wide m6A landscape in chondrogenic differentiation of SMSCs and combined RNA-seq and MeRIP-seq in SMSCs by the interference of METTL3.
Results: The expression of m6A regulators were regulated in the chondrogenesis of SMSCs, only METTL3 is the most significant factor. In addition, after the knockdown of METTL3, MeRIP-seq and RNA-seq technology were applied to analyze the transcriptome level in SMSCs. 832 DEGs displayed significant changes, consisting of 438 upregulated genes and 394 downregulated genes. DEGs were enriched in signaling pathways regulating the glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate and ECM-receptor interaction via Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. The findings of this study indicate a difference in transcripts of MMP3, MMP13, and GATA3 containing consensus m6A motifs required for methylation by METTL3. Further, the reduction of METTL3 decreased the expression of MMP3, MMP13, and GATA3.
Conclusion: These findings confirm the molecular mechanisms of METTL3-mediated m6A post-transcriptional change in the modulation of SMSCs differentiating into chondrocytes, thus highlighting the potential therapeutic effect of SMSCs for cartilage regeneration.

Keywords:  COL2A1, collagen type II: alpha 1; Chondrogenesis; GATA3, GATA binding protein 3; METTL3; METTL3, methyltransferase like 3; MMP13, matrix metallopeptidase 13; MMP3, matrix metallopeptidase 3; MeRIP-sequencing; N6-methyladenosine; SMSCs; SMSCs, synovium-derived mesenchymal stem cells; SOX9, SRP (sex determining region Y)-box 9; m6A, N6-methyladenosine

DOI:  https://doi.org/10.1016/j.reth.2023.01.005
Anal Chim Acta. 2023 Mar 22. pii: S0003-2670(23)00123-X. [Epub ahead of print]1247 340902

Target-promoted specific activation of m6A-DNAzyme for SPEXPAR-amplified and highly sensitive non-label electrochemical assay of FTO demethylase.

Huahui Gao, Xinmei Song, Qirong Chen, Ruo Yuan, Yun Xiang.

  The demethylase of fat mass and obesity related protein (FTO) is critical to regulate the dynamic N6-methyladenosine (m6A) modification of eukaryotic mRNAs, and its overexpression has found to be closely related to the initiation of several cancers. On the basis of a target-promoted specific activation of DNAzyme strategy coupled with self-primer exponential amplification reaction (SPEXPAR) cycles and DNA supersandwich assemblies, the highly sensitive and label-free electrochemical FTO assay approach is established. The modification of the catalytic core nucleobase of the DNAzyme probe by m6A can inhibit its cleavage activity. The presence of target FTO catalyzes the elimination of the methyl group to restore the DNAzyme activity, which cleaves the hairpin substrates to trigger the SPEXPAR for yielding many ssDNAs. The capture of these DNAs on the sensor electrode leads to the initiation of supersandwich assembly formation of long dsDNAs. Tremendous electrochemical signal probe of [Ru(NH3)6]Cl3 are then absorbed on these dsDNAs to produce highly amplified catalytic currents with the assistance of K3[Fe(CN)6] for detecting trace FTO with 63.1 fM detection limit. Furthermore, the sensor can be employed for selective assay of FTO in cell lysates, revealing the great potential of this sensing strategy for biomedical and biological study applications.

Keywords:  DNA supersandwich; Electrochemical biosensor; FTO demethylase; Self-primer exponential amplification reaction

DOI:  https://doi.org/10.1016/j.aca.2023.340902
Nucleic Acids Res. 2023 Feb 16. pii: gkad081. [Epub ahead of print]

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

Jue Ju, Tomohiko Aoyama, Yuka Yashiro, Seisuke Yamashita, Hidehito Kuroyanagi, Kozo Tomita.

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.

DOI: https://doi.org/10.1093/nar/gkad081
Front Oncol. 2023 ;13 1013054

Expression and significance of m6A-RNA-methylation in oral cancer and precancerous lesion.

Zhiming Qin, Jiaying Bai, Huiying He, Binbin Li.

   Background: Oral potentially malignant disorders (OPMDs) and oral squamous cell carcinoma (OSCC) are a series of related pathologic and molecular events involving simple epithelial hyperplasia, mild to severe dysplasia and canceration. N6-methyladenosine RNA methylation, as the most common modification of both coding mRNA and non-coding ncRNA in eukaryotes, participates in the regulation of the occurrence and development of various malignant tumors in human. However, its role in oral epithelial dysplasia (OED) and OSCC remain unclear.
Materials and methods: In this study, multiple public databases were used for bioinformatics analysis of 23 common m6A methylation regulators in head and neck squamous cell carcinoma (HNSCC). Protein expressions of IGF2BP2 and IGF2BP3 were verified accordingly in clinical cohort samples of OED and OSCC.
Results: Patients with high expression of FTO、HNRNPC、HNRNPA2B1、LRPPRC、IGF2BP1、IGF2BP2、IGF2BP3 had a poor prognosis. IGF2BP2 had a relatively high mutation rate in HNSCC, and its expression was significantly positively correlated with tumor purity, and significantly negatively correlated with the infiltration level of B cells and CD8+T cells. The expression of IGF2BP3 was significantly positively correlated with tumor purity and CD4+T cells. Immunohistochemistrically, the expression of IGF2BP2 and IGF2BP3 in oral simple epithelial hyperplasia, OED and OSCC increased gradually. Both were strongly expressed in OSCC.
Conclusion: IGF2BP2 and IGF2BP3 were the potential biological prognostic indicators of OED and OSCC.

Keywords:  IGF2BP2; IGF2BP3; N6-methyladenosine; oral epithelial dysplasia; oral squamous cell carcinoma

DOI:  https://doi.org/10.3389/fonc.2023.1013054
Cell Death Dis. 2023 Feb 15. 14(2): 125

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

Min Yang, Renxiong Wei, Sheng Zhang, Sang Hu, Xiaoxiao Liang, Zhiqiang Yang, Chong Zhang, Yufeng Zhang, Lin Cai, Yuanlong Xie.

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.

DOI: https://doi.org/10.1038/s41419-023-05646-x
Clin Transl Med. 2023 Feb;13(2): e1198

ALKBH5-mediated m6 A demethylation of TIRAP mRNA promotes radiation-induced liver fibrosis and decreases radiosensitivity of hepatocellular carcinoma.

Yuhan Chen, Peitao Zhou, Yixun Deng, Xinni Cai, Mingrui Sun, Yining Sun, Dehua Wu.

   BACKGROUND: Radiation-induced hepatic stellate cell (HSC) activation promotes radiation-induced liver fibrosis (RILF), a complication for hepatocellular carcinoma (HCC) radiotherapy. The demethylase alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) decreases N6-methyladenylate methylation (m6 A) modification of RNA, while its role in regulating RILF pathogenesis and HCC radiosensitivity remains unknown.
METHODS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-sequencing (RNA-seq) were used to screen target genes regulated by ALKBH5. HSC with altered ALKBH5 expression was used to assess irradiation-induced HSC activation and the effect of HSC on recruitment and polarisation of monocytes. Key cytokines in medium from irradiated HSC-educated monocytes were identified by cytokine array detection. The effects of blocking ALKBH5 and key cytokines on RILF and HCC radiosensitivity were also evaluated.
RESULTS: Radiation-induced ALKBH5 expression in HSC mediated m6 A demethylation of toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) mRNA and activated its downstream NF-κB and JNK/Smad2 pathways to promote HSC activation. Additionally, ALKBH5 regulated CCL5 secretion by irradiated HSC to promote monocyte recruitment and M2 macrophage polarisation. Notably, polarised monocytes secreted CCL20 to up-regulate ALKBH5 expression in HSC, and reduce HCC radiosensitivity by activating ALKBH5/TIRAP axis in HCC cells. ALKBH5 knockdown-combined CCR6 (CCL20 receptor) inhibitor significantly alleviated RILF and improved HCC radiosensitivity in mice. HCC patients with high ALKBH5 and TIRAP expression were prone to radiation-induced liver injury and poor tumour response to radiotherapy.
CONCLUSIONS: Collectively, irradiation up-regulates ALKBH5 in HSC to mediate monocyte recruitment and M2 polarisation and form positive feedback to promote RILF and reduce HCC radiosensitivity. The dual roles of ALKBH5 as a microenvironmental regulator and radiosensitisation target provide new ideas for RILF prevention and radiosensitisation of HCC.

Keywords:  N6-methyladenylate methylation; hepatic stellate cells; monocytes; radiation-induced liver injury; radiotherapy

DOI:  https://doi.org/10.1002/ctm2.1198
Biochem Pharmacol. 2023 Feb 11. pii: S0006-2952(23)00047-3. [Epub ahead of print] 115456

YTHDC1 maintains trophoblasts function by promoting degradation of mA6-modified circMPP1.

Dan Wang, Hongbo Guan, Yajun Xia.

  N6-methyladenosine (m6A) is the most abundant mRNA internal modification in eukaryotic mRNAs. This study focuses on the effect of circMPP1 on placental villi function and the molecular mechanism. First, differentially expressed circular RNAs (circRNAs) in placenta tissues of large-for-gestational-age(LGA) neonates were screened by m6A-circRNA Epitranscriptomic Microarray and bioinformatics analyses. The abnormal expression of circMPP1 in placental tissues and cell lines was validated by RT-qPCR. In-vitro and in-vivo functional experiments were performed to evaluate the role of circMPP1 in placental impairment and fetal dysplasia. The interacting proteins of circMPP1 were identified and validated using RNA pull-down, RNA immunoprecipitation, fluorescence in situ hybridization, and immunofluorescence experiments. Protein interactions and expression levels were detected by Co-immunoprecipitation and western blot analysis. The m6A modification in circMPP1 was verified by methylated RNA immunoprecipitation assay. Bioinformatics analyses showed that circMPP1 was highly expressed in tissues with disordered placental function. In-vitro and in-vivo functional experiments showed that circMPP1 inhibited the function of placental villi. Further mechanism analyses showed that circMPP1 activated the NF-kappa B and MAPK3 signaling pathways. In addition, the m6A "reader" protein YTHDC1 was found to reduce circMPP1 expression via m6A modification. In conclusion, this study demonstrates that YTHDC1 maintains trophoblasts function by promoting degradation of m6A-mediated circMPP1.

Keywords:  Large-for-gestational-age; MAPK; NF-kappa B; YTHDC1; circMPP1; m6A

DOI:  https://doi.org/10.1016/j.bcp.2023.115456
bioRxiv. 2023 Feb 01. pii: 2023.01.30.526234. [Epub ahead of print]

m 6 A RNA methylation orchestrates transcriptional dormancy during developmental pausing.

Evelyne Collignon, Brandon Cho, Julie Fothergill-Robinson, Giacomo Furlan, Robert L Ross, Patrick A Limbach, Miguel Ramalho-Santos.

Embryos across metazoan lineages can enter reversible states of developmental pausing, or diapause, in response to adverse environmental conditions. The molecular mechanisms that underlie this remarkable dormant state remain largely unknown. Here we show that m 6 A RNA methylation by Mettl3 is required for developmental pausing in mice by maintaining dormancy of paused embryonic stem cells and blastocysts. Mettl3 enforces transcriptional dormancy via two interconnected mechanisms: i) it promotes global mRNA destabilization and ii) suppresses global nascent transcription by specifically destabilizing the mRNA of the transcriptional amplifier and oncogene N-Myc, which we identify as a critical anti-pausing factor. Our findings reveal Mettl3 as a key orchestrator of the crosstalk between transcriptomic and epitranscriptomic regulation during pausing, with implications for dormancy in stem cells and cancer.

DOI: https://doi.org/10.1101/2023.01.30.526234
Respir Res. 2023 Feb 14. 24(1): 50

Decreased ubiquitin modifying enzyme A20 associated with hyper-responsiveness to ovalbumin challenge following intrauterine growth restriction.

Xuefeng Xu, Fei Zheng, Shanshan Xu, Minfei Hu, Chengcheng Hang, Lingke Liu, Chencong Shen, Weizhong Gu, Lizhong Du.

   BACKGROUND: Intrauterine growth restriction (IUGR) is strongly correlated with an increased risk of asthma later in life. Farm dust protects mice from developing house dust mite-induced asthma, and loss of ubiquitin modifying enzyme A20 in lung epithelium would abolish this protective effect. However, the mechanisms of A20 in the development of asthma following IUGR remains unknown.
METHODS: An IUGR rat model induced by maternal nutrient restriction was used for investigating the role of A20 in the response characteristics of IUGR rats to ovalbumin (OVA) challenge. The ubiquitination of proteins and N6-methyladenosine (m6A) modifications were used to further assess the potential mechanism of A20.
RESULTS: IUGR can reduce the expression of A20 protein in lung tissue of newborn rats and continue until 10 weeks after birth. OVA challenging can increase the expression of A20 protein in lung tissue of IUGR rats, but its level was still significantly lower than the control OVA group. The differentially ubiquitinated proteins in lung tissues were also observed in IUGR and normal newborn rats. Furthermore, this ubiquitination phenomenon continued from the newborn to adulthood. In the detected RNA methylations, m6A abundance of the motif GGACA was the highest. The higher abundances of m6A modification of A20 mRNA from IUGR were negatively correlated with the trend of A20 protein levels.
CONCLUSION: These findings indicate A20 as a key regulator during the development of asthma following IUGR, providing further insight into the prevention of asthma induced by environmental factors.

Keywords:  A20; Asthma; Intrauterine growth restriction; RNA modification; Ubiquitination

DOI:  https://doi.org/10.1186/s12931-023-02360-2
Front Genet. 2023 ;14 1113515

N6-methyladenine regulator-mediated RNA methylation modification patterns in immune microenvironment regulation of osteoarthritis.

Yong Gu, Zhengming Wang, Rui Wang, Yunshang Yang, Peijian Tong, Shuaijie Lv, Long Xiao, Zhirong Wang.

  Background: Osteoarthritis is a common chronic degenerative disease, and recently, an increasing number of studies have shown that immunity plays an important role in the progression of osteoarthritis, which is exacerbated by local inflammation. The role of N6-methyladenine (m6A) modification in immunity is being explored. However, the role of m6A modification in regulating the immune microenvironment of osteoarthritis remains unknown. In this study, we sought to discuss the association between the N6-methyladenine (m6A) modification and the immune microenvironment of osteoarthritis. Methods: First, the data and gene expression profiles of 139 samples, including 33 healthy samples and 106 osteoarthritis samples, were obtained from the Genetics osteoARthritis and Progression (GARP) study. Then the differences in m6A regulators between healthy individuals and osteoarthritis patients were analyzed. The correlation between m6A regulators and immune characteristics was also investigated by single-sample gene set enrichment analysis (ssGSEA). Principal component analysis (PCA), Gene Set Variation Analysis (GSVA) enrichment analysis, weighted gene coexpression network analysis (WGCNA), and Associated R packages were used to identify the m6A phenotype and its biological functions. Results: A total of 23 m6A regulators were involved in this study. We found a close correlation between most m6A regulators in all samples as well as in osteoarthritis samples. VIRMA and LRPPRC were the most highly correlated m6A regulators and showed a positive correlation, whereas VIRMA and RBM15B were the most negatively correlated. M6A regulators are associated with osteoarthritis immune characteristics. For example, MDSC cell abundance was strongly correlated with RBM15B and HNRNPC. Meanwhile, RBM15B and HNRNPC were important effectors of natural killer cell immune responses. IGFBP3 is an important regulator of cytolytic activity immune function. We performed an unsupervised consensus cluster analysis of the osteoarthritis samples based on the expression of 23 m6A regulators. Three different m6A subtypes of osteoarthritis were identified, including 27 samples in subtype C1, 21 samples in subtype C2, and 58 samples in subtype C3. Different m6A subtypes have unique biological pathways and play different roles in the immune microenvironment of osteoarthritis. Conclusion: The m6A modification plays a crucial role in the diversity and complexity of the immune microenvironment in osteoarthritis.

Keywords:  N6-methyladenine (m6A); immune; methylation; microenvironment; osteoarthritis

DOI:  https://doi.org/10.3389/fgene.2023.1113515
Genomics Proteomics Bioinformatics. 2023 Feb 10. pii: S1672-0229(23)00034-7. [Epub ahead of print]

Identifying RNA Modifications by Direct RNA Sequencing Reveals Complexity of Epitranscriptomic Dynamics in Rice.

Feng Yu, Huanhuan Qi, Li Gao, Sen Luo, Rebecca Njeri Damaris, Yinggen Ke, Wenhua Wu, Pingfang Yang.

  Transcriptome analysis based on high-throughput sequencing of a cDNA library has been widely applied for functional genomic studies. However, the cDNA dependence of most RNA sequencing techniques constrains their ability to detect base modifications on RNA, which is an important element for the post-transcriptional regulation of gene expression. To comprehensively profile the N6-methyladenosine (m6A) and N5-methylcytosine (m5C) modifications on RNA, direct RNA sequencing (DRS) using the latest Oxford Nanopore Technology was applied to analyze the transcriptome of six tissues in rice. Approximately 94 million reads were generated, with an average length ranging from 619 to 1013 nt, and a total of 45,707 transcripts across 34,763 genes were detected. Expression profiles of transcripts at the isoform level were quantified among tissues. Transcriptome-wide mapping of m6A and m5C demonstrated that both modifications exhibited tissue-specific characteristics. The transcripts with m6A modification tended to be modified by m5C, and the transcripts with modifications presented higher expression levels along with shorter poly(A) tail than transcripts without modification, suggesting the complexity of gene expression regulation. Gene ontology analysis demonstrated that m6A- and m5C-modified transcripts are involved in central metabolic pathways related to the life cycle, with modifications on the target genes selected in a tissue-specific manner. Furthermore, most modified sites were located within quantitative trait loci that control important agronomic traits, highlighting the value of cloning functional loci. The results provide new insights into the expression regulation complexity and data resource of the transcriptome and epitranscriptome, improving our understanding of the rice genome.

Keywords:  Direct RNA resequencing; N(5)-methylcytosine; N(6)-methyladenosine; Polyadenylated transcriptome; Rice

DOI:  https://doi.org/10.1016/j.gpb.2023.02.002
Genome Res. 2023 Feb 14. pii: gr.276696.122. [Epub ahead of print]

Evaluation of N6-methyldeoxyadenosine antibody-based genomic profiling in eukaryotes.

Brian M Debo, Benjamin J Mallory, Andrew B Stergachis.

Low-level DNA N6-methyldeoxyadenosine (DNA-m6A) has recently been reported across various eukaryotes. Although anti-m6A antibody-based approaches are commonly used to measure DNA-m6A levels, this approach is known to be confounded by DNA secondary structures, RNA contamination, and bacterial contamination. To evaluate for these confounding features, we introduce an approach for systematically validating the selectivity of antibody-based DNA-m6A methods and use a highly selective anti-DNA-m6A antibody to reexamine patterns of DNA-m6A in C. reinhardtii, A. thaliana, and D. melanogaster Our findings raise caution about the use of antibody-based methods for endogenous m6A quantification and mapping in eukaryotes.

DOI: https://doi.org/10.1101/gr.276696.122
Am J Cancer Res. 2023 ;13(1): 276-292

DOT1L promotes cell proliferation and invasion by epigenetically regulating STAT5B in renal cell carcinoma.

Yanguang Hou, Shiyu Huang, Jiachen Liu, Lei Wang, Yan Yuan, Hao Liu, Xiaodong Weng, Zhiyuan Chen, Juncheng Hu, Xiuheng Liu.

DOT1L, the only histone H3 lysine 79 methyltransferase, has a prominent effect on promoting the progression of various malignancies, yet the functional contribution of DOT1L to renal cell carcinoma (RCC) progression remains unclear. DOT1L is overexpressed in RCC and linked to poor clinical outcomes. Chemical (SGC0946) or genetic suppression of DOT1L attenuates the growth and invasion of renal cancer cells and results in S-phase arrest. STAT5B expression was suppressed after DOT1L knockdown, and STAT5B overexpression rescued the DOT1L silencing-induced decrease in cell proliferation. DOT1L was found to epigenetically promote the transcription of STAT5B via H3K79me2, and CDK6 acted as a downstream effector of STAT5B to mediate cell cycle arrest. Our study confirmed that DOT1L promotes STAT5B expression in a histone methyltransferase-dependent manner. Downregulation of DOT1L inhibited RCC proliferation and invasion. Thus, targeting DOT1L might be a potential therapeutic intervention for RCC.

Keywords: CDK6; DOT1L; Renal cell carcinoma; STAT5B; histone methylation
Pediatr Surg Int. 2023 Feb 15. 39(1): 126

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

Zhaorong Huang, Caiyun Luo, Xinwei Hou, Daiyue Yu, Yuqian Su, Xinxin Li, Yinyan Luo, Guoying Liao, Jianhua Mu, Kai Wu.

   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.

Keywords:  Hirschsprung disease (HSCR); METTL3; Methyltransferase; Neural crest cells; YAP; m6A

DOI:  https://doi.org/10.1007/s00383-023-05421-1
J Gene Med. 2023 Feb 13. e3481

Methyltransferase-like 3 modulates osteogenic differentiation of adipose-derived stem cells in osteoporotic rats.

Daowen Luo, Shuanglin Peng, Qing Li, Pengcheng Rao, Gang Tao, Lang Wang, Jingang Xiao.

   BACKGROUND: Osteoporosis (OP) is a metabolic bone disease involving reduced bone mass. Adipose-derived stem cells (ASCs) play an important role in bone regeneration. Emerging evidence suggests that Methyltransferase-like 3(METTL3) plays a significant role in bone development and metabolism. Therefore, this study investigates changes to METTL3 in the osteogenic differentiation of adipose stem cells in osteoporotic rats (OP-ASCs) and explores ways to enhance their osteogenic ability.
METHODS: An animal model of osteoporosis was established by removing both ovaries in rats (OVX). Real-time PCR (qPCR) and western blotting (WB) were performed to detect the expression of METTL3 and bone-related molecules, including runt-related transcription factor 2(Runx2) and osteopontin (Opn). Furthermore, ALP staining was used to confirm the osteogenic potential of stem cells. Mettl3 small interfering RNA (siRNA) and Mettl3 overexpression lentivirus were used to assess the role of METTL3 in osteogenic differentiation of ASCs and OP-ASCs.
RESULTS: The osteogenic differentiation capacity and Mettl3 expression significantly decreased in OP-ASCs. Moreover, Mettl3 silencing down-regulated the osteogenic ability of ASCs, and overexpression of Mettl3 recovered the impaired osteogenic capacity in OP-ASCs invitro.
CONCLUSION: The Mettl3 expression levels and osteogenic potential of OP-ASCs decreased. However, overexpression of METTL3 rescued osteogenic ability of OP-ASCs, providing a new target for treatment of osteoporotic bone defects.

Keywords:  Adipose-derived stem cells; METTL3; Osteogenic differentiation; Osteoporosis

DOI:  https://doi.org/10.1002/jgm.3481
Genomics Proteomics Bioinformatics. 2023 Feb 13. pii: S1672-0229(23)00035-9. [Epub ahead of print]

RNA Methylome Reveals the m6A-mediated Regulation of Flavor Metabolites in Tea Leaves under Solar-withering.

Chen Zhu, Shuting Zhang, Chengzhe Zhou, Caiyun Tian, Biying Shi, Kai Xu, Linjie Huang, Yun Sun, Yuling Lin, Zhongxiong Lai, Yuqiong Guo.

  The epitranscriptomic mark N6-methyladenosine (m6A), which is the predominant internal modification in RNAs, is important for plant responses to diverse stresses. Multiple environmental stresses caused by the tea-withering process can greatly influence the accumulation of specialized metabolites and the formation of tea flavor. However, the effects of the m6A-mediated regulatory mechanism on flavor-related metabolic pathways in tea leaves remain relatively uncharacterized. We performed an integrated RNA methylome and transcriptome analysis to explore the m6A-mediated regulatory mechanism and its effects on flavonoid and terpenoid metabolism under solar-withering conditions. Dynamic changes in global m6A levels in tea leaves were mainly controlled by two m6A erasers (CsALKBH4A and CsALKBH4B) during solar-withering treatments. Differentially methylated peak-associated genes following solar-withering treatments with different shading rates were assigned to terpenoid biosynthesis and spliceosome pathways. Further analyses indicated that CsALKBH4-driven RNA demethylations can directly affect the accumulation of volatile terpenoids by mediating the stability and abundance of terpenoid biosynthesis-related transcripts and also indirectly influence the flavonoid, catechin, and theaflavin contents by triggering alternative splicing-mediated regulation. Our findings revealed a novel layer of epitranscriptomic gene regulation in tea flavor-related metabolic pathways and established a link between the m6A-mediated regulatory mechanism and the formation of tea flavor under solar-withering conditions.

Keywords:  Camellia sinensis; Epitranscriptome; RNA methylation; Secondary metabolite; Withering

DOI:  https://doi.org/10.1016/j.gpb.2023.02.003
Nat Commun. 2023 Feb 16. 14(1): 863

Nsun2 coupling with RoRγt shapes the fate of Th17 cells and promotes colitis.

Wen-Lan Yang, Weinan Qiu, Ting Zhang, Kai Xu, Zi-Juan Gu, Yu Zhou, Heng-Ji Xu, Zhong-Zhou Yang, Bin Shen, Yong-Liang Zhao, Qi Zhou, Ying Yang, Wei Li, Peng-Yuan Yang, Yun-Gui Yang.

T helper 17 (Th17) cells are a subset of CD4+ T helper cells involved in the inflammatory response in autoimmunity. Th17 cells secrete Th17 specific cytokines, such as IL-17A and IL17-F, which are governed by the master transcription factor RoRγt. However, the epigenetic mechanism regulating Th17 cell function is still not fully understood. Here, we reveal that deletion of RNA 5-methylcytosine (m5C) methyltransferase Nsun2 in mouse CD4+ T cells specifically inhibits Th17 cell differentiation and alleviates Th17 cell-induced colitis pathogenesis. Mechanistically, RoRγt can recruit Nsun2 to chromatin regions of their targets, including Il17a and Il17f, leading to the transcription-coupled m5C formation and consequently enhanced mRNA stability. Our study demonstrates a m5C mediated cell intrinsic function in Th17 cells and suggests Nsun2 as a potential therapeutic target for autoimmune disease.

DOI: https://doi.org/10.1038/s41467-023-36595-w
FASEB J. 2023 03;37(3): e22802

Transcriptome-wide high-throughput m6 A sequencing of differential m6 A methylation patterns in the decidual tissues from RSA patients.

Yong Luo, Jin Chen, Ying Cui, Fang Fang, Ziyu Zhang, Lili Hu, Xiaoyong Chen, Zengming Li, Liping Li, Lina Chen.

  Recurrent spontaneous abortion (RSA) is characterized by two or more consecutive pregnancy losses in the first trimester of pregnancy, experienced by 5% of women during their reproductive age. As a complex pathological process, the etiology of RSA remains poorly understood. Recent studies have established that gene expression changes dramatically in human endometrial stromal cells (ESCs) during decidualization. N6-methyladenosine (m6 A) modification is the most prevalent epigenetic modification of mRNA in eukaryotic cells and it is closely related to the occurrence and development of many pathophysiological phenomena. In this study, we first confirmed that high levels of m6 A mRNA methylation in decidual tissues are associated with RSA. Then, we used m6 A-modified RNA immunoprecipitation sequence (m6 A-seq) and RNA sequence (RNA-seq) to identify the differentially expressed m6 A methylation in decidual tissues from RSA patients and identified the key genes involved in abnormal decidualization by bioinformatics analysis. Using m6 A-seq, we identified a total of 2169 genes with differentially expressed m6 A methylation, of which 735 m6 A hypermethylated genes and 1434 m6 A hypomethylated genes were identified. Further joint analysis of m6 A-seq and RNA-seq revealed that 133 genes were m6 A modified with mRNA expression. GO and KEGG analyses indicated that these unique genes were mainly enriched in environmental information processing pathways, including the cytokine-cytokine receptor interaction and PI3K-Akt signaling pathway. In summary, this study uncovered the transcriptome-wide m6 A modification pattern in decidual tissue of RSA, which provides a theoretical basis for further research into m6 A modification and new therapeutic strategies for RSA.

Keywords:  RNA-seq; decidualization; m6A methylation; m6A-seq; recurrent spontaneous abortion

DOI:  https://doi.org/10.1096/fj.202201232RRRR
EMBO Rep. 2023 Feb 16. e56325

METTL14 modulates glycolysis to inhibit colorectal tumorigenesis in p53-wild-type cells.

Yichao Hou, Xintian Zhang, Han Yao, Lidan Hou, Qingwei Zhang, Enwei Tao, Xiaoqiang Zhu, Shanshan Jiang, Yimeng Ren, Xialu Hong, Shiyuan Lu, Xiaoxu Leng, Yile Xie, Yaqi Gao, Yu Liang, Ting Zhong, Bohan Long, Jing-Yuan Fang, Xiangjun Meng.

  The frequency of p53 mutations in colorectal cancer (CRC) is approximately 40-50%. A variety of therapies are being developed to target tumors expressing mutant p53. However, potential therapeutic targets for CRC expressing wild-type p53 are rare. In this study, we show that METTL14 is transcriptionally activated by wild-type p53 and suppresses tumor growth only in p53-wild-type (p53-WT) CRC cells. METTL14 deletion promotes both AOM/DSS and AOM-induced CRC growth in mouse models with the intestinal epithelial cell-specific knockout of METTL14. Additionally, METTL14 restrains aerobic glycolysis in p53-WT CRC, by repressing SLC2A3 and PGAM1 expression via selectively promoting m6 A-YTHDF2-dependent pri-miR-6769b/pri-miR-499a processing. Biosynthetic mature miR-6769b-3p and miR-499a-3p decrease SLC2A3 and PGAM1 levels, respectively, and suppress malignant phenotypes. Clinically, METTL14 only acts as a beneficial prognosis factor for the overall survival of p53-WT CRC patients. These results uncover a new mechanism for METTL14 inactivation in tumors and, most importantly, reveal that the activation of METTL14 is a critical mechanism for p53-dependent cancer growth inhibition, which could be targeted for therapy in p53-WT CRC.

Keywords:  METTL14; aerobic glycolysis; colorectal cancer; m6A; wild-type p53

DOI:  https://doi.org/10.15252/embr.202256325
Int J Med Sci. 2023 ;20(2): 254-261

m6A modifications of circular RNAs in ischemia-induced retinal neovascularization.

Yedi Zhou, Bingyan Li, Zicong Wang, Wei Tan, Jingling Zou, Haixiang Zhou, Yuting Cai, Jie Liu, Yan He, Shigeo Yoshida, Yun Li.

  Ischemia-induced pathological neovascularization in the retina is a leading cause of blindness in various age groups. The purpose of the current study was to identify the involvement of circular RNAs (circRNAs) methylated by N6-methyladenosine (m6A), and predict their potential roles in oxygen-induced retinopathy (OIR) in mice. Methylation assessment via microarray analysis indicated that 88 circRNAs were differentially modified by m6A methylation, including 56 hyper-methylated circRNAs and 32 hypo-methylated circRNAs. Gene ontology enrichment analysis predicted that the enriched host genes of the hyper-methylated circRNAs were involved in cellular process, cellular anatomical entity, and protein binding. Host genes of the hypo-methylated circRNAs were enriched in the regulation of cellular biosynthetic process, the nucleus, and binding. According to the Kyoto Encyclopedia of Genes and Genomes analysis, those host genes were involved in the pathways of selenocompound metabolism, salivary secretion, and lysine degradation. MeRIP-qPCR verified significant alterations in m6A methylation levels of mmu_circRNA_33363, mmu_circRNA_002816, and mmu_circRNA_009692. In conclusion, the study revealed the m6A modification alterations in OIR retinas, and the findings above shed light on the potential roles of m6A methylation in circRNA regulatory functions in the pathogenesis of ischemia-induced pathological retinal neovascularization.

Keywords:  RNA methylation; circRNA; m6A; oxygen-induced retinopathy; retinal neovascularization

DOI:  https://doi.org/10.7150/ijms.79409