bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2022‒10‒30
fifty-one papers selected by
Sk Ramiz Islam
Saha Institute of Nuclear Physics
  1. Senescent neutrophils-derived exosomal piRNA-17560 promotes chemoresistance and EMT of breast cancer via FTO-mediated m6A demethylation.
  2. The Role of m6A Modification and m6A Regulators in Esophageal Cancer.
  3. Function and Prognostic Value of N6-Methyladenosine-modified RNAs in Lung Adenocarcinoma.
  4. Reduction of Methyltransferase-like 3-Mediated RNA N6-Methyladenosine Exacerbates the Development of Psoriasis Vulgaris in Imiquimod-Induced Psoriasis-like Mouse Model.
  5. Research progress of m6A methylation in prostate cancer.
  6. The feedback loop of METTL14 and USP38 regulates cell migration, invasion and EMT as well as metastasis in bladder cancer.
  7. N6-methyladenosine demethylase FTO enhances chemo-resistance in colorectal cancer through SIVA1-mediated apoptosis.
  8. Interplay between the m6A Epitranscriptome and Tumor Metabolism: Mechanisms and Therapeutic Implications.
  9. m6A Methyltransferase KIAA1429 Regulates the Cisplatin Sensitivity of Gastric Cancer Cells via Stabilizing FOXM1 mRNA.
  10. PRKAA1, stabilized by FTO in an m6A-YTHDF2-dependent manner, promotes cell proliferation and glycolysis of gastric cancer by regulating the redox balance.
  11. The m6A reader IGF2BP2 regulates glutamine metabolism and represents a therapeutic target in acute myeloid leukemia.
  12. Emerging role of N6-methyladenosine RNA methylation in lung diseases.
  13. MTDeepM6A-2S: A two-stage multi-task deep learning method for predicting RNA N6-methyladenosine sites of Saccharomyces cerevisiae.
  14. The Role of N6-Methyladenosine Modification in Microvascular Dysfunction.
  15. [Cluster classification and clinical prognostic modeling based on m6A RNA methylation regulators in liver cancer].
  16. LncRNA FENDRR with m6A RNA methylation regulates hypoxia-induced pulmonary artery endothelial cell pyroptosis by mediating DRP1 DNA methylation.
  17. WTAP mediates the anti-inflammatory effect of Astragalus mongholicus polysaccharide on THP-1 macrophages.
  18. M6A RNA Methylation Mediates NOD1/NF-kB Signaling Activation in the Liver of Piglets Challenged with Lipopolysaccharide.
  19. Simultaneous Determination of Methylated Nucleosides by HILIC-MS/MS Revealed Their Alterations in Urine from Breast Cancer Patients.
  20. [Research advances on the effects of RNA N6-methyladenosine modification in the relevant pathophysiological processes of wound repair].
  21. METTL3 acetylation impedes cancer metastasis via fine-tuning its nuclear and cytosolic functions.
  22. m6A modification confers thermal vulnerability to HPV E7 oncotranscripts via reverse regulation of its reader protein IGF2BP1 upon heat stress.
  23. Development and validation of a m6A -regulated prognostic signature in lung adenocarcinoma.
  24. METTL3-IGF2BP3-axis mediates the proliferation and migration of pancreatic cancer by regulating spermine synthase m6A modification.
  25. Mettl14 mutation restrains liver regeneration by attenuating mitogens derived from non-parenchymal liver cells.
  26. Identification of a novel prognostic signature correlated with epithelial-mesenchymal transition, N6-methyladenosine modification, and immune infiltration in colorectal cancer.
  27. Insights into the mechanism underlying crystalline silica-induced pulmonary fibrosis via transcriptome-wide m6A methylation profile.
  28. Effects of Moderate-Intensity Continuous Training and High-Intensity Interval Training on Testicular Oxidative Stress, Apoptosis and m6A Methylation in Obese Male Mice.
  29. IGF2BP2 serves as a core m6A regulator in head and neck squamous cell carcinoma.
  30. Downregulation of the m6A reader protein YTHDC1 leads to islet β-cell failure and diabetes.
  31. Acetaminophen changes the RNA m6A levels and m6A-related proteins expression in IL-1β-treated chondrocyte cells.
  32. The N6-methyladenosine methyltransferase METTL16 enables erythropoiesis through safeguarding genome integrity.
  33. Absolute quantification of single-base m6A methylation in the mammalian transcriptome using GLORI.
  34. Modulation of DNA/RNA Methylation by Small-Molecule Modulators and Their Implications in Cancer.
  35. The blue light receptor CRY1 interacts with FIP37 to promote N6-methyladenosine RNA modification and photomorphogenesis in Arabidopsis.
  36. Feedforward loop between IMP1 and YAP/TAZ promotes tumorigenesis and malignant progression in glioblastoma.
  37. Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution.
  38. DNA methylation regulator-mediated modification pattern defines tumor microenvironment immune infiltration landscape in colon cancer.
  39. Modulation of DNA/RNA Methylation Signaling Mediating Metabolic Homeostasis in Cancer.
  40. METTL3-modified lncRNA-SNHG8 binds to PTBP1 to regulate ALAS2 expression to increase oxidative stress and promote myocardial infarction.
  41. Identification of N7-methylguanosine related subtypes and construction of prognostic model in gastric cancer.
  42. Targeting tumour-intrinsic N7-methylguanosine tRNA modification inhibits MDSC recruitment and improves anti-PD-1 efficacy.
  43. RM2Target: a comprehensive database for targets of writers, erasers and readers of RNA modifications.
  44. METTL3 regulates m6A methylation of PTCH1 and GLI2 in Sonic hedgehog signaling to promote tumor progression in SHH-medulloblastoma.
  45. ADAR regulates APOL1 via A-to-I RNA editing by inhibition of MDA5 activation in a paradoxical biological circuit.
  46. Genome-Wide DNA Methylation and Gene Expression Profiling Characterizes Molecular Subtypes of Esophagus Squamous Cell Carcinoma for Predicting Patient Survival and Immunotherapy Efficacy.
  47. Comprehensive analysis of key m5C modification-related genes in type 2 diabetes.
  48. OTUB2 exerts tumor-suppressive roles via STAT1-mediated CALML3 activation and increased phosphatidylserine synthesis.
  49. ECE2 is a prognostic biomarker associated with m6A modification and involved in immune infiltration of lung adenocarcinoma.
  50. Single-molecule identification of the target RNAs of different RNA binding proteins simultaneously in cells.
  51. Locally correlated kinetics of post-replication DNA methylation reveals processivity and region specificity in DNA methylation maintenance.
  1. Cell Death Dis. 2022 Oct 27. 13(10): 905
    Senescent neutrophils-derived exosomal piRNA-17560 promotes chemoresistance and EMT of breast cancer via FTO-mediated m6A demethylation.
    Baochi Ou, Yuan Liu, Zongxuan Gao, Jun Xu, Yunwen Yan, Yongxiang Li, Jingjie Zhang.
      Cellular senescence is characterized by a tumor-suppressive program as well as a pro-inflammatory secretome. Neutrophils constitute significant compositions of malignancies and play key roles in tumor development. However, the role of senescent neutrophils in cancer progression is presently unexplored. Here, we demonstrate that neutrophils display enhanced senescence in breast cancer patients receiving chemotherapy. The senescent neutrophils produce increased number of exosomes, which confer drug resistance to tumor cells in vitro and in vivo. Mechanistically, senescent neutrophils-derived exosomal piRNA-17560 enhances the expression of fat mass and obesity-associated protein (FTO) in breast cancer cells. The upregulation of FTO further strengthens ZEB1 transcripts stability and expression by decreasing N6-methyladenosine (m6A) RNA methylation, leading to chemoresistance and epithelial-mesenchymal transition (EMT) of tumor cells. Clinically, the level of exosomal piR-17560 correlates with poor chemotherapy response in patients with breast cancer. In addition, YTHDF2 is essential for the posttranscriptional regulation of ZEB1 by piRNA-17560/FTO signaling. Senescent neutrophils secret exosomal piR-17560 in a STAT3-dependent manner. Altogether, this study suggests that senescent neutrophils-derived exosomal piR-17560 confers chemoresistance to tumor cells and senescent neutrophils may serve as a potential therapeutic target in breast cancer.
    DOI:  https://doi.org/10.1038/s41419-022-05317-3
  2. Cancers (Basel). 2022 Oct 20. pii: 5139. [Epub ahead of print]14(20):
    The Role of m6A Modification and m6A Regulators in Esophageal Cancer.
    Yuekao Li, Chaoxu Niu, Na Wang, Xi Huang, Shiru Cao, Saijin Cui, Tianyu Chen, Xiangran Huo, Rongmiao Zhou.
      N6-methyladenosine (m6A) modification, the most prevalent RNA modification, is involved in all aspects of RNA metabolism, including RNA processing, nuclear export, stability, translation and degradation. Therefore, m6A modification can participate in various physiological functions, such as tissue development, heat shock response, DNA damage response, circadian clock control and even in carcinogenesis through regulating the expression or structure of the gene. The deposition, removal and recognition of m6A are carried out by methyltransferases, demethylases and m6A RNA binding proteins, respectively. Aberrant m6A modification and the dysregulation of m6A regulators play critical roles in the occurrence and development of various cancers. The pathogenesis of esophageal cancer (ESCA) remains unclear and the five-year survival rate of advanced ESCA patients is still dismal. Here, we systematically reviewed the recent studies of m6A modification and m6A regulators in ESCA and comprehensively analyzed the role and possible mechanism of m6A modification and m6A regulators in the occurrence, progression, remedy and prognosis of ESCA. Defining the effect of m6A modification and m6A regulators in ESCA might be helpful for determining the pathogenesis of ESCA and providing some ideas for an early diagnosis, individualized treatment and improved prognosis of ESCA patients.
    Keywords:  esophageal cancer; m6A modification; m6A regulators; occurrence; prognosis; progression; treatment
    DOI:  https://doi.org/10.3390/cancers14205139
  3. J Gene Med. 2022 Oct 25. e3454
    Function and Prognostic Value of N6-Methyladenosine-modified RNAs in Lung Adenocarcinoma.
    Jiayuan Liu, Zhi Zheng, Fang Tian, Jinlong Zhong.
      Aberrant regulation of N6-methyladenosine (m6A) modification is reportedly vital for cancer progression, including lung adenocarcinoma (LUAD). However, current studies mainly focus on the function and mechanism of m6A-modified regulators, such as m6A writers (METTL3 and METTL14), erasers (ALKBH5 and FTO), and readers (YTHDF1 and YTHDF2). The landscape, function, and prognostic value of RNAs by m6A-modified have not been fully clarified until now. Hence, the present study identified 57 RNAs with significantly different m6A-methylation levels in LUAD tissues using epitranscriptomic microarray analysis. Among the 57 RNAs, 28 and 29 were hypermethylated and hypomethylated, respectively. The m6A-methylation level increased in mRNA and lncRNA but decreased in sRNA. After pathway enrichment analyses, RNA metabolism-associated pathways such as nucleotide metabolism were enriched in total and m6A-hypermethylated mRNAs. Further, lncRNA networks were built using miRNet tools, revealing that the immune system was closed to m6A-modified lncRNAs. To evaluate the prognostic value of mRNAs with hypermethylated or hypomethylated, we calculated the risk scores, and constructed signatures to predict the survival time of patients with LUAD using multicox regression analysis. In addition, hypermethylated-mRNA and hypomethylated-mRNA signatures were established. The survival plotter showed that these two signatures effectively predicted the survival time of patients with LUAD. These results support the evidence for understanding the expression, function, and potential prognostic values of m6A-modified RNAs, possibly promoting effective therapies for patients with LUAD.
    Keywords:  N6-methyladenosine; biomarkers; lncRNAs; lung cancer; mRNA
    DOI:  https://doi.org/10.1002/jgm.3454
  4. Int J Mol Sci. 2022 Oct 21. pii: 12672. [Epub ahead of print]23(20):
    Reduction of Methyltransferase-like 3-Mediated RNA N6-Methyladenosine Exacerbates the Development of Psoriasis Vulgaris in Imiquimod-Induced Psoriasis-like Mouse Model.
    Yanan Wang, Jiuzuo Huang, Hongzhong Jin.
      N6-methyladenosine (m6A) methylation is the most pervasive and intensively studied mRNA modification, which regulates gene expression in different physiological processes, such as cell proliferation, differentiation, and inflammation. Studies of aberrant m6A in human diseases such as cancer, obesity, infertility, neuronal disorders, immune diseases, and inflammation are rapidly evolving. However, the regulatory mechanism and physiological significance of m6A methylation in psoriasis vulgaris are still poorly understood. In this study, we found that m6A methylation and Methyltransferase-like 3 (METTL3) were both downregulated in psoriatic skin lesions and were negatively correlated with Psoriasis Area and Severity Index (PASI) scores. Inhibiting m6A methylation by knocking down Mettl3 promoted the development of psoriasis and increased its severity in imiquimod-induced psoriasis-like model mice. Our results indicate a critical role of METTL3- mediated m6A methylation in the pathogenesis of psoriasis vulgaris.
    Keywords:  METTL3; N6-methyladenosine; RNA modification; m6A methylation; psoriasis; psoriasis vulgaris
    DOI:  https://doi.org/10.3390/ijms232012672
  5. Asian J Androl. 2022 Oct 28.
    Research progress of m6A methylation in prostate cancer.
    Shou-Yi Zhang, Yu Zeng.
      N6-methyladenosine (m6A) is a ubiquitous RNA modification in mammals. This modification is "written" by methyltransferases and then "read" by m6A-binding proteins, followed by a series of regulation, such as alternative splicing, translation, RNA stability, and RNA translocation. At last, the modification is "erased" by demethylases. m6A modification is essential for normal physiological processes in mammals and is also a very important epigenetic modification in the development of cancer. In recent years, cancer-related m6A regulation has been widely studied, and various mechanisms of m6A regulation in cancer have also been recognized. In this review, we summarize the changes of m6A modification in prostate cancer and discuss the effect of m6A regulation on prostate cancer progression, aiming to profile the potential relevance between m6A regulation and prostate cancer development. Intensive studies on m6A regulation in prostate cancer may uncover the potential role of m6A methylation in the cancer diagnosis and cancer therapy.
    Keywords:  N6-methyladenosine methylation; epigenetic; molecular biology; prostate cancer
    DOI:  https://doi.org/10.4103/aja202265
  6. PLoS Genet. 2022 Oct;18(10): e1010366
    The feedback loop of METTL14 and USP38 regulates cell migration, invasion and EMT as well as metastasis in bladder cancer.
    Ji Huang, Weimin Zhou, Chao Hao, Qiuming He, Xinhua Tu.
      BACKGROUND: Bladder cancer (BCa) is one of the most prevalent malignancies globally. Previous study has reported the inhibitory effect of methyltransferase-like 14 (METTL14) on BCa tumorigenesis, but its role in the cell migration, invasion and epithelial-mesenchymal transition (EMT) in BCa remains unknown.MATERIALS AND METHODS: Quantitative real-time PCR (RT-qPCR) and western blot were applied to measure RNA and protein expression respectively. Cell migration, invasion and EMT were evaluated by wound healing, Transwell, and immunofluorescence (IF) assays as well as western blot of EMT-related proteins. In vivo experiments were performed to analyze metastasis of BCa. Mechanism investigation was also conducted to study METTL14-mediated regulation of BCa progression.
    RESULTS: METTL14 overexpression prohibits BCa cell migration, invasion in vitro and tumor metastasis in vivo. METTL14 stabilizes USP38 mRNA by inducing N6-methyladenosine (m6A) modification and enhances USP38 mRNA stability in YTHDF2-dependent manner. METTL14 represses BCa cell migration, invasion and EMT via USP38. Additionally, miR-3165 inhibits METTL14 expression to promote BCa progression.
    CONCLUSIONS: Our study demonstrated that METTL14 suppresses BCa progression and forms a feedback loop with USP38. In addition, miR-3165 down-regulates METTL14 expression to promote BCa progression. The findings may provide novel insight into the underlying mechanism of METTL14 in BCa progression.
    DOI:  https://doi.org/10.1371/journal.pgen.1010366
  7. Mol Ther. 2022 Oct 28. pii: S1525-0016(22)00624-4. [Epub ahead of print]
    N6-methyladenosine demethylase FTO enhances chemo-resistance in colorectal cancer through SIVA1-mediated apoptosis.
    Ziyou Lin, Arabella H Wan, Lei Sun, Heng Liang, Yi Niu, Yuan Deng, Shijia Yan, Qiao-Ping Wang, Xianzhang Bu, Xiaolei Zhang, Kunhua Hu, Guohui Wan, Weiling He.
      N6-methyladenosine (m6A) is the most pervasive RNA modification and is recognized as a novel epigenetic regulation in RNA metabolism. Although the m6A modification involves various physiological processes, its roles in drug-resistance in colorectal cancer (CRC) still remain unknown. We analyzed the RNA expression profile of m6A/A (%) with MRM mass spectrometry in human 5-fluorouracil (5-FU) resistant CRC tissues, and used the m6A RNA immunoprecipitation assay to validate the m6A-regulated target. Our results have shown that the m6A demethylase FTO was up-regulated in human primary and 5-FU resistant CRC. Depletion of FTO decreased cell growth, colony formation and metastasis in 5-FU resistant CRC cells in vitro and in vivo. Mechanistically, we identified SIVA1, a critical apoptotic gene, as a key downstream target of the FTO-mediated m6A demethylation. The m6A demethylation of SIVA1 at the CDS region induced its mRNA degradation via a YTHDF2-dependent mechanism. The SIVA1 levels were negatively correlated with the FTO levels in clinical CRC tissues. Notably, inhibition of FTO significantly reduced the tolerance of 5-FU in 5-FU-resistant CRC cells via the FTO-SIVA1 axis, whereas SIVA1-depletion could restore the m6A-dependent 5-FU sensitivity in CRC cells. In summary, our findings demonstrate a critical role of FTO as an m6A demethylase enhancing chemo-resistance in CRC cells, and suggest that FTO inhibition may restore the sensitivity of chemo-resistant CRC cells to 5-FU.
    Keywords:  Colorectal cancer; FTO; N6-methyladenosine; SIVA1; chemo-resistance
    DOI:  https://doi.org/10.1016/j.ymthe.2022.10.012
  8. Biomedicines. 2022 Oct 15. pii: 2589. [Epub ahead of print]10(10):
    Interplay between the m6A Epitranscriptome and Tumor Metabolism: Mechanisms and Therapeutic Implications.
    Asa Sergei Fong, Yasi Pan, Jun Yu, Chi Chun Wong.
      N6-methyladenosine (m6A) modification of messenger RNA (mRNA) influences the stability and translation of the transcripts into functional proteins. Recent studies reveal the role of m6A modifications in regulating the metabolism of basic biomolecules such as glucose, lipids and amino acids. Such mechanisms are not only important for physiological functions of normal cells but also prove to be pivotal for the pathogenesis of cancers by driving dysregulated metabolism. M6A writers, readers and erasers function co-operatively to promote aberrant glucose, lipid and amino acid metabolism in cancer cells, which in turn support increased proliferative and metastatic potential. Better understanding of the relationship between m6A and metabolism in malignancy may unravel novel therapeutic targets as well as biomarkers in cancer. In this review, we summarize the recent evidence demonstrating the interplay between m6A modification and cancer metabolism and their therapeutic implications.
    Keywords:  N6-methyladenosine; amino acid; cancer; glucose; lipid; metabolism
    DOI:  https://doi.org/10.3390/biomedicines10102589
  9. Cancers (Basel). 2022 Oct 14. pii: 5025. [Epub ahead of print]14(20):
    m6A Methyltransferase KIAA1429 Regulates the Cisplatin Sensitivity of Gastric Cancer Cells via Stabilizing FOXM1 mRNA.
    Zhongcheng Zhu, Yuan Zhou, Yongheng Chen, Zhongyi Zhou, Wenxue Liu, Linyi Zheng, Qian Pei, Fengbo Tan, Haiping Pei, Yuqiang Li.
      Although cisplatin is frequently used to treat gastric cancer, the resistance is the main obstacle for effective treatment. mRNA modification, N6-methyladenosine (m6A), is involved in the tumorigenesis of many types of cancer. As one of the largest m6A methyltransferase complex components, KIAA1429 bridges the catalytic m6A methyltransferase components, such as METTL3. In gastric cancer, KIAA1429 was reported to promote cell proliferation. However, whether KIAA1429 is involved in the resistance of gastric cancer to cisplatin remains unclear. Here, we generated cisplatin resistant gastric cancer cell lines, and compared the m6A content between resistant cells and wild type cells. The m6A content as well as KIAA1429 expression are higher in resistant cells. Interestingly, the expression of KIAA1429 was significantly increased after cisplatin treatment. We then used shRNA to knockdown KIAA1429 and found that resistant cells responded more to cisplatin treatment after KIAA1429 depletion, while overexpression of KIAA1429 decreased the sensitivity. Moreover, we identified a putative p65 binding site on the promoter area of KIAA1429 and ChIP assay confirmed the binding. p65 depletion decreased the expression of KIAA1429. YTHDF1 is the most abundant m6A "reader" that interacts with m6A modified mRNA. Mechanistically, YTHDF1 was recruited to the 3'-untranslated Region (3'-UTR) of transcriptional factor, FOXM1 by KIAA1429 and stabilized FOXM1 mRNA. More importantly, KIAA1429 knockdown increased the sensitivity of resistant cells to cisplatin in vivo. In conclusion, our results demonstrated that KIAA1429 facilitated cisplatin resistance by stabilizing FOXM1 mRNA in gastric cancer cells.
    Keywords:  YTHDF1; drug resistance; m6A modification; mRNA stability; p65
    DOI:  https://doi.org/10.3390/cancers14205025
  10. Neoplasma. 2022 Oct 28. pii: 220714N714. [Epub ahead of print]
    PRKAA1, stabilized by FTO in an m6A-YTHDF2-dependent manner, promotes cell proliferation and glycolysis of gastric cancer by regulating the redox balance.
    Yangmei Zhang, Xichang Zhou, Xue Cheng, Xiou Hong, Xiaowei Jiang, Guilong Jing, Kai Chen, Yang Li.
      Gastric carcinoma (GC) is the fourth most common malignancy worldwide and the second cause of death of all malignancies worldwide. AMPK catalytic subunit α1 (PRKAA1) is involved in various types of cancer and PRKAA1 knockdown significantly decreased the invasiveness of GC cells. Fat mass and obesity-associated protein (FTO)-regulation of m6A has been shown to be involved in different diseases including cancer. However, the role of PRKAA1 and m6A modification in GC remains to be elucidated. PRKAA1 was silenced or overexpressed to study the role of PRKAA1 in regulating cell viability, colony formation, and glycolysis. Levels of lactic acid, GSH, and NADP+/NADPH were measured using commercial kits. Extracellular acidification rates were determined by an extracellular flux analyzer. RNA immunoprecipitation was performed to test m6A levels and the interaction between PRKAA1-3'-UTR and YTHDF2. Quantitative RT-PCR and immunoblots were applied to measure mRNA or protein levels, respectively. PRKAA1 silencing inhibited cell growth, colony formation, and glycolysis but enhanced apoptosis, while the PRKAA1 overexpression promoted cell growth, colony formation, and glycolysis but inhibited apoptosis of GC cells. Data also indicated that PRKAA1 regulated cell growth and apoptosis by regulating the redox balance. Mechanism study demonstrated that FTO regulated PRKAA1 mRNA m6A modification and stability. Clinical samples analysis demonstrated that PRKAA1 and FTO expression were increased in GC patients and positively correlated with each other. FTO increased levels of PRKAA1 by regulating its mRNA m6A modification and stability. PRKAA1, in turn, promoted cell viability, colony formation, and glycolysis but inhibited apoptosis of GC cells by promoting the redox balance.
    DOI:  https://doi.org/10.4149/neo_2022_220714N714
  11. Cancer Cell. 2022 Oct 26. pii: S1535-6108(22)00495-0. [Epub ahead of print]
    The m6A reader IGF2BP2 regulates glutamine metabolism and represents a therapeutic target in acute myeloid leukemia.
    Hengyou Weng, Feng Huang, Zhaojin Yu, Zhenhua Chen, Emily Prince, Yalin Kang, Keren Zhou, Wei Li, Jiacheng Hu, Chen Fu, Tursunjan Aziz, Hongzhi Li, Jingwen Li, Ying Yang, Li Han, Subo Zhang, Yuelong Ma, Mingli Sun, Huizhe Wu, Zheng Zhang, Mark Wunderlich, Sean Robinson, Daniel Braas, Johanna Ten Hoeve, Bin Zhang, Guido Marcucci, James C Mulloy, Keda Zhou, Hong-Fang Tao, Xiaolan Deng, David Horne, Minjie Wei, Huilin Huang, Jianjun Chen.
      N6-Methyladenosine (m6A) modification and its modulators play critical roles and show promise as therapeutic targets in human cancers, including acute myeloid leukemia (AML). IGF2BP2 was recently reported as an m6A binding protein that enhances mRNA stability and translation. However, its function in AML remains largely elusive. Here we report the oncogenic role and the therapeutic targeting of IGF2BP2 in AML. High expression of IGF2BP2 is observed in AML and associates with unfavorable prognosis. IGF2BP2 promotes AML development and self-renewal of leukemia stem/initiation cells by regulating expression of critical targets (e.g., MYC, GPT2, and SLC1A5) in the glutamine metabolism pathways in an m6A-dependent manner. Inhibiting IGF2BP2 with our recently identified small-molecule compound (CWI1-2) shows promising anti-leukemia effects in vitro and in vivo. Collectively, our results reveal a role of IGF2BP2 and m6A modification in amino acid metabolism and highlight the potential of targeting IGF2BP2 as a promising therapeutic strategy in AML.
    Keywords:  GPT2; IGF2BP2; MYC; SLC1A5; acute myeloid leukemia; glutamine metabolism; leukemia stem cells; m(6)A modification; mitochondria oxygen consumption; targeted therapy
    DOI:  https://doi.org/10.1016/j.ccell.2022.10.004
  12. Exp Biol Med (Maywood). 2022 Oct 22. 15353702221128564
    Emerging role of N6-methyladenosine RNA methylation in lung diseases.
    Limin Xu, Lingyan Zhou, Chenxin Yan, Liqin Li.
      In recent years, with the increase of air pollution, smoking, aging, and respiratory infection, the incidence rate and mortality of lung diseases are increasing annually, which has become a major hazard to human health. N6-methyladenosine (m6A) RNA methylation is the most abundant modifications in eukaryotes, and such modified RNA can be specifically recognized and combined by m6A recognition proteins and then mediate RNA splicing, maturation, enucleation, degradation, and translation. More and more studies have revealed that the m6A modification is involved in the pathogenesis and development of some diseases; however, the mechanisms of m6A in lung diseases are poorly understood. In this review, we summarize the latest progress in the biological function of m6A modifications in lung diseases and discuss the potential therapeutic and prognostic strategies. The dysregulation of global m6A levels and m6A regulators may affect the occurrence and development of asthma, chronic obstructive pulmonary disease, lung cancer, and other lung diseases through inflammation and immune function. In lung cancer, this modification has an important impact on malignant cell proliferation, migration, invasion, and drug resistance. In addition, abnormally changed m6A-modified proteins in lung cancer tissue samples and circulating tumor cells (CTCs) may be used as diagnostic and prognostic markers of lung cancer. Models composed of multiple m6A regulators can be used to evaluate the risk prediction or prognosis of asthma and pulmonary fibrosis. In general, the in-depth study of m6A modifications is a frontier direction in disease research. It provides novel insights for understanding of the molecular mechanisms underlying disease occurrence, development, and drug resistance, as well as for the development of effective novel therapeutics.
    Keywords:  N6-methyladenosine; asthma; chronic obstructive pulmonary disease; idiopathic pulmonary fibrosis; lung cancer
    DOI:  https://doi.org/10.1177/15353702221128564
  13. Front Microbiol. 2022 ;13 999506
    MTDeepM6A-2S: A two-stage multi-task deep learning method for predicting RNA N6-methyladenosine sites of Saccharomyces cerevisiae.
    Hong Wang, Shihao Zhao, Yinchu Cheng, Shoudong Bi, Xiaolei Zhu.
      N6-methyladenosine (m6A) is one of the most important RNA modifications, which is involved in many biological activities. Computational methods have been developed to detect m6A sites due to their high efficiency and low costs. As one of the most widely utilized model organisms, many methods have been developed for predicting m6A sites of Saccharomyces cerevisiae. However, the generalization of these methods was hampered by the limited size of the benchmark datasets. On the other hand, over 60,000 low resolution m6A sites and more than 10,000 base resolution m6A sites of Saccharomyces cerevisiae are recorded in RMBase and m6A-Atlas, respectively. The base resolution m6A sites are often obtained from low resolution results by post calibration. In view of these, we proposed a two-stage deep learning method, named MTDeepM6A-2S, to predict RNA m6A sites of Saccharomyces cerevisiae based on RNA sequence information. In the first stage, a multi-task model with convolutional neural network (CNN) and bidirectional long short-term memory (BiLSTM) deep framework was built to not only detect the low resolution m6A sites but also assign a reasonable probability for the predicted site. In the second stage, a transfer-learning strategy was used to build the model to predict the base resolution m6A sites from those low resolution m6A sites. The effectiveness of our model was validated on both training and independent test sets. The results show that our model outperforms other state-of-the-art models on the independent test set, which indicates that our model holds high potential to become a useful tool for epitranscriptomics analysis.
    Keywords:  BiLSTM; N6-methyladenosine site; computational methods; multi-task learning; post-transcriptional modification
    DOI:  https://doi.org/10.3389/fmicb.2022.999506
  14. Cells. 2022 Oct 11. pii: 3193. [Epub ahead of print]11(20):
    The Role of N6-Methyladenosine Modification in Microvascular Dysfunction.
    Ye-Ran Zhang, Jiang-Dong Ji, Jia-Nan Wang, Ying Wang, Hong-Jing Zhu, Ru-Xu Sun, Qing-Huai Liu, Xue Chen.
      Microvascular dysfunction (MVD) has long plagued the medical field despite improvements in its prevention, diagnosis, and intervention. Microvascular lesions from MVD increase with age and further lead to impaired microcirculation, target organ dysfunction, and a mass of microvascular complications, thus contributing to a heavy medical burden and rising disability rates. An up-to-date understanding of molecular mechanisms underlying MVD will facilitate discoveries of more effective therapeutic strategies. Recent advances in epigenetics have revealed that RNA methylation, an epigenetic modification, has a pivotal role in vascular events. The N6-methylation of adenosine (m6A) modification is the most prevalent internal RNA modification in eukaryotic cells, which regulates vascular transcripts through splicing, degradation, translation, as well as translocation, thus maintaining microvascular homeostasis. Conversely, the disruption of the m6A regulatory network will lead to MVD. Herein, we provide a review discussing how m6A methylation interacts with MVD. We also focus on alterations of the m6A regulatory network under pathological conditions. Finally, we highlight the value of m6A regulators as prognostic biomarkers and novel therapeutic targets, which might be a promising addition to clinical medicine.
    Keywords:  N6-methyladenosine modification; angiogenesis; epigenetics; microvascular dysfunctions
    DOI:  https://doi.org/10.3390/cells11203193
  15. Zhonghua Gan Zang Bing Za Zhi. 2022 Sep 20. 30(9): 962-969
    [Cluster classification and clinical prognostic modeling based on m6A RNA methylation regulators in liver cancer].
    F Y Liu, X M Feng, X L Ji, X L Su.
      Objective: Cluster classification based on m6A methylation regulators and construct prognostic evaluation model. Methods: Utilizing consensus cluster to classify the liver cancer samples form TCGA based on the expression of 13 m6A methylation regulators, and verify the function and prognostic significance of the clustered subtypes. Marker genes were further screened to construct a risk prediction model for evaluating the prognosis of liver cancer patients. Results: The two clustered subtypes based on m6A methylation regulators showed significant differences in the prognosis value of liver cancer patients (P=0.048), and 38 prognostic markers related to m6A methylation in liver cancer were screened from the subgroup with poor prognosis. Two m6A regulatory genes, YTHDF1 and YTHDF2, are proved with adverse prognosis by univariate cox analysis (P<0.05, Hazard ratio>1). We used Lasso regression method to build risk assessment model and effectively predicted the prognosis status of liver cancer patients within 4 years (4-year AUC=0.685, 3-year AUC=0.669). Moreover, the assessment model was validated in another dataset of Asia liver cancer patients. Conclusion: The study provided ideas for studying m6A methylation in liver cancer, and the risk prediction model can be used to evaluate the short-term prognosis of liver cancer patients.
    DOI:  https://doi.org/10.3760/cma.j.cn501113-20200727-00428
  16. Mol Med. 2022 Oct 25. 28(1): 126
    LncRNA FENDRR with m6A RNA methylation regulates hypoxia-induced pulmonary artery endothelial cell pyroptosis by mediating DRP1 DNA methylation.
    Xiaoying Wang, Qian Li, Siyu He, June Bai, Cui Ma, Lixin Zhang, Xiaoyu Guan, Hao Yuan, Yiying Li, Xiangrui Zhu, Jian Mei, Feng Gao, Daling Zhu.
      BACKGROUND: Pyroptosis is a form of programmed cell death involved in the pathophysiological progression of hypoxic pulmonary hypertension (HPH). Emerging evidence suggests that N6-methyladenosine (m6A)-modified transcripts of long noncoding RNAs (lncRNAs) are important regulators that participate in many diseases. However, whether m6A modified transcripts of lncRNAs can regulate pyroptosis in HPH progression remains unexplored.METHODS: The expression levels of FENDRR in hypoxic pulmonary artery endothelial cells (HPAECs) were detected by using quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization (FISH). Western blot, Lactate dehydrogenase (LDH) release assay, Annexin V-FITC/PI double staining, Hoechst 33342/PI fluorescence staining and Caspase-1 activity assay were used to detect the role of FENDRR in HPAEC pyroptosis. The relationship between FENDRR and dynamin-related protein 1 (DRP1) was explored using bioinformatics analysis, Chromatin Isolation by RNA Purification (CHIRP), Electrophoretic mobility shift assay (EMSA) and Methylation-Specific PCR (MSP) assays. RNA immunoprecipitation (RIP) and m6A dot blot were used to detect the m6A modification levels of FENDRR. A hypoxia-induced mouse model of pulmonary hypertension (PH) was used to test preventive effect of conserved fragment TFO2 of FENDRR.
    RESULTS: We found that FENDRR was significantly downregulated in the nucleus of hypoxic HPAECs. FENDRR overexpression inhibited hypoxia-induced HPAEC pyroptosis. Additionally, DRP1 is a downstream target gene of FENDRR, and FENDRR formed an RNA-DNA triplex with the promoter of DRP1, which led to an increase in DRP1 promoter methylation that decreased the transcriptional level of DRP1. Notably, we illustrated that the m6A reader YTHDC1 plays an important role in m6A-modified FENDRR degradation. Additionally, conserved fragment TFO2 of FENDEE overexpression prevented HPH in vivo.
    CONCLUSION: In summary, our results demonstrated that m6A-induced decay of FENDRR promotes HPAEC pyroptosis by regulating DRP1 promoter methylation and thereby provides a novel potential target for HPH therapy.
    Keywords:  Dynamin-related protein 1; Pulmonary artery endothelial cells; Pyroptosis; lncRNA FENDRR; m6A RNA methylation
    DOI:  https://doi.org/10.1186/s10020-022-00551-z
  17. Front Pharmacol. 2022 ;13 1023878
    WTAP mediates the anti-inflammatory effect of Astragalus mongholicus polysaccharide on THP-1 macrophages.
    Haijiao Long, Haiyue Lin, Pan Zheng, Lianjie Hou, Ming Zhang, Shuyun Lin, Kai Yin, Guojun Zhao.
      Background: Astragalus mongholicus polysaccharides (APS) have anti-inflammatory, antioxidant and immunomodulatory effects. Recent studies have demonstrated the epigenetic regulation of N6-methyladenosine (m6A) in the development of inflammation. However, the effect of APS on m6A modification is unclear. Here, for the first time, we investigate the mechanism of m6A modification in APS regulation of THP-1 macrophage inflammation. Methods: We treated LPS-induced THP-1 macrophages with APS at different concentrations and times, and detected IL-6 mRNA and protein levels by quantitative real-time PCR (qRT-PCR) and western blot, respectively. The m6A modification level was detected by m6A quantification kit. The proteins that regulate m6A modification were screened by western blot. Wilms' tumor 1-associating protein (WTAP) was overexpressed in APS-treated THP-1 macrophages and the m6A modification level and IL-6 expressions were detected. Results: These findings confirmed that APS significantly abolished LPS-induced IL-6 levels in THP-1 macrophages. Meanwhile, APS reduced m6A modification levels and WTAP gene expression in THP-1 macrophages. Further overexpression of WTAP can significantly reverse APS-induced m6A modification level and IL-6 expression. Mechanistically, APS regulates IL-6 expression through WTAP-mediated p65 nuclear translocation. Conclusion: Overall, our study suggested that WTAP mediates the anti-inflammatory effect of APS by regulating m6A modification levels in THP-1 macrophages. This study reveals a new dimension of APS regulation of inflammation at the epigenetic level.
    Keywords:  APS; IL-6; M6A; WTAP; macrophage inflammation
    DOI:  https://doi.org/10.3389/fphar.2022.1023878
  18. Antioxidants (Basel). 2022 Sep 30. pii: 1954. [Epub ahead of print]11(10):
    M6A RNA Methylation Mediates NOD1/NF-kB Signaling Activation in the Liver of Piglets Challenged with Lipopolysaccharide.
    Menghui Xu, Ruhao Zhuo, Shengxiang Tao, Yaxu Liang, Chunru Liu, Qingyang Liu, Tian Wang, Xiang Zhong.
      N6-methyladenosine (m6A) is the most abundant internal modification that widely participates in various immune and inflammatory responses; however, its regulatory mechanisms in the inflammation of liver induced by lipopolysaccharide in piglets remain largely unknown. In the present study, piglets were intraperitoneally injected with 80 μg/kg LPS or an equal dose of sterile saline. Results indicated that LPS administration increased activities of serum alanine aminotransferase (ALT), induced M1 macrophage polarization and promoted secretion of inflammatory cytokines, and finally led to hepatic lesions in piglets. The NOD1/NF-κB signaling pathway was activated in the livers of the LPS group. Moreover, the total m6A level was significantly elevated after LPS treatment. MeRIP-seq showed that 1166 and 1344 transcripts contained m6A methylation in control and LPS groups, respectively. The m6A methylation sites of these transcripts mainly distributes in the 5' untranslated region (5'UTR), the coding sequence (CDS), and the 3' untranslated region (3'UTR). Interestingly, these genes were mostly enriched in the NF-κB signaling pathway, and LPS treatment significantly changed the m6A modification in NOD1, RIPK2, NFKBIA, NFKBIB, and TNFAIP3 mRNAs. In addition, knockdown of METTL3 or overexpression of FTO both changed gene levels in the NOD1/NF-κB pathway, suggesting that activation of this pathway was regulated by m6A RNA methylation. Moreover, the alteration of m6A RNA methylation profile may be associated with the increase of reactive oxygen species (ROS), HIF-1α, and MAT2A. In conclusion, LPS activated the NOD1/NF-κB pathway at post-transcriptional regulation through changing m6A RNA methylation, and then promoted the overproduction of proinflammatory cytokines, ultimately resulting in liver inflammation and damage.
    Keywords:  NF-κB; NOD1; immune; inflammation; m6A modification; oxidative stress; piglet
    DOI:  https://doi.org/10.3390/antiox11101954
  19. Metabolites. 2022 Oct 14. pii: 973. [Epub ahead of print]12(10):
    Simultaneous Determination of Methylated Nucleosides by HILIC-MS/MS Revealed Their Alterations in Urine from Breast Cancer Patients.
    Zhihao Fang, Yiqiu Hu, Xiujuan Hong, Xiaoxiao Zhang, Tao Pan, Chi Pan, Shu Zheng, Cheng Guo.
      RNA methylation plays a vital role in the pathogenesis of a variety of diseases including cancer, and aberrant levels of modified nucleosides in RNA were revealed to be related to cancer. Urine is a favored source for biomarker discovery due to the non-invasion to patients. Herein, we developed a sensitive hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method combined with stable isotope dilution for accurate quantification of methylated nucleosides in human urine. With this method, we successfully quantified ten methylated nucleosides in urine samples collected from healthy controls and breast cancer patients. We found N6-methyladenosine (m6A), 2'-O-methyladenosine (Am), N1-methyladenosine (m1A), N6,2'-O-dimethyladenosine (m6Am), N1-methylguanosine (m1G), 2'-O-methylguanosine (Gm), 5-methylcytidine (m5C) and 2'-O-methylcytidine (Cm) were all decreased in early-stage breast cancer patients, and a nomogram prediction model was constructed. Locally advanced breast cancer patients exhibited elevated levels of urinary 2'-O-methylated nucleosides in comparison to early-stage breast cancer patients. Together, we developed a robust method for the simultaneous determination of methylated nucleosides in human urine, and the results revealed an association between the contents of urinary methylated nucleosides and the occurrence of breast cancer, which may stimulate future studies about the regulatory roles of these methylated nucleosides in the initiation and progression of breast cancer.
    Keywords:  HILIC–MS/MS; biomarker; breast cancer; methylated nucleosides; urine
    DOI:  https://doi.org/10.3390/metabo12100973
  20. Zhonghua Shao Shang Za Zhi. 2022 Oct 20. 38(10): 989-993
    [Research advances on the effects of RNA N6-methyladenosine modification in the relevant pathophysiological processes of wound repair].
    X X Liu, D W Liu.
      N6-methyladenosine (m6A) exists widely in eukaryotes as a post-transcriptional modification. This modification is dynamically and reversibly regulated by methyltransferases and demethylases, and is involved in regulating biological effects through m6A binding proteins. Recent studies have elucidated that m6A is involved in embryonic skin morphogenesis, wound repair, and pathophysiological processes such as inflammatory response, angiogenesis, and fibrosis. This review summarizes the role of m6A and its related proteins in the related pathophysiological processes of wound repair, so as to provide a new theoretical basis for the treatment strategy of wound repair.
    DOI:  https://doi.org/10.3760/cma.j.cn501120-20210804-00267
  21. Nat Commun. 2022 Oct 26. 13(1): 6350
    METTL3 acetylation impedes cancer metastasis via fine-tuning its nuclear and cytosolic functions.
    Yuanpei Li, Xiaoniu He, Xiao Lu, Zhicheng Gong, Qing Li, Lei Zhang, Ronghui Yang, Chengyi Wu, Jialiang Huang, Jiancheng Ding, Yaohui He, Wen Liu, Ceshi Chen, Bin Cao, Dawang Zhou, Yufeng Shi, Juxiang Chen, Chuangui Wang, Shengping Zhang, Jian Zhang, Jing Ye, Han You.
      The methyltransferase like 3 (METTL3) has been generally recognized as a nuclear protein bearing oncogenic properties. We find predominantly cytoplasmic METTL3 expression inversely correlates with node metastasis in human cancers. It remains unclear if nuclear METTL3 is functionally distinct from cytosolic METTL3 in driving tumorigenesis and, if any, how tumor cells sense oncogenic insults to coordinate METTL3 functions within these intracellular compartments. Here, we report an acetylation-dependent regulation of METTL3 localization that impacts on metastatic dissemination. We identify an IL-6-dependent positive feedback axis to facilitate nuclear METTL3 functions, eliciting breast cancer metastasis. IL-6, whose mRNA transcript is subjected to METTL3-mediated m6A modification, promotes METTL3 deacetylation and nuclear translocation, thereby inducing global m6A abundance. This deacetylation-mediated nuclear shift of METTL3 can be counterbalanced by SIRT1 inhibition, a process that is further enforced by aspirin treatment, leading to ablated lung metastasis via impaired m6A methylation. Intriguingly, acetylation-mimetic METTL3 mutant reconstitution results in enhanced translation and compromised metastatic potential. Our study identifies an acetylation-dependent regulatory mechanism determining the subcellular localization of METTL3, which may provide mechanistic clues for developing therapeutic strategies to combat breast cancer metastasis.
    DOI:  https://doi.org/10.1038/s41467-022-34209-5
  22. Cell Rep. 2022 Oct 25. pii: S2211-1247(22)01402-4. [Epub ahead of print]41(4): 111546
    m6A modification confers thermal vulnerability to HPV E7 oncotranscripts via reverse regulation of its reader protein IGF2BP1 upon heat stress.
    Lingfang Wang, Guankai Zhan, Yasen Maimaitiyiming, Yingfeng Su, Shitong Lin, Jinfeng Liu, Kunhui Su, Jiebo Lin, Shizhen Shen, Wentao He, Fenfen Wang, Jiafeng Chen, Siqi Sun, Yite Xue, Jiaxin Gu, Xiaojing Chen, Jian Zhang, Lu Zhang, Qianqian Wang, Kao-Jung Chang, Shih-Hwa Chiou, Mikael Björklund, Hua Naranmandura, Xiaodong Cheng, Chih-Hung Hsu.
      Human papillomavirus (HPV)-induced carcinogenesis critically depends on the viral early protein 7 (E7), making E7 an attractive therapeutic target. Here, we report that the E7 messenger RNA (mRNA)-containing oncotranscript complex can be selectively targeted by heat treatment. In HPV-infected cells, viral E7 mRNA is modified by N6-methyladenosine (m6A) and stabilized by IGF2BP1, a cellular m6A reader. Heat treatment downregulates E7 mRNA and protein by destabilizing IGF2BP1 without the involvement of canonical heat-shock proteins and reverses HPV-associated carcinogenesis in vitro and in vivo. Mechanistically, heat treatment promotes IGF2BP1 aggregation only in the presence of m6A-modified E7 mRNA to form distinct heat-induced m6A E7 mRNA-IGF2BP1 granules, which are resolved by the ubiquitin-proteasome system. Collectively, our results not only show a mutual regulation between m6A RNA and its reader but also provide a heat-treatment-based therapeutic strategy for HPV-associated malignancies by specifically downregulating E7 mRNA-IGF2BP1 oncogenic complex.
    Keywords:  CP: Cancer; CP: Molecular biology; HPV; N6-methyladenosine; cervical cancer; heat stress; human papillomavirus E7 oncotranscripts; hyperthermia; m6A; m6A reader IGF2BP1; phase separation; protein degradation
    DOI:  https://doi.org/10.1016/j.celrep.2022.111546
  23. Front Oncol. 2022 ;12 947808
    Development and validation of a m6A -regulated prognostic signature in lung adenocarcinoma.
    Yaxin Chen, Lei Xia, Yuxuan Peng, Gang Wang, Liyun Bi, Xue Xiao, Cui Li, Weimin Li.
      Lung adenocarcinoma (LUAD) is the most frequent subtype of lung cancer, with a 5-year survival rate of less than 20%. N6-methyladenosine (m6A) is the most prevalent RNA epigenetic modification in eukaryotic cells, and post-transcriptionally regulates gene expression and function by affecting RNA metabolism. The alterations of functionally important m6A sites have been previously shown to play vital roles in tumor initiation and progression, but little is known about the extent to which m6A-regulated genes play in prognostic performance for patients with LUAD. Here, we presented an overview of the m6A methylome in LUAD tissues using transcriptome-wide m6A methylation profiles, and found that differentially methylated transcripts were significantly enriched in tumor-related processes, including immune response, angiogenesis and cell-substrate adhesion. Joint analysis of m6A modification and gene expression suggested that 300 genes were regulated by m6A. Furthermore, we developed a m6A-regulated prognosis-associated signature (m6A-PPS) by performing a multi-step process. The m6A-PPS model, a 15-gene set, was qualified for prognosis prediction for LUAD patients. By regrouping the patients with this model, the OS of the high-risk group was shorter than that of the low-risk group across all datasets. Importantly, patients with high m6A-PPS scores respond better to immunotherapeutic. Our results provide a valuable resource for understanding the important role of epitranscriptomic modifications in the pathogenesis of LUAD, and obtain potential prognostic biomarkers.
    Keywords:  epitranscriptomic; lung adenocarcinoma; m6A; prognosis; signature
    DOI:  https://doi.org/10.3389/fonc.2022.947808
  24. Front Oncol. 2022 ;12 962204
    METTL3-IGF2BP3-axis mediates the proliferation and migration of pancreatic cancer by regulating spermine synthase m6A modification.
    Zhenyun Guo, Xiang Zhang, Chengjie Lin, Yue Huang, Yun Zhong, Hailing Guo, Zhou Zheng, Shangeng Weng.
      Spermine synthase (SMS) is an enzyme participating in polyamine synthesis; however, its function and role in pancreatic cancer remains elusive. Here we report that SMS is upregulated in pancreatic cancer and predicts a worse overall survival and significantly promotes the proliferation and migration of pancreatic cancer cells. Excessive SMS reduces the accumulation of spermidine by converting spermidine into spermine, which activates the phosphorylation of serine/threonine kinase (AKT) and epithelial-mesenchymal transition (EMT) signaling pathway, thereby inhibiting pancreatic cancer cell proliferation and invasion. Moreover, SMS was identified as the direct target of both methyltransferase like 3 (METTL3) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly bind to the m6A modification sites of SMS and inhibit mRNA degradation. Knockdown of METTL3 or IGF2BP3 significantly reduced the SMS protein expression and inhibited the migration of pancreatic cancer. We propose a novel regulatory mechanism in which the METTL3-IGF2BP3 axis mediates the mRNA degradation of SMS in an m6A-dependent manner to regulate spermine/spermidine conversion, which regulates AKT phosphorylation and EMT activation, thereby inducing tumor progression and migration in pancreatic cancer.
    Keywords:  AKT; EMT; m6A; pancreatic cancer; polyamine; spermine synthase
    DOI:  https://doi.org/10.3389/fonc.2022.962204
  25. BMB Rep. 2022 Oct 26. pii: 5715. [Epub ahead of print]
    Mettl14 mutation restrains liver regeneration by attenuating mitogens derived from non-parenchymal liver cells.
    Insook Yang, Seung Yeon Oh, Suin Jang, Il Yong Kim, You Me Kim, Jae-Sung Sung.
      Liver regeneration is a well-known systemic homeostatic phenomenon. Recently, the N6-methyladenosine (m6A) modification pathway has been associated with liver regeneration and hepatocellular carcinoma. m6A methyltransferases, such as methyltransferase 3 (METTL3) and methyltransferase 14 (METTL14), are associated with the hepatocyte-specific-regenerative pathway. To illustrate the role of METTL14, secreted from non-parenchymal liver cells, in the initiation phase of liver regeneration, we performed 70% partial hepatectomy (PH) in Mettl14 heterozygous (HET) and wild-type (WT) mice. Next, we analyzed the ratio of liver weight to body weight and the expression of mitogenic stimulators derived from non-parenchymal liver cells. We also evaluated the expression of cell cycle-related genes and the hepatocyte proliferation rate by MKI67-immunostaining. During regeneration after PH, the weight ratio was lower in Mettl14 HET mice compared to WT mice. The expression levels of hepatocyte growth factor (HGF) and tumor necrosis factor (TNF)-α, mitogens derived from non-parenchymal liver cells that stimulate the cell cycle, as well as the expression of cyclin B1 and D1, which regulate the cell cycle, and the number of MKI67-positive cells, which indicate proliferative hepatocyte in the late G1-M phase, were significantly reduced in Mettl14 HET mice 72h after PH. Our findings demonstrate that global Mettl14 mutation may interrupt the homeostasis of liver regeneration after an acute injury like PH by restraining certain mitogens, such as HGF and TNF-α, derived from sinusoidal endothelial cells, stellate cells, and Kupffer cells. These results provide new insights into the role of Mettl14 in the clinical translation of liver disease.
  26. Cancer Med. 2022 Oct 25.
    Identification of a novel prognostic signature correlated with epithelial-mesenchymal transition, N6-methyladenosine modification, and immune infiltration in colorectal cancer.
    Xiao Qu, Honghong Tan, Jingxian Mao, Mengxue Yang, Jian Xu, Xuebing Yan, Wenjuan Wu.
      OBJECTIVE: Colorectal cancer (CRC) is a commonly diagnosed human malignancy worldwide. Both epithelial-mesenchymal transition (EMT) and N6-methyladenosine (m6A) modification play a crucial role in CRC development. This study aimed to construct a prognostic signature based on the genes related to EMT and m6A modification.METHOD: Firstly, the mRNA expression profiling of CRC tissues was analyzed using TCGA and GEO databases. The prognostic hub genes related to EMT and m6A modification were selected using weighted correlation network and cox regression analysis. The prognostic signature was constructed based on hub genes, followed by validation in three external cohorts. Finally, the expression of the representative hub gene was detected in clinical samples, and its biological role was investigated using assays in vivo and in vitro.
    RESULTS: A prognostic signature was constructed using the following genes: YAP1, FAM3C, NUBPL, GLO1, JARID2, NFKB1, CDKN1B, HOOK1, and GIPC2. The signature effectively stratified the clinical outcome of CRC patients in the training cohort and two validation cohorts. The subgroup analysis demonstrated the signature could identify high-risk population from CRC patients within stage I-II or III-IV, female, male and elder patients. The signature was correlated with the infiltration of some immune cells (such as macrophage and regulatory T cells) and gene mutation counts. Finally, the hub gene GIPC2 was found to be downregulated in CRC tissues and most CRC cells lines. GIPC2 overexpression inhibited the malignant characteristics of CRC cells in vitro and in vivo through upregulating E-cadherin and downregulating N-cadherin, Vimentin, and Snail, while the opposite results were observed for GIPC2 knockdown in CRC cells.
    CONCLUSION: Our present study for the first time constructed a novel prognostic signature related to EMT, m6A modification, and immune infiltration for CRC risk stratification. In addition, GIPC2 is identified as a promising clinical biomarker or therapeutical target for CRC.
    Keywords:  N6-methyladenosine modification; colorectal cancer; epithelial-mesenchymal transition; immunity and biomarker
    DOI:  https://doi.org/10.1002/cam4.5384
  27. Ecotoxicol Environ Saf. 2022 Oct 25. pii: S0147-6513(22)01055-7. [Epub ahead of print]247 114215
    Insights into the mechanism underlying crystalline silica-induced pulmonary fibrosis via transcriptome-wide m6A methylation profile.
    Yingdie Zhang, Pei Gu, Yujia Xie, Lieyang Fan, Xiaojie You, Shiyu Yang, Yuxin Yao, Weihong Chen, Jixuan Ma.
      Silicosis is one of the most severe interstitial lung fibrosis diseases worldwide, caused by crystalline silica exposure. While the mechanisms and pathogenesis underlying silicosis remained unknown. N6-methyladenosine (m6A) methylation has received significant attention in a variety of human diseases. However, whether m6A methylation is involved in silicosis has not been clarified. In this study, we conducted methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and transcriptome sequencing (RNA-Seq) to profile the m6A modification in normal and silicosis mouse models (n = 3 pairs). The global levels of m6A methylation were further assessed by m6A RNA methylation quantification kits, and the major regulators of m6A RNA methylation were verified by qRT-PCR. Our results showed that long-term exposure to crystalline silica led to silicosis, accompanied by increasing levels of m6A methylation. Upregulation of METTL3 and downregulation of ALKBH5, FTO, YTHDF1, and YTHDF3 might contribute to aberrant m6A modification. Compared with controls, 359 genes showed differential m6A methylation peaks in silicosis (P < 0.05 and FC ≥ 2). Among them, 307 genes were hypermethylated, and 52 genes were hypomethylated. RNA-Seq analysis revealed 1091 differentially expressed genes between the two groups, 789 genes were upregulated and 302 genes were downregulated in the lungs of silicosis mice (P < 0.05 and FC ≥ 2). In the conjoint analysis of MeRIP-Seq and RNA-Seq, we identified that 18 genes showed significant changes in both m6A modification and mRNA expression. The functional analysis further noted that these 18 m6A-mediated mRNAs regulated pathways that were closely related to "phagosome", "antigen processing and presentation", and "apoptosis". All findings suggested that m6A methylation played an essential role in the formation of silicosis. Our discovery with multi-omics approaches not only gives clues for the epigenetic mechanisms underlying the pathogenesis of silicosis but also provides novel and viable strategies for the prevention and treatment of silicosis.
    Keywords:  Crystalline silica; M(6)A RNA methylation; Pulmonary fibrosis; Silicosis
    DOI:  https://doi.org/10.1016/j.ecoenv.2022.114215
  28. Antioxidants (Basel). 2022 Sep 21. pii: 1874. [Epub ahead of print]11(10):
    Effects of Moderate-Intensity Continuous Training and High-Intensity Interval Training on Testicular Oxidative Stress, Apoptosis and m6A Methylation in Obese Male Mice.
    Zujie Xu, Ying Qin, Binbin Lv, Zhenjun Tian, Bing Zhang.
      Exercise is an effective way to improve reproductive function in obese males. Oxidative stress and apoptosis are important pathological factors of obesity-related male infertility. Accumulating studies have demonstrated that N6-methyladenosine (m6A) methylation is associated with obesity and testicular reproductive function. Our study aimed to investigate and compare the effect of 8 weeks of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) on testicular oxidative stress, apoptosis and m6A methylation in obese male mice. Male C57BL/6 mice were randomly allocated into the four groups: normal diet (ND) group, high-fat diet (HFD) group, high-fat diet with moderate-intensity continuous training (HFD-MICT) group and high-fat diet with high-intensity interval training (HFD-HIIT) group. Mice in the HFD-MICT and HFD-HIIT groups were subjected to 8 weeks of MICT or HIIT treadmill protocols after 12 weeks of HFD feeding. We found that MICT and HIIT increased the protein expression of Nrf2, HO-1 and NQO-1 in the testes of obese mice, and HIIT increased it more than MICT. The Bax/Bcl-2 ratio, Cleaved Caspase-3 protein expression and TUNEL-positive cells were consistently up-regulated in the testes of obese mice, but MICT and HIIT restrained these HFD-induced effects. In addition, HFDs increased m6A levels and the gene expression of METTL3, YTHDF2 and FTO in the testes, but these effects were reversed by MICT and HIIT. However, HIIT was more effective than MICT in reducing m6A methylation in the testes of obese mice. These results demonstrate that both MICT and HIIT protected against HFD-induced oxidative stress, apoptosis and m6A methylation in testicular tissues; as a result, testicular morphological and functional impairment improved. In particular, HIIT was more beneficial than MICT in increasing the mRNA expression of steroidogenic enzymes and testicular antioxidant capacity and decreasing m6A methylation in the testes of HFD-fed mice.
    Keywords:  HIIT; MICT; N6-methyladenosine (m6A) methylation; apoptosis; obesity; oxidative stress; testicular
    DOI:  https://doi.org/10.3390/antiox11101874
  29. Biosci Rep. 2022 Oct 25. pii: BSR20221311. [Epub ahead of print]
    IGF2BP2 serves as a core m6A regulator in head and neck squamous cell carcinoma.
    Yuan Hu, Jiexin Chen, Muyuan Liu, Qin Feng, Hanwei Peng.
      Methylation of N6 adenosine (m6A) plays a crucial role in the development and progression of cancers. Its modification is regulated by three types of m6A-related regulators (methyltransferases (writers), demethylases (erasers), and RNA binding proteins (readers)). Till now, the functions and roles of these regulators in head and neck squamous cell carcinoma (HNSC) remain largely unexplored. Therefore, we utilized the open HNSC dataset in Cancer Genome Atlas (TCGA), 4 different cell lines and our HNSC patient samples (n = 40) to explore the clinical significance of 19 m6A regulators, and selected the most significant prognosis-related regulator. Authentic analyses based on online websites were also used in the study (Oncomine, Human Protein Atlas (HPA), cBioPortal, LinkedOmics, String, etc.). From the results, general overexpression of m6A regulators was observed in pan-cancer, especially in HNSC. IGF2BP2 was recognized as the hub m6A regulator, which was an independent, unfavorable prognostic factor in HNSC. Its mRNA and protein expression in HNSC were significantly upregulated. Gene mutation types of IGF2BP2 in HNSC (32%) were mainly mRNA High or Amplification, which represented the high expression of IGF2BP2. And these mutations were associated with a poor prognosis. In functional analysis, IGF2BP2 was negatively correlated to tumor immune infiltration in HNSC. Finally, HMGA2 might interact with the IGF2BP2 in HNSC. In conclusion, IGF2BP2 serves as a core m6A regulator among all regulators in HNSC, which has a high expression and predicts the poor prognosis of HNSC patients independently. IGF2BP2 might bring a new direction for HNSC treatment in the future.
    Keywords:  Clinical significance; Methylation of N6 adenosine; Squamous Cell Carcinoma of Head and Neck
    DOI:  https://doi.org/10.1042/BSR20221311
  30. Metabolism. 2022 Oct 24. pii: S0026-0495(22)00217-7. [Epub ahead of print] 155339
    Downregulation of the m6A reader protein YTHDC1 leads to islet β-cell failure and diabetes.
    Xinzhi Li, Ying Yang, Zheng Chen.
      N6-methyladenosine (m6A) methyltransferase writer proteins (METTL3/METTL14) have been shown to regulate β-cell function and diabetes. However, whether and which m6A reader proteins regulate β-cell function and the pathogenesis of diabetes are not largely unknown. In this study, we showed that YTHDC1 (YTH domain-containing protein 1), a key m6A nuclear reader protein, plays an essential role in maintaining β-cell function. YTHDC1 is downregulated in islet β cells in type 2 diabetes, which is due to lipotoxicity and chronic inflammation. β-Cell specific deletion of Ythdc1 results in β-cell failure and diabetes, which is likely due to the decreased expression of β-cell specific transcription factors and insulin secretion-related genes. Taken together, YTHDC1 is required for maintaining β-cell function, and the downregulation of YTHDC1 leads to β-cell failure and diabetes.
    Keywords:  Diabetes; Hyperglycemia; Insulin secretion; Islet β cells; YTHDC1
    DOI:  https://doi.org/10.1016/j.metabol.2022.155339
  31. BMC Mol Cell Biol. 2022 Oct 27. 23(1): 45
    Acetaminophen changes the RNA m6A levels and m6A-related proteins expression in IL-1β-treated chondrocyte cells.
    Jie Gao, Yan Li, Zijin Liu, Dong Wang, Huawu Zhang.
      BACKGROUND: Acetaminophen is commonly recommended for the early analgesia of osteoarthritis. However, the molecular mechanism by which it acts remains unknown. The aim of this study is to investigate the effect of acetaminophen on inflammation and extracellular matrix degradation in human chondrocytes, and the possible molecular mechanisms involved in its effect.METHODS: The normal chondrocyte cell line C28/I2 was treated with interleukin-1β to mimic the inflammatory state. Acetaminophen and the methylation inhibitor (cycloleucine) were used to treat interleukin-1β-induced C28/I2 cells. The expression of RNA N6-methyladenosine -related proteins was detected by RT-qPCR and western blot. The total RNA N6-methyladenosine level was measured by dot blot analysis and enzyme linked immunosorbent assay. The levels of interleukin-6, interleukin-8 and anti-tumor necrosis factor-α were measured by enzyme linked immunosorbent assay. The extracellular matrix synthesis and degradation were examined by western blot.
    RESULTS: After interleukin-1β stimulated C28/I2 cells, the intracellular RNA N6-methyladenosine level increased, and the expression of regulatory proteins also changed, mainly including the increased expression of methyltransferase like 3 and the downregulated expression of AlkB family member 5. The use of cycloleucine inhibited interleukin-1β-induced inflammation and extracellular matrix degradation by inhibiting RNA N6-methyladenosine modification. In contrast, acetaminophen treatment counteracted interleukin-1β-induced changes in RNA N6-methyladenosine levels and regulatory protein expression. Furthermore, acetaminophen treatment of interleukin-1β-induced C28/I2 cells inhibited the secretion of interleukin-6, interleukin-8 and anti-tumor necrosis factor-α, down-regulated the expression of matrix metalloproteinase-13 and Collagen X, and up-regulated the expression of collagen II and aggrecan. In addition, AlkB family member 5 overexpression activated interleukin-1β-induced chondrocyte viability and suppressed inflammation and extracellular matrix degradation.
    CONCLUSION: Acetaminophen affects inflammatory factors secretion and extracellular matrix synthesis of human chondrocytes by regulating RNA N6-methyladenosine level and N6-methyladenosine-related protein expression. Stimulation of the normal chondrocyte cell line C28/I2 with the cytokine IL-1β (10 μM) mimics the inflammatory state in vitro. Acetaminophen (Ace, 50 μg/mL) changes the m6A related proteins expression and the total RNA m6A levels in IL-1β-treated chondrocyte cells. Furthermore, regulation of RNA m6A levels (by methylation inhibitor Cyc and/or Ace) affects IL-1β-induced inflammatory cytokines secretion and extracellular matrix synthesis in C28/I2 cells.
    Keywords:  ALKBH5; Acetaminophen; Chondrocyte cell; Extracellular matrix; Inflammatory factors; Interleukin-1β; RNA N6-methyladenosine modification
    DOI:  https://doi.org/10.1186/s12860-022-00444-3
  32. Nat Commun. 2022 Oct 28. 13(1): 6435
    The N6-methyladenosine methyltransferase METTL16 enables erythropoiesis through safeguarding genome integrity.
    Masanori Yoshinaga, Kyuho Han, David W Morgens, Takuro Horii, Ryosuke Kobayashi, Tatsuaki Tsuruyama, Fabian Hia, Shota Yasukura, Asako Kajiya, Ting Cai, Pedro H C Cruz, Alexis Vandenbon, Yutaka Suzuki, Yukio Kawahara, Izuho Hatada, Michael C Bassik, Osamu Takeuchi.
      During erythroid differentiation, the maintenance of genome integrity is key for the success of multiple rounds of cell division. However, molecular mechanisms coordinating the expression of DNA repair machinery in erythroid progenitors are poorly understood. Here, we discover that an RNA N6-methyladenosine (m6A) methyltransferase, METTL16, plays an essential role in proper erythropoiesis by safeguarding genome integrity via the control of DNA-repair-related genes. METTL16-deficient erythroblasts exhibit defective differentiation capacity, DNA damage and activation of the apoptotic program. Mechanistically, METTL16 controls m6A deposition at the structured motifs in DNA-repair-related transcripts including Brca2 and Fancm mRNAs, thereby upregulating their expression. Furthermore, a pairwise CRISPRi screen revealed that the MTR4-nuclear RNA exosome complex is involved in the regulation of METTL16 substrate mRNAs in erythroblasts. Collectively, our study uncovers that METTL16 and the MTR4-nuclear RNA exosome act as essential regulatory machinery to maintain genome integrity and erythropoiesis.
    DOI:  https://doi.org/10.1038/s41467-022-34078-y
  33. Nat Biotechnol. 2022 Oct 27.
    Absolute quantification of single-base m6A methylation in the mammalian transcriptome using GLORI.
    Cong Liu, Hanxiao Sun, Yunpeng Yi, Weiguo Shen, Kai Li, Ye Xiao, Fei Li, Yuchen Li, Yongkang Hou, Bo Lu, Wenqing Liu, Haowei Meng, Jinying Peng, Chengqi Yi, Jing Wang.
      N6-methyladenosine (m6A) is the most abundant RNA modification in mammalian cells and the best-studied epitranscriptomic mark. Despite the development of various tools to map m6A, a transcriptome-wide method that enables absolute quantification of m6A at single-base resolution is lacking. Here we use glyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI) to develop an absolute m6A quantification method that is conceptually similar to bisulfite-sequencing-based quantification of DNA 5-methylcytosine. We apply GLORI to quantify the m6A methylomes of mouse and human cells and reveal clustered m6A modifications with differential distribution and stoichiometry. In addition, we characterize m6A dynamics under stress and examine the quantitative landscape of m6A modification in gene expression regulation. GLORI is an unbiased, convenient method for the absolute quantification of the m6A methylome.
    DOI:  https://doi.org/10.1038/s41587-022-01487-9
  34. Subcell Biochem. 2022 ;100 557-579
    Modulation of DNA/RNA Methylation by Small-Molecule Modulators and Their Implications in Cancer.
    Ayushi Verma, Abhipsa Sinha, Dipak Datta.
      Chromatin is an organized complex of DNA, histone proteins, and RNA. Chromatin modifications include DNA methylation, RNA methylation, and histone acetylation and methylation. The methylation of chromatin complexes predominantly alters the regulation of gene expression, and its deregulation is associated with several human diseases including cancer. Cancer is a disease characterized by dynamic changes in the genetic and epigenetic architecture of a cell. Altered DNA methylation by DNA methyltransferases (DNMTs) and m6A RNA methylation facilitate tumor initiation and progression and thus serve as critical targets for cancer therapy. Small-molecule modulators of these epigenetic targets are at the hotspots of current cancer drug discovery research. Indeed, recent studies have led to the discovery of several chemical modulators against these targets, some of which have already gained approval for cancer therapy while others are undergoing clinical trials. In this chapter, we will focus on the role of small-molecule modulators in regulating DNA/RNA methylation and their implications in cancer.
    Keywords:  DNA methylation; DNMTs; Drug discovery; Epigenetics; RNA methylation; Small-molecule modulator
    DOI:  https://doi.org/10.1007/978-3-031-07634-3_17
  35. New Phytol. 2022 Oct 28.
    The blue light receptor CRY1 interacts with FIP37 to promote N6-methyladenosine RNA modification and photomorphogenesis in Arabidopsis.
    Jiaxin Yang, Lan Li, Xin Li, Ming Zhong, Xinmei Li, Lina Qu, Hui Zhang, Dongying Tang, Xuanming Liu, Chongsheng He, Xiaoying Zhao.
      Light is a particularly important environmental cue that regulates a variety of diverse plant developmental processes, such as photomorphogenesis. Blue light promotes photomorphogenesis mainly through the activation of the photoreceptor Cryptochrome 1 (CRY1). However, the mechanism underling the CRY1-mediated regulation of growth is not fully understood. Here, we found that blue light induced N6 -methyladenosine (m6 A) RNA modification during photomorphogenesis partially via CRY1. CRY1 mediates blue light-induced expression of FIP37 which is a component of m6 A writer. Moreover, we showed that CRY1 physically interacted with FIP37 in vitro and in vivo, and mediated blue light-activation of FIP37 binding to RNA. Furthermore, CRY1 and FIP37 modulated the m6 A on photomorphogenesis-related genes PIF3, PIF4 and PIF5, thereby accelerating decay of their transcripts. Genetically, FIP37 repressed hypocotyl elongation under blue light, and fip37 mutation could partially rescue the short hypocotyl phenotype of CRY1-overexpression plants. Together, our results indicated a new insight of CRY1 signal in modulating m6 A methylation and stability of PIFs, and established an essential molecular link between m6 A modification and determination of photomorphogenesis in plants.
    Keywords:  Arabidopsis; Cryptochrome 1; FIP37; blue light; m6A
    DOI:  https://doi.org/10.1111/nph.18583
  36. Cancer Sci. 2022 Oct 29.
    Feedforward loop between IMP1 and YAP/TAZ promotes tumorigenesis and malignant progression in glioblastoma.
    Jia Yang, Xujia Wu, Jia Wang, Xing Guo, Junju Chen, Xuesong Yang, Jian Zhong, Xixi Li, Zhong Deng.
      YAP/TAZ have been identified as master regulators in malignant phenotypes of glioblastoma (GBM), however, YAP/TAZ transcriptional disruptor in GBM treatment remains ineffective. Whether posttranscriptional disregulation of YAP/TAZ improving GBM outcome is currently unknown. Here, we report that insulin-like growth factor 2 (IGF2) mRNA-binding proteins 1 (IGF2BP1 or IMP1) is upregulated in mesenchymal GBM compared to proneural GBM, and correlates to patient worse outcome. Overexpression of IMP1 in proneural glioma stem-like cells (GSCs) promotes, while IMP1 knockdown in mesenchymal GSCs attenuates, tumorigenesis and mesenchymal signatures. IMP1 binds to and stabilizes m6A-YAP mRNA, leading to activation of YAP/TAZ signaling depends on its m6A recognition and binding domain. On the other hand, TAZ functions as enhancer for IMP1 expression. Collectively, our data reveal a feedforward loop between IMP1 and YAP/TAZ maintaining GBM/GSC tumorigenesis and malignant progression, and a promising molecular target in GBM.
    Keywords:  Glioblastoma; IMP1; YAP/TAZ pathway; malignant progression
    DOI:  https://doi.org/10.1111/cas.15636
  37. Nat Biotechnol. 2022 Oct 27.
    Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution.
    Qing Dai, Li-Sheng Zhang, Hui-Lung Sun, Kinga Pajdzik, Lei Yang, Chang Ye, Cheng-Wei Ju, Shun Liu, Yuru Wang, Zhong Zheng, Linda Zhang, Bryan T Harada, Xiaoyang Dou, Iryna Irkliyenko, Xinran Feng, Wen Zhang, Tao Pan, Chuan He.
      Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.
    DOI:  https://doi.org/10.1038/s41587-022-01505-w
  38. Front Genet. 2022 ;13 1008644
    DNA methylation regulator-mediated modification pattern defines tumor microenvironment immune infiltration landscape in colon cancer.
    Shijin Yuan, Yuzhen Gao, Yan Xia, Zhuo Wang, Xian Wang.
      Emerging evidence implies a non-negligible role of DNA methylation in tumor immunity, however, its comprehensive impact on tumor microenvironment (TME) formation and immune activation remains unclear. In this study, we integrated 24 DNA methylation regulators among 754 colon cancer patients to distinguish different modification patterns via an unsupervised clustering method, and explore their TME immune characteristics. Three DNA methylation modification patterns with distinct prognosis and biological behaviors were identified, consistent with three known phenotypes of immune-inflamed, immune-excluded, and immune-desert. We then determined a DNA methylation gene signature and constructed a DNA methylation score (DMS) to quantify modification patterns individually through principal component analysis algorithms. DMS-low group had characteristics of specific molecular subtypes, including microsatellite instability, CpG island methylator phenotype positive, and mutant BRAF, presented by increased mutation burden, activation of DNA damage repair and immune-related pathways, highly TME immune cells infiltration, and hence, a preferable prognosis. Further, low DMS was also demonstrated to be correlated to better response and prolonged survival of anti-PD-L1 antibody, indicating that DMS could be considered as an effective predictive tool for immunotherapy. In conclusion, our work presented a landscape of different DNA methylation modification patterns, and their vital role in the formation of TME diversity and complexity, which could help to enhance understanding of TME immune infiltration characteristics and more importantly, guide immunotherapy strategies more effectively and personalized.
    Keywords:  DNA methylation; biomarker; colon cancer; immunotherapy; tumor microenvironment
    DOI:  https://doi.org/10.3389/fgene.2022.1008644
  39. Subcell Biochem. 2022 ;100 201-237
    Modulation of DNA/RNA Methylation Signaling Mediating Metabolic Homeostasis in Cancer.
    Pallawi Choubey, Harshdeep Kaur, Kushagra Bansal.
      Nucleic acid methylation is a fundamental epigenetic mechanism that impinges upon several cellular attributes, including metabolism and energy production. The dysregulation of deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) methylation can lead to metabolic rewiring in the cell, which in turn facilitates tumor development. Here, we review the current knowledge on the interplay between DNA/RNA methylation and metabolic programs in cancer cells. We also discuss the mechanistic role of these pathways in tumor development and progression.
    Keywords:  5mC; Cancer; CpG; DNA methylation; RNA methylation; Tumor metabolism; Warburg effect; m6A
    DOI:  https://doi.org/10.1007/978-3-031-07634-3_7
  40. Mol Cell Biochem. 2022 Oct 25.
    METTL3-modified lncRNA-SNHG8 binds to PTBP1 to regulate ALAS2 expression to increase oxidative stress and promote myocardial infarction.
    Juan Tang, Qi-Xia Tang, Shan Liu.
      Myocardial infarction (MI) is one of the important factors leading to death in today's society. Therefore, to study the related mechanism of MI and reduce myocardial ischemia-reperfusion injury is an important link to reduce MI injury. MI mice in vivo and cell model in vitro were constructed. The cardiac function and MI area of mice were detected, and myocardial tissue injury was detected by HE staining. ALAS2 expression in mice myocardial tissue was detected by IHC. The expressions of lncRNA-SNHG8, METTL3, PTBP1 and ALAS2 in myocardial tissue or cardiomyocytes were detected by qRT-PCR assay. MTT assay was used to measured viability of cardiomyocytes. The oxidative stress level in myocardial tissue or cardiomyocytes was detected by ELISA assay and ROS assay. RIP-qPCR and RNA pulldown assays determined the interaction between METTL3 and lncRNA-SNHG8, as well as PTBP1 and ALAS2. lncRNA-SNHG8 knockdown in MI mice was reduced myocardial infarction size, alleviated myocardial tissue injury and oxidative stress, and inhibited ALAS2 expression in myocardial tissue. RNA pulldown and RIP assays showed that lncRNA-SNHG8 binged with PTBP1 and PTBP1 interacted with ALAS2 mRNA. Knockdown of lncRNA-SNHG8, METTL3 or PTBP1 in MI cells enhanced viability of myocardial cells, attenuated ROS release and MDA level, increased SOD level, alleviated oxidative stress. ALAS overexpression attenuated the corresponding effect of knockdown of lncRNA-SNHG8 and/or PTBP1 on MI cells. In sum, our paper is demonstrated for the first time that METTL3 can promote lncRNA-SNHG8 through m6A modification, thereby regulating ALAS2 to induce oxidative stress and aggravate myocardial injury.
    Keywords:  ALAS2; METTL3; Myocardial infarction; Oxidative stress; PTBP1; lncRNA-SNHG8
    DOI:  https://doi.org/10.1007/s11010-022-04570-6
  41. Front Immunol. 2022 ;13 984149
    Identification of N7-methylguanosine related subtypes and construction of prognostic model in gastric cancer.
    Xiaoxiao Li, Hao Dong, Ling Chen, Yujie Wang, Zhibin Hao, Yingyi Zhang, Yuan Jiao, Zhiyue Zhao, Xiaobo Peng, Xianbao Zhan.
      Background: N7-methylguanosine (m7G), one of the most common post-transcriptional modifications, can be present in tRNA, mRNA, and miRNA to mediate the progression of various tumors. However, the possible role of m7G in gastric cancer (GC) is still unknown.Materials and Methods: In this study, SNVs (single nucleotide variations), CNVs (copy number variations), and methylation of m7G-related genes (m7GRGs) were analyzed. The relationship between them and the expression of m7GRGs and prognosis of GC patients was explored. Based on 13 prognostic-related m7GRGs, 567 GC samples were classified into three subtypes using the ConsensusClusterPlus package. we compared survival status, clinical traits, immune cell infiltration, immune checkpoints, tumor microenvironment (TME), tumor immune dysfunction and exclusion (TIDE), and potential biological pathways among the three subtypes. Then, patients were again grouped into different genetic subtypes based on the DEGs among the three subtypes. In addition, a prognostic m7GRG_Score was constructed using five risk genes applicable to patients of any age, gender and stage. We also assessed tumor mutational burden (TMB), microsatellite instability (MSI), cancer stem cell (CSC) index, sensitivity of antineoplastic drugs, efficacy of anti-PD-1 and anti-CTLA4 immunotherapy between high and low m7GRG_Score groups. Finally, we established a nomogram based on m7GRG_Score and tumor stage to enhance the clinical application of the model. miRNAs and lncRNAs that could regulate expression of risk genes were searched.
    Results: SNVs, CNVs, and methylation of m7GRGs were associated with m7GRGs expression. However, they did not significantly affect the survival of GC patients. Our results also confirmed that patients in subtypes B and C and low m7GRG_Score groups had longer survival time, better clinical stage, more immune cell infiltration, fewer immune escape and dysfunction compared to subtype A and high m7GRG_Score groups. A low m7GRG_score was featured with increased microsatellite instability-high (MSI-H), TMB, and efficacy of immunotherapy.
    Conclusion: The m7GRG_Score model may become a beneficial tool for predicting prognosis and guiding personalized treatment in GC patients. These findings will improve our knowledge of m7G in GC and provide new methods for more effective treatment strategies.
    Keywords:  N7-methylguanosine; gastric cancer; immune infiltration; immunotherapy; molecule subtypes; prognosis; tumor microenvironment
    DOI:  https://doi.org/10.3389/fimmu.2022.984149
  42. Gut. 2022 Oct 25. pii: gutjnl-2022-327230. [Epub ahead of print]
    Targeting tumour-intrinsic N7-methylguanosine tRNA modification inhibits MDSC recruitment and improves anti-PD-1 efficacy.
    Haining Liu, Xuezhen Zeng, Xuxin Ren, Yifan Zhang, Manling Huang, Li Tan, Zihao Dai, Jiaming Lai, Wenxuan Xie, Zebin Chen, Sui Peng, Lixia Xu, Shuling Chen, Shunli Shen, Ming Kuang, Shuibin Lin.
      OBJECTIVE: Intrahepatic cholangiocarcinoma (ICC) exhibits very low response rate to immune checkpoint inhibitors (ICIs) and the underlying mechanism is largely unknown. We investigate the tumour immune microenvironment (TIME) of ICCs and the underlying regulatory mechanisms with the aim of developing new target to inhibit tumour growth and improve anti-programmed cell death protein-1 (PD-1) efficacy.DESIGN: Tumour tissues from patients with ICC together with hydrodynamic ICC mouse models were employed to identify the key cell population in TIME of ICCs. Functional analysis and mechanism studies were performed using cell culture, conditional knockout mouse model and hydrodynamic transfection ICC model. The efficacy of single or combined therapy with anti-PD-1 antibody, gene knockout and chemical inhibitor were evaluated in vivo.
    RESULTS: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are enriched in advanced ICCs and significantly correlated with N7-methylguanosine tRNA methyltransferase METTL1. Using diverse in vivo cancer models, we demonstrate the crucial immunomodulator function of METTL1 in regulation of PMN-MDSC accumulation in TIME and ICC progression. Mechanistically, CXCL8 in human and Cxcl5 in mouse are key translational targets of METTL1 that facilitate its function in promoting PMN-MDSC accumulation in TIME and ICC progression in vivo. Co-blockade of METTL1 and its downstream chemokine pathway enhances the anti-PD-1 efficacy in ICC preclinical mouse models.
    CONCLUSIONS: Our data uncover novel mechanisms underlying chemokine regulation and TIME shaping at the layer of messenger RNA translation level and provide new insights for development of efficient cancer immunotherapeutic strategies.
    Keywords:  CANCER IMMUNOBIOLOGY; CHEMOKINES; CHOLANGIOCARCINOMA; IMMUNOTHERAPY; MOLECULAR MECHANISMS
    DOI:  https://doi.org/10.1136/gutjnl-2022-327230
  43. Nucleic Acids Res. 2022 Oct 27. pii: gkac945. [Epub ahead of print]
    RM2Target: a comprehensive database for targets of writers, erasers and readers of RNA modifications.
    Xiaoqiong Bao, Yin Zhang, Huiqin Li, Yuyan Teng, Lixia Ma, Zhihang Chen, Xiaotong Luo, Jian Zheng, An Zhao, Jian Ren, Zhixiang Zuo.
      RNA modification is a dynamic and reversible process regulated by a series of writers, erasers and readers (WERs). Abnormal changes of WERs will disrupt the RNA modification homeostasis of their target genes, leading to the dysregulation of RNA metabolisms such as RNA stability and translation, and consequently to diseases such as cancer. A public repository hosting the regulatory relationships between WERs and their target genes will help in understanding the roles of RNA modifications in various physiological and pathological conditions. Previously, we developed a database named 'm6A2Target' to host targets of WERs in m6A, one of the most prevalent RNA modifications in eukaryotic cells. To host all RNA modification (RM)-related WER-target associations, we hereby present an updated database, named 'RM2Target' (http://rm2target.canceromics.org/). In this update, RM2Target encompasses 1 619 653 WER-target associations for nine RNA modifications in human and mouse, including m6A, m6Am, m5C, m5U, m1A, m7G, pseudouridine, 2'-O-Me and A-to-I. Extensive annotations of target genes are available in RM2Target, including but not limited to basic gene information, RNA modifications, RNA-RNA/RNA-protein interactions and related diseases. Altogether, we expect that RM2Target will facilitate further downstream functional and mechanistic studies in the field of RNA modification research.
    DOI:  https://doi.org/10.1093/nar/gkac945
  44. Cell Rep. 2022 Oct 25. pii: S2211-1247(22)01386-9. [Epub ahead of print]41(4): 111530
    METTL3 regulates m6A methylation of PTCH1 and GLI2 in Sonic hedgehog signaling to promote tumor progression in SHH-medulloblastoma.
    Zhi-Wei Zhang, Xufei Teng, Fu Zhao, Chunhui Ma, Jing Zhang, Ling-Feng Xiao, Yaning Wang, Mengqi Chang, Yongji Tian, Chunde Li, Zhang Zhang, Shuhui Song, Wei-Min Tong, Pinan Liu, Yamei Niu.
      SHH subgroup medulloblastoma (SHH-MB) is one of the most common malignant pediatric tumors that arises in the cerebellum. Previously, we showed that RNA m6A methylation participates in regulation of cerebellar development. Here we investigate whether dysregulated m6A methylation contributes to tumorigenesis of SHH-MB. We show that high expression of m6A methyltransferase METTL3 associates with worse survival in the patients with SHH-MB. A large number of hypermethylated transcripts are identified in SHH-MB tumor cells by m6A-seq. We find that METTL3 promotes tumor progression via activating Sonic hedgehog signaling. Mechanistically, METTL3 methylates PTCH1 and GLI2 RNAs and further regulates their RNA stability and translation. Importantly, targeting METTL3 by depleting METTL3 expression or treatment with its catalytic inhibitor STM2457 restrains tumor progression. Collectively, this study shows a critical function for METTL3 and m6A methylation in SHH-MB, indicative of a potential role of METTL3 as therapeutic target in SHH-MB.
    Keywords:  CP: Cancer; CP: Molecular biology; METTL3; RNA m(6)A methylation; hedgehog signaling pathway; medulloblastoma
    DOI:  https://doi.org/10.1016/j.celrep.2022.111530
  45. Proc Natl Acad Sci U S A. 2022 Nov;119(44): e2210150119
    ADAR regulates APOL1 via A-to-I RNA editing by inhibition of MDA5 activation in a paradoxical biological circuit.
    Nephrotic Syndrome Study Network (NEPTUNE)
      APOL1 risk variants are associated with increased risk of kidney disease in patients of African ancestry, but not all individuals with the APOL1 high-risk genotype develop kidney disease. As APOL1 gene expression correlates closely with the degree of kidney cell injury in both cell and animal models, the mechanisms regulating APOL1 expression may be critical determinants of risk allele penetrance. The APOL1 messenger RNA includes Alu elements at the 3' untranslated region that can form a double-stranded RNA structure (Alu-dsRNA) susceptible to posttranscriptional adenosine deaminase acting on RNA (ADAR)-mediated adenosine-to-inosine (A-to-I) editing, potentially impacting gene expression. We studied the effects of ADAR expression and A-to-I editing on APOL1 levels in podocytes, human kidney tissue, and a transgenic APOL1 mouse model. In interferon-γ (IFN-γ)-stimulated human podocytes, ADAR down-regulates APOL1 by preventing melanoma differentiation-associated protein 5 (MDA5) recognition of dsRNA and the subsequent type I interferon (IFN-I) response. Knockdown experiments showed that recognition of APOL1 messenger RNA itself is an important contributor to the MDA5-driven IFN-I response. Mathematical modeling suggests that the IFN-ADAR-APOL1 network functions as an incoherent feed-forward loop, a biological circuit capable of generating fast, transient responses to stimuli. Glomeruli from human kidney biopsies exhibited widespread editing of APOL1 Alu-dsRNA, while the transgenic mouse model closely replicated the edited sites in humans. APOL1 expression in mice was inversely correlated with Adar1 expression under IFN-γ stimuli, supporting the idea that ADAR regulates APOL1 levels in vivo. ADAR-mediated A-to-I editing is an important regulator of APOL1 expression that could impact both penetrance and severity of APOL1-associated kidney disease.
    Keywords:  ADAR; APOL1; RNA editing; innate immunity; kidney disease
    DOI:  https://doi.org/10.1073/pnas.2210150119
  46. Cancers (Basel). 2022 Oct 11. pii: 4970. [Epub ahead of print]14(20):
    Genome-Wide DNA Methylation and Gene Expression Profiling Characterizes Molecular Subtypes of Esophagus Squamous Cell Carcinoma for Predicting Patient Survival and Immunotherapy Efficacy.
    Yulong Zheng, Qiqi Gao, Xingyun Su, Cheng Xiao, Bo Yu, Shenglin Huang, Yifeng Sun, Sheng Wu, Yixin Wo, Qinghua Xu, Nong Xu, Hui Yu.
      BACKGROUND: Immunotherapy is recently being used to treat esophageal squamous cell carcinoma (ESCC); however, response and survival benefits are limited to a subset of patients. A better understanding of the molecular heterogeneity and tumor immune microenvironment in ESCC is needed for improving disease management.METHODS: Based on the DNA methylation and gene expression profiles of ESCC patients, we identify molecular subtypes of patients and construct a predictive model for subtype classification. The clinical value of molecular subtypes for the prediction of immunotherapy efficacy is assessed in an independent validation cohort of Chinese ESCC patients who receive immunotherapy.
    RESULTS: We identify two molecular subtypes of ESCC (S1 and S2) that are associated with distinct immune-related pathways, tumor microenvironment and clinical outcomes. Accordingly, S2 subtype patients had a poorer prognosis. A 15-gene expression signature is developed to classify molecular subtypes with an overall accuracy of 94.7% (89/94, 95% CI: 0.880-0.983). The response rate of immunotherapy is significantly higher in the S1 subtype than in the S2 subtype patients (68.75% vs. 25%, p = 0.028). Finally, potential target drugs, including mitoxantrone, are identified for treating patients of the S2 subtype.
    CONCLUSIONS: Our findings demonstrated that the identified molecular subtypes constitute a promising prognostic and predictive biomarker to guide the clinical care of ESCC patients.
    Keywords:  esophagus squamous cell carcinoma; gene expression signature; immune checkpoint inhibitors; molecular subtype; prognosis
    DOI:  https://doi.org/10.3390/cancers14204970
  47. Front Genet. 2022 ;13 1015879
    Comprehensive analysis of key m5C modification-related genes in type 2 diabetes.
    Yaxian Song, Yan Jiang, Li Shi, Chen He, Wenhua Zhang, Zhao Xu, Mengshi Yang, Yushan Xu.
      Background: 5-methylcytosine (m5C) RNA methylation plays a significant role in several human diseases. However, the functional role of m5C in type 2 diabetes (T2D) remains unclear. Methods: The merged gene expression profiles from two Gene Expression Omnibus (GEO) datasets were used to identify m5C-related genes and T2D-related differentially expressed genes (DEGs). Least-absolute shrinkage and selection operator (LASSO) regression analysis was performed to identify optimal predictors of T2D. After LASSO regression, we constructed a diagnostic model and validated its accuracy. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to confirm the biological functions of DEGs. Gene Set Enrichment Analysis (GSEA) was used to determine the functional enrichment of molecular subtypes. Weighted gene co-expression network analysis (WGCNA) was used to select the module that correlated with the most pyroptosis-related genes. Protein-protein interaction (PPI) network was established using the STRING database, and hub genes were identified using Cytoscape software. The competitive endogenous RNA (ceRNA) interaction network of the hub genes was obtained. The CIBERSORT algorithm was applied to analyze the interactions between hub gene expression and immune infiltration. Results: m5C-related genes were significantly differentially expressed in T2D and correlated with most T2D-related DEGs. LASSO regression showed that ZBTB4 could be a predictive gene for T2D. GO, KEGG, and GSEA indicated that the enriched modules and pathways were closely related to metabolism-related biological processes and cell death. The top five genes were identified as hub genes in the PPI network. In addition, a ceRNA interaction network of hub genes was obtained. Moreover, the expression levels of the hub genes were significantly correlated with the abundance of various immune cells. Conclusion: Our findings may provide insights into the molecular mechanisms underlying T2D based on its pathophysiology and suggest potential biomarkers and therapeutic targets for T2D.
    Keywords:  5-methylcytosine; bioinformatics; differentially expressed gene; immune microenvironment; pyroptosis; type 2 diabetes
    DOI:  https://doi.org/10.3389/fgene.2022.1015879
  48. Cell Rep. 2022 Oct 25. pii: S2211-1247(22)01422-X. [Epub ahead of print]41(4): 111561
    OTUB2 exerts tumor-suppressive roles via STAT1-mediated CALML3 activation and increased phosphatidylserine synthesis.
    Wan Chang, Qingyu Luo, Xiaowei Wu, Yabing Nan, Pengfei Zhao, Lingqiang Zhang, Aiping Luo, Wenjie Jiao, Qiong Zhu, Yesheng Fu, Zhihua Liu.
      Oral and esophageal squamous cell carcinomas (SCCs) are associated with high mortality, yet the molecular mechanisms underlying these malignancies are largely unclear. We show that DNA hypermethylation of otubain 2 (OTUB2), a previously recognized oncogene, drives tongue and esophageal SCC initiation and drug resistance. Mechanistically, OTUB2 promotes the deubiquitination and phosphorylation of signal transducer and activator of transcription 1 (STAT1) and subsequently regulates the transcription of calmodulin-like protein 3 (CALML3). Activation of CALML3-mediated mitochondrial calcium signaling promotes oxidative phosphorylation (OXPHOS) and the synthesis of phosphatidylserine (PS). In mouse models, orally administered soybean-derived PS inhibits SCC initiation in cells with low OTUB2 expression and increases their sensitivity to chemotherapy. Our study indicates that the OTUB2/STAT1/CALML3/PS axis plays tumor-suppressive roles and shows the potential of PS administration as a strategy for the treatment and prevention of tongue and esophageal SCCs.
    Keywords:  CALML3; CP: Cancer; OTUB2; OXPHOS; STAT1; calcium signaling; chemoresistance; lipid metabolism; phosphatidylserine; squamous cell carcinoma; tumorigenesis
    DOI:  https://doi.org/10.1016/j.celrep.2022.111561
  49. Front Endocrinol (Lausanne). 2022 ;13 1013238
    ECE2 is a prognostic biomarker associated with m6A modification and involved in immune infiltration of lung adenocarcinoma.
    Yao-Hua Zhang, Jing Zeng, Xu-Sheng Liu, Yan Gao, Xue-Yan Kui, Xiao-Yu Liu, Yu Zhang, Zhi-Jun Pei.
      Background: The targeted therapy for lung cancer relies on prognostic genes and requires further research. No research has been conducted to determine the effect of endothelin-converting enzyme 2 (ECE2) in lung cancer.Methods: We analyzed the expression of ECE2 in lung adenocarcinoma (LUAD) and normal adjacent tissues and its relationship with clinicopathological characteristics from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus database (GEO). Immunohistochemical staining was used to further validate the findings. GO/KEGG enrichment analysis and gene set enrichment analysis (GSEA) of ECE2 co-expression were performed using R software. Data from TIMER, the GEPIA database, and TCGA were analyzed to determine the relationship between ECE2 expression and LUAD immune infiltration. To investigate the relationship between ECE2 expression levels and LUAD m6A modification, TCGA data and GEO data were analyzed.
    Results: ECE2 is highly expressed in various cancers including LUAD. ECE2 showed high accuracy in distinguishing tumor and normal sample results. The expression level of ECE2 in LUAD was significantly correlated with tumor stage and prognosis. GO/KEGG enrichment analysis showed that ECE2 was closely related to mitochondrial gene expression, ATPase activity and cell cycle. GSEA analysis showed that ECE2-related differential gene enrichment pathways were related to mitotic cell cycle, MYC pathway, PLK1 pathway, DNA methylation pathway, HIF1A pathway and Oxidative stress-induced cellular senescence. Analysis of the TIMER, GEPIA database, and TCGA datasets showed that ECE2 expression levels were significantly negatively correlated with B cells, CD4+ cells, M2 macrophages, neutrophils, and dendritic cells. TCGA and GEO datasets showed that ECE2 was significantly associated with m6A modification-related genes HNRNPC, IGF2BP1, IGF2BP3 and RBM1.
    Conclusion: ECE2 is associated with m6A modification and immune infiltration and is a prognostic biomarker in LUAD.
    Keywords:  ECE2; immune infiltration; lung adenocarcinoma; m6A modification; prognostic biomarker
    DOI:  https://doi.org/10.3389/fendo.2022.1013238
  50. Genes Dev. 2022 Oct 27.
    Single-molecule identification of the target RNAs of different RNA binding proteins simultaneously in cells.
    Mathieu N Flamand, Ke Ke, Renee Tamming, Kate D Meyer.
      RNA-binding proteins (RBPs) regulate nearly every aspect of mRNA processing and are important regulators of gene expression in cells. However, current methods for transcriptome-wide identification of RBP targets are limited, since they examine only a single RBP at a time and do not provide information on the individual RNA molecules that are bound by a given RBP. Here, we overcome these limitations by developing TRIBE-STAMP, an approach for single-molecule detection of the target RNAs of two RNA binding proteins simultaneously in cells. We applied TRIBE-STAMP to the cytoplasmic m6A reader proteins YTHDF1, YTHDF2, and YTHDF3 and discovered that individual mRNA molecules can be bound by more than one YTHDF protein throughout their lifetime, providing new insights into the function of YTHDF proteins in cells. TRIBE-STAMP is a highly versatile approach that enables single-molecule analysis of the targets of RBP pairs simultaneously in the same cells.
    Keywords:  RNA binding proteins; STAMP; TRIBE; TRIBE-STAMP; YTHDF; m6A; m6A readers
    DOI:  https://doi.org/10.1101/gad.349983.122
  51. J R Soc Interface. 2022 Oct;19(195): 20220415
    Locally correlated kinetics of post-replication DNA methylation reveals processivity and region specificity in DNA methylation maintenance.
    Honglei Ren, Robert B Taylor, Timothy L Downing, Elizabeth L Read.
      DNA methylation occurs predominantly on cytosine-phosphate-guanine (CpG) dinucleotides in the mammalian genome, and the methylation landscape is maintained over mitotic cell division. It has been posited that coupling of maintenance methylation activity among neighbouring CpGs is critical to stability over cellular generations; however, the mechanism is unclear. We used mathematical models and stochastic simulation to analyse data from experiments that probe genome-wide methylation of nascent DNA post-replication in cells. We find that DNA methylation maintenance rates on individual CpGs are locally correlated, and the degree of this correlation varies by genomic regional context. By using theory of protein diffusion along DNA, we show that exponential decay of methylation rate correlation with genomic distance is consistent with enzyme processivity. Our results provide quantitative evidence of genome-wide methyltransferase processivity in vivo. We further developed a method to disentangle different mechanistic sources of kinetic correlations. From the experimental data, we estimate that an individual methyltransferase methylates neighbour CpGs processively if they are 36 basepairs apart, on average. But other mechanisms of coupling dominate for longer inter-CpG distances. Our study demonstrates that quantitative insights into enzymatic mechanisms can be obtained from replication-associated, cell-based genome-wide measurements, by combining data-driven statistical analyses with hypothesis-driven mathematical modelling.
    Keywords:  DNA methylation; processivity; stochastic modelling
    DOI:  https://doi.org/10.1098/rsif.2022.0415
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