bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2023–07–02
forty-two papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics
  1. METTL3-mediated m6A RNA modification promotes corneal neovascularization by upregulating the canonical Wnt pathway during HSV-1 infection.
  2. A cancer-associated METTL14 mutation induces aberrant m6A modification, affecting tumor growth.
  3. Circular RNA circ_KIAA1429 accelerates hepatocellular carcinoma progression via the miR-133a-3p/high mobility group AT-hook 2 (HMGA2) axis in an m6A-dependent manner.
  4. Novel insights into mutual regulation between N6-methyladenosine modification and LncRNAs in tumors.
  5. Expression profiling of N6-methyladenosine-modified mRNA in PC12 cells in response to unconjugated bilirubin.
  6. The mRNA methyltransferase Mettl3 modulates cytokine mRNA stability and limits functional responses in mast cells.
  7. ALKBH5/YTHDF2-mediated m6A modification of circAFF2 enhances radiosensitivity of colorectal cancer by inhibiting Cullin neddylation.
  8. Epigenetic modification of m6A regulator proteins in cancer.
  9. K235 acetylation couples with PSPC1 to regulate the m6A demethylation activity of ALKBH5 and tumorigenesis.
  10. Globally reduced N6-methyladenosine (m6A) in C9ORF72-ALS/FTD dysregulates RNA metabolism and contributes to neurodegeneration.
  11. METTL16 promotes osteosarcoma progression by downregulating VPS33B in an m6 A-dependent manner.
  12. METTL3 Boosts Mitochondrial Fission and Induces Cardiac Fibrosis by Enhancing LncRNA GAS5 Methylation.
  13. Cells at early and late stages of infection with Senecavirus A: Comparative analysis of N6-methyladenosine modification on mRNAs.
  14. An Exploration of the Coherent Effects between METTL3 and NDUFA10 on Alzheimer's Disease.
  15. Comprehensive analysis of differences in N6-methyladenosine RNA methylomes in Helicobacter pylori infection.
  16. YTHDF2 negatively correlates with tumor immune infiltration in small cell lung cancer.
  17. High glucose induces tau hyperphosphorylation in hippocampal neurons via inhibition of ALKBH5-mediated Dgkh m6A demethylation: a potential mechanism for diabetic cognitive dysfunction.
  18. METTL3-Modulated circUHRF2 Promotes Colorectal Cancer Stemness and Metastasis through Increasing DDX27 mRNA Stability by Recruiting IGF2BP1.
  19. Intron retention decreases METTL3 expression by inhibiting mRNA export to the cytoplasm.
  20. Long noncoding RNA AI662270 promotes kidney fibrosis through enhancing METTL3-mediated m6 A modification of CTGF mRNA.
  21. VIRMA facilitates intrahepatic cholangiocarcinoma progression through epigenetic augmentation of TMED2 and PARD3B mRNA stabilization.
  22. N6-methyladenosine methylation mediates non-coding RNAs modification in microplastic-induced cardiac injury.
  23. Regulation and functions of non-m6A mRNA modifications.
  24. Sperm Rhoa m6A modification mediates intergenerational transmission of paternally acquired hippocampal neuronal senescence and cognitive deficits after combined exposure to environmental cadmium and high-fat diet in mice.
  25. N6-methyladenosine RNA methylation in liver diseases: from mechanism to treatment.
  26. m6A mRNA Methylation Analysis Provides Novel Insights into Pigmentation in Sheep Skin.
  27. m6A Regulates the Stability of Cellular Transcripts Required for Efficient KSHV Lytic Replication.
  28. Loss of Mettl3 enhances liver tumorigenesis by inducing hepatocyte dedifferentiation and hyperproliferation.
  29. Targeting choroidal vasculopathy via up-regulation of tRNA-derived fragment tRF-22 expression for controlling progression of myopia.
  30. The m6A modulator-mediated cytarabine sensitivity and immune cell infiltration signature in acute myeloid leukemia.
  31. QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.
  32. Adenosine Methylation Level of miR-125a-5p Promotes Anti-PD-1 Therapy Escape through the Regulation of IGSF11/VSIG3 Expression.
  33. Single-cell and spatial transcriptomics reveal 5-methylcytosine RNA methylation regulators immunologically reprograms tumor microenvironment characterizations, immunotherapy response and precision treatment of clear cell renal cell carcinoma.
  34. LncRNA MIR4435-2HG drives cancer progression by modulating cell cycle regulators and mTOR signaling in stroma-enriched subtypes of urothelial carcinoma of the bladder.
  35. Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers.
  36. NUSAP1-LDHA-Glycolysis-Lactate feedforward loop promotes Warburg effect and metastasis in pancreatic ductal adenocarcinoma.
  37. METTL3-mediated m6A methylation orchestrates mRNA stability and dsRNA contents to equilibrate γδ T1 and γδ T17 cells.
  38. Schisandrin treatment suppresses the proliferation, migration, invasion, and inflammatory responses of fibroblast-like synoviocytes from rheumatoid arthritis patients and attenuates synovial inflammation and joint destruction in CIA mice.
  39. RP11-296E3.2 acts as an important molecular chaperone for YBX1 and promotes colorectal cancer proliferation and metastasis by activating STAT3.
  40. SOX1 acts as a tumor hypnotist rendering nasopharyngeal carcinoma cells refractory to chemotherapy.
  41. PRMT5 reduces immunotherapy efficacy in triple-negative breast cancer by methylating KEAP1 and inhibiting ferroptosis.
  42. The differences in immune features and genomic profiling between squamous cell carcinoma and adenocarcinoma - A multi-center study in Chinese patients with uterine cervical cancer.
  1. Cell Signal. 2023 Jun 23. pii: S0898-6568(23)00198-5. [Epub ahead of print] 110784
    METTL3-mediated m6A RNA modification promotes corneal neovascularization by upregulating the canonical Wnt pathway during HSV-1 infection.
    Wenzhe Wang, Wei Ye, Si Chen, Yun Tang, Deyan Chen, Yan Lu, Zhiwei Wu, Zhenping Huang, Yirui Ge.
       BACKGROUND: Corneal neovascularization (CNV) is a symptom of herpes simplex keratitis (HSK), which can result in blindness. The corneal angiogenesis brought on by herpes simplex virus type 1 (HSV-1) is strongly affected by vascular endothelial growth factor A (VEGFA). The N6-methyladenosine (m6A) modification catalyzed by methyltransferase-like 3 (METTL3) is a crucial epigenetic regulatory process for angiogenic properties. However, the roles of METTL3 and m6A in HSK-induced CNV remain unknown. Here, we investigated these roles in vitro and in vivo.
    METHODS: A PCR array in HSV-1-infected human umbilical vein endothelial cells (HUVECs) was used to screen for METTL3 among the epitranscriptomic genes. Tube formation and scratch assays were conducted to investigate cell migration capacity. The global mRNA m6A abundance was evaluated using a dot blot assay. Gene expression was assessed by RT-qPCR, western blotting, and fluorescence immunostaining. In addition, bioinformatic analysis was conducted to identify the downstream molecules of METTL3 in HUVECs. METTL3 knockdown and STM2457 treatment clarified the specific underlying molecular mechanisms affecting HSV-1-induced angiogenesis in vitro. An acute HSK mouse model was established to examine the effects of METTL3 knockdown or inhibition using STM2457 on pathological angiogenic development in vivo.
    RESULTS: METTL3 was highly upregulated in HSV-1-infected HUVECs and led to increased m6A levels. METTL3 knockdown or inhibition by STM2457 further reduced m6A levels and VEGFA expression and impaired migration and tube formation capacity in HUVECs after HSV-1 infection. Mechanistically, METTL3 regulated LRP6 expression through post-transcriptional mRNA modification in an m6A-dependent manner, increasing its stability, upregulating VEGFA expression, and promoting angiogenesis in HSV-1-infected HUVECs. Furthermore, METTL3 knockdown or inhibition by STM2457 reduced CNV in vivo.
    CONCLUSION: Our findings revealed that METTL3 promotes pathological angiogenesis through canonical Wnt and VEGF signaling in vitro and in vivo, providing potential pharmacological targets for preventing the progression of CNV in HSK.
    Keywords:  Corneal neovascularization; HSV-1; Herpes simplex keratitis; METTL3, m(6)A modification; VEGFA
    DOI:  https://doi.org/10.1016/j.cellsig.2023.110784
  2. Cell Rep. 2023 Jun 23. pii: S2211-1247(23)00699-X. [Epub ahead of print]42(7): 112688
    A cancer-associated METTL14 mutation induces aberrant m6A modification, affecting tumor growth.
    Kotaro Miyake, Pedro Henrique Costa Cruz, Izumi Nagatomo, Yuki Kato, Daisuke Motooka, Shingo Satoh, Yuichi Adachi, Yoshito Takeda, Yukio Kawahara, Atsushi Kumanogoh.
      The methyltransferase-like 3 (METTL3)-/METTL14-containing complex predominantly catalyzes N6-methyladenosine (m6A) modification, which affects mRNA stability. Although the METTL14 R298P mutation is found in multiple cancer types, its biological effects are not completely understood. Here, we show that the heterozygous R298P mutation promotes cancer cell proliferation, whereas the homozygous mutation reduces proliferation. Methylated RNA immunoprecipitation sequencing analysis indicates that the R298P mutation reduces m6A modification at canonical motifs. Furthermore, this mutation induces m6A modification at aberrant motifs, which is evident only in cell lines harboring the homozygous mutation. The aberrant recognition of m6A modification sites alters the methylation efficiency at surrounding canonical motifs. One example is c-MET mRNA, which is highly methylated at canonical motifs close to the aberrantly methylated sites. Consequently, c-MET mRNA is severely destabilized, reducing c-Myc expression and suppressing cell proliferation. These data suggest that the METTL14 R298P mutation affects target recognition for m6A modification, perturbing gene expression patterns and cell growth.
    Keywords:  CP: Cancer; CP: Molecular biology; METTL14; MeRIP-seq; RNA methylation; c-MET; c-Myc; endometrial cancer; m6A; mutational hotspot
    DOI:  https://doi.org/10.1016/j.celrep.2023.112688
  3. Hum Cell. 2023 Jun 27.
    Circular RNA circ_KIAA1429 accelerates hepatocellular carcinoma progression via the miR-133a-3p/high mobility group AT-hook 2 (HMGA2) axis in an m6A-dependent manner.
    Chun-Peng Zhang, Xin-Ying Huang.
      Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide with high mortality rate, and the N6-methyladenosine (m6A) epigenetic modifications have been reported to be closely associated with the pathogenesis of HCC, but the detailed molecular mechanisms by which m6A regulates HCC progression have not been fully delineated. In this study, we evidenced that the m6A methyltransferase-like 3 (METTL3)-mediated m6A modification contributed to HCC aggressiveness through modulating a novel circ_KIAA1429/miR-133a-3p/HMGA2 axis. Specifically, circ_KIAA1429 was aberrantly overexpressed in HCC tissues and cells, and the expression levels of circ_KIAA1429 was positively regulated by METTL3 in HCC cells in a m6A-dependent manner. Then, functional experiments confirmed that deletion of both circ_KIAA1429 and METTL3 suppressed HCC cell proliferation, migration and cell mitosis in vitro and in vivo, and conversely, circ_KIAA1429 overexpression had opposite effects to accelerate HCC development. Furthermore, the downstream mechanisms by which circ_KIAA1429 regulated HCC progression were uncovered, and we validated that silencing of circ_KIAA1429 restrained the malignant phenotypes in HCC cells through modulating the miR-133a-3p/high mobility group AT-hook 2 (HMGA2) axis. To summarize, our study firstly investigated the involvement of a novel METTL3/m6A/circ_KIAA1429/miR-133a-3p/HMGA2 axis in regulating HCC development, which provided novel indicators for HCC diagnosis, therapy and prognosis.
    Keywords:  Circular RNA; Hepatocellular carcinoma; MicroRNA; N6-Methyladenosine
    DOI:  https://doi.org/10.1007/s13577-023-00933-3
  4. Cancer Cell Int. 2023 Jun 26. 23(1): 127
    Novel insights into mutual regulation between N6-methyladenosine modification and LncRNAs in tumors.
    Nan Zhang, Yifei Sun, Zongqin Mei, Zuoshun He, Shiyan Gu.
      N6-methyladenosine (m6A), one of the most common RNA methylation modifications, has emerged in recent years as a new layer of the regulatory mechanism controlling gene expression in eukaryotes. As a reversible epigenetic modification, m6A not only occurs on mRNAs but also on Long non-coding RNAs (LncRNAs). As we all known, despite LncRNAs cannot encode proteins, they affect the expression of proteins by interacting with mRNAs or miRNAs, thus playing important roles in the occurrence and development of a variety of tumors. Up to now, it has been widely accepted that m6A modification on LncRNAs affects the fate of the corresponding LncRNAs. Interestingly, levels and functions of m6A modifications are also mediated by LncRNAs through affecting the m6A methyltransferases (METTL3, METTL14, WTAP, METTL16, etc.), demethylases (FTO, ALKBH5) and methyl-binding proteins (YTHDFs, YTHDCs, IGF2BPs, HNRNPs, etc.), which are collectively referred to as "m6A regulators". In this review, we summarized the mutual regulation mechanisms between N6-methyladenosine modification and LncRNAs in cancer progression, metastasis, invasion and drug resistance. In detail, we focus on the specific mechanisms of m6A modification, which is mediated by methyltransferases and demethylases, involves in the regulation of LncRNA levels and functions in the first part. And section two intensively displays the mediation roles of LncRNAs in m6A modification via changing the regulatory proteins. At last part, we described the interaction effects between LncRNAs and methyl-binding proteins of m6A modification during various tumor occurrence and development.
    Keywords:  Demethylases; LncRNAs; Methyl-binding proteins; Methyltransferases; N6-methyladenosine
    DOI:  https://doi.org/10.1186/s12935-023-02955-1
  5. Mol Biol Rep. 2023 Jun 28.
    Expression profiling of N6-methyladenosine-modified mRNA in PC12 cells in response to unconjugated bilirubin.
    Jinfu Zhou, Sining Liao, Chenran Zhang, Jinying Luo, Guilin Li, Huangyuan Li.
       BACKGROUND: Abnormal methylation of N6-methyladenosine (m6A) is reportedly associated with central nervous system disorders. However, the role of m6A mRNA methylation in unconjugated bilirubin (UCB) neurotoxicity requires further research.
    METHODS: Rat pheochromocytoma PC12 cells treated with UCB were used as in vitro models. After the PC12 cells were treated with UCB (0, 12, 18, and 24 µM) for 24 h, the total RNA m6A levels were measured using an m6A RNA methylation quantification kit. The expression of m6A demethylases and methyltransferases was detected through western blotting. We determined the m6A mRNA methylation profile in PC12 cells exposed to UCB (0 and 18 µM) for 24 h using methylated RNA immunoprecipitation sequencing (MeRIP-seq).
    RESULTS: Compared with the control group, UCB (18 and 24 µM) treatment decreased the expression of the m6A demethylase ALKBH5 and increased the expression of the methyltransferases METTL3 and METTL14, which resulted in an increase in the total m6A levels in PC12 cells. Furthermore, 1533 m6A peaks were significantly elevated and 1331 peaks were reduced in the UCB (18 µM)-treated groups compared with those in the control group. Genes with differential m6A peaks were mainly enriched in protein processing in the endoplasmic reticulum, ubiquitin-mediated proteolysis, cell cycle, and endocytosis. Through combined analysis of the MeRIP-seq and RNA sequencing data, 129 genes with differentially methylated m6A peaks and differentially expressed mRNA levels were identified.
    CONCLUSION: Our study suggests that the modulation of m6A methylation modifications plays a significant role in UCB neurotoxicity.
    Keywords:  Methylated RNA immunoprecipitation sequencing; Methylation; N 6-methyladenosine; Neurotoxicity; Unconjugated bilirubin; mRNA profile
    DOI:  https://doi.org/10.1007/s11033-023-08576-1
  6. Nat Commun. 2023 Jun 29. 14(1): 3862
    The mRNA methyltransferase Mettl3 modulates cytokine mRNA stability and limits functional responses in mast cells.
    Cristina Leoni, Marian Bataclan, Taku Ito-Kureha, Vigo Heissmeyer, Silvia Monticelli.
      Mast cells are central players in allergy and asthma, and their dysregulated responses lead to reduced quality of life and life-threatening conditions such as anaphylaxis. The RNA modification N6-methyladenosine (m6A) has a prominent impact on immune cell functions, but its role in mast cells remains unexplored. Here, by optimizing tools to genetically manipulate primary mast cells, we reveal that the m6A mRNA methyltransferase complex modulates mast cell proliferation and survival. Depletion of the catalytic component Mettl3 exacerbates effector functions in response to IgE and antigen complexes, both in vitro and in vivo. Mechanistically, deletion of Mettl3 or Mettl14, another component of the methyltransferase complex, lead to the enhanced expression of inflammatory cytokines. By focusing on one of the most affected mRNAs, namely the one encoding the cytokine IL-13, we find that it is methylated in activated mast cells, and that Mettl3 affects its transcript stability in an enzymatic activity-dependent manner, requiring consensus m6A sites in the Il13 3'-untranslated region. Overall, we reveal that the m6A machinery is essential in mast cells to sustain growth and to restrain inflammatory responses.
    DOI:  https://doi.org/10.1038/s41467-023-39614-y
  7. Clin Transl Med. 2023 07;13(7): e1318
    ALKBH5/YTHDF2-mediated m6A modification of circAFF2 enhances radiosensitivity of colorectal cancer by inhibiting Cullin neddylation.
    Yingjie Shao, Zhenhua Liu, Xing Song, Rui Sun, You Zhou, Dachuan Zhang, Huihui Sun, Junchao Huang, Chenxi Wu, Wendong Gu, Xiao Zheng, Jingting Jiang.
       BACKGROUND: Circular RNA (circRNA) and N6-methyladenosine (m6A) play a critical role in tumour occurrence and development, including colorectal cancer (CRC). However, little is known about the interaction between circRNA and m6A in the radiosensitivity of CRC. Here, we investigated the role of a novel m6A-regulated circRNA in CRC.
    METHODS: Differentially expressed circRNAs from radiosensitive and radioresistant CRC tissues were screened. Modifications of the selected circRNAs were examined by methylated RNA immunoprecipitation assay. Finally, the selected circRNAs were subjected to radiosensitivity assay.
    RESULTS: We identified that circAFF2 is closely related to both radiosensitivity and m6A in CRC. CircAFF2 was highly expressed in patients with radiosensitive rectal cancer, and patients with high expression of circAFF2 had a better prognosis. In addition, circAFF2 can enhance the radiosensitivity of CRC cells both in vitro and in vivo. The regulation of circAFF2 involves ALKBH5-mediated demethylation, followed by its recognition and degradation via YTHDF2. Rescue experiments revealed that circAFF2 could reverse the radiosensitivity induced by ALKBH5 or YTHDF2. Mechanistically, circAFF2 binds with CAND1, promotes the binding of CAND1 to Cullin1 and inhibits its neddylation, subsequently impacting the radiosensitivity of CRC.
    CONCLUSION: We identified and characterised circAFF2 as a novel m6A-modified circRNA and validated the ALKBH5/YTHDF2/circAFF2/Cullin-NEDD8 axis as a potential radiotherapy target for CRC.
    Keywords:  ALKBH5; YTHDF2; circAFF2; m6A; radiosensitivity
    DOI:  https://doi.org/10.1002/ctm2.1318
  8. Mol Cancer. 2023 Jun 30. 22(1): 102
    Epigenetic modification of m6A regulator proteins in cancer.
    Yumin Wang, Yan Wang, Harsh Patel, Jichao Chen, Jinhua Wang, Zhe-Sheng Chen, Hongquan Wang.
      Divergent N6-methyladenosine (m6A) modifications are dynamic and reversible posttranscriptional RNA modifications that are mediated by m6A regulators or m6A RNA methylation regulators, i.e., methyltransferases ("writers"), demethylases ("erasers"), and m6A-binding proteins ("readers"). Aberrant m6A modifications are associated with cancer occurrence, development, progression, and prognosis. Numerous studies have established that aberrant m6A regulators function as either tumor suppressors or oncogenes in multiple tumor types. However, the functions and mechanisms of m6A regulators in cancer remain largely elusive and should be explored. Emerging studies suggest that m6A regulators can be modulated by epigenetic modifications, namely, ubiquitination, SUMOylation, acetylation, methylation, phosphorylation, O-GlcNAcylation, ISGylation, and lactylation or via noncoding RNA action, in cancer. This review summarizes the current roles of m6A regulators in cancer. The roles and mechanisms for epigenetic modification of m6A regulators in cancer genesis are segregated. The review will improve the understanding of the epigenetic regulatory mechanisms of m6A regulators.
    Keywords:  Cancer; N6-methyladenosine methylation; RNA modification; m6A methylation enzymes; m6A regulators
    DOI:  https://doi.org/10.1186/s12943-023-01810-1
  9. Nat Commun. 2023 06 27. 14(1): 3815
    K235 acetylation couples with PSPC1 to regulate the m6A demethylation activity of ALKBH5 and tumorigenesis.
    Xiao-Lan Zhang, Xin-Hui Chen, Binwu Xu, Min Chen, Song Zhu, Nan Meng, Ji-Zhong Wang, Huifang Zhu, De Chen, Jin-Bao Liu, Guang-Rong Yan.
      N6-methyladenosine (m6A) modification plays important roles in bioprocesses and diseases. AlkB homolog 5 (ALKBH5) is one of two m6A demethylases. Here, we reveal that ALKBH5 is acetylated at lysine 235 (K235) by lysine acetyltransferase 8 and deacetylated by histone deacetylase 7. K235 acetylation strengthens the m6A demethylation activity of ALKBH5 by increasing its recognition of m6A on mRNA. RNA-binding protein paraspeckle component 1 (PSCP1) is a regulatory subunit of ALKBH5 and preferentially interacts with K235-acetylated ALKBH5 to recruit and facilitate the recognition of m6A mRNA by ALKBH5, thereby promoting m6A erasure. Mitogenic signals promote ALKBH5 K235 acetylation. K235 acetylation of ALKBH5 is upregulated in cancers and promotes tumorigenesis. Thus, our findings reveal that the m6A demethylation activity of ALKBH5 is orchestrated by its K235 acetylation and regulatory subunit PSPC1 and that K235 acetylation is necessary for the m6A demethylase activity and oncogenic roles of ALKBH5.
    DOI:  https://doi.org/10.1038/s41467-023-39414-4
  10. Nat Neurosci. 2023 Jun 26.
    Globally reduced N6-methyladenosine (m6A) in C9ORF72-ALS/FTD dysregulates RNA metabolism and contributes to neurodegeneration.
    Yini Li, Xiaoyang Dou, Jun Liu, Yu Xiao, Zhe Zhang, Lindsey Hayes, Rong Wu, Xiujuan Fu, Yingzhi Ye, Bing Yang, Lyle W Ostrow, Chuan He, Shuying Sun.
      Repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we show that N6-methyladenosine (m6A), the most prevalent internal mRNA modification, is downregulated in C9ORF72-ALS/FTD patient-derived induced pluripotent stem cell (iPSC)-differentiated neurons and postmortem brain tissues. The global m6A hypomethylation leads to transcriptome-wide mRNA stabilization and upregulated gene expression, particularly for genes involved in synaptic activity and neuronal function. Moreover, the m6A modification in the C9ORF72 intron sequence upstream of the expanded repeats enhances RNA decay via the nuclear reader YTHDC1, and the antisense RNA repeats can also be regulated through m6A modification. The m6A reduction increases the accumulation of repeat RNAs and the encoded poly-dipeptides, contributing to disease pathogenesis. We further demonstrate that, by elevating m6A methylation, we could significantly reduce repeat RNA levels from both strands and the derived poly-dipeptides, rescue global mRNA homeostasis and improve survival of C9ORF72-ALS/FTD patient iPSC-derived neurons.
    DOI:  https://doi.org/10.1038/s41593-023-01374-9
  11. J Cell Physiol. 2023 Jun 25.
    METTL16 promotes osteosarcoma progression by downregulating VPS33B in an m6 A-dependent manner.
    Jun Cheng, Zhihao Xu, Wei Tan, Jinpeng He, Boyu Pan, Yan Zhang, Youwen Deng.
      N6-methyladenosine (m6 A) is one of the main epitranscriptomic modifications that accelerates the progression of malignant tumors by modifying RNA. Methyltransferase-like 16 (METTL16) is a newly identified methyltransferase that has been found to play an important oncogenic role in a few malignancies; however, its function in osteosarcoma (OS) remains unclear. In this study, METTL16 was found to be upregulated in OS tissues, and associated with poor prognosis in OS patients. Functionally, METTL16 substantially promoted OS cell proliferation, migration, and invasion in vitro and OS growth in vivo. Mechanistically, vacuolar protein sorting protein 33b (VPS33B) was identified as the downstream target of METTL16, which induced m6 A modification of VPS33B and impaired the stability of the VPS33B transcript, thereby degrading VPS33B. In addition, VPS33B was found to be downregulated in OS tissues, VPS33B knockdown markedly attenuated shMETTL16-mediated inhibition on OS progression. Finally, METTL16/VPS33B might facilitate OS progression through PI3K/AKT pathway. In summary, this study revealed an important role for the METTL16-mediated m6 A modification in OS progression, implying it as a promising target for OS treatment.
    Keywords:  METTL16; N6-methyladenosine (m6A); PI3K/Akt pathway; VPS33B; osteosarcoma
    DOI:  https://doi.org/10.1002/jcp.31068
  12. Pharmacol Res. 2023 Jun 26. pii: S1043-6618(23)00196-2. [Epub ahead of print] 106840
    METTL3 Boosts Mitochondrial Fission and Induces Cardiac Fibrosis by Enhancing LncRNA GAS5 Methylation.
    Bin Tu, Kai Song, Yang Zhou, He Sun, Zhi-Yan Liu, Li-Chan Lin, Ji-Fei Ding, Ji-Ming Sha, Yan Shi, Jing-Jing Yang, Rui Li, Ye Zhang, Jian-Yuan Zhao, Hui Tao.
      Dysregulated mitochondrial metabolism occurs in several pathological processes characterized by cell proliferation and migration. Nonetheless, the role of mitochondrial fission is not well appreciated in cardiac fibrosis, which is accompanied by enhanced fibroblast proliferation and migration. We investigated the causes and consequences of mitochondrial fission in cardiac fibrosis using cultured cells, animal models, and clinical samples. Increased METTL3 expression caused excessive mitochondrial fission, resulting in the proliferation and migration of cardiac fibroblasts that lead to cardiac fibrosis. Knockdown of METTL3 suppressed mitochondrial fission, inhibiting fibroblast proliferation and migration for ameliorating cardiac fibrosis. Elevated METTL3 and N6-methyladenosine (m6A) levels were associated with low expression of long non-coding RNA GAS5. Mechanistically, METTL3-mediated m6A methylation of GAS5 induced its degradation, dependent of YTHDF2. GAS5 could interact with mitochondrial fission marker Drp1 directly; overexpression of GAS5 suppressed Drp1-mediated mitochondrial fission, inhibiting cardiac fibroblast proliferation and migration. Knockdown of GAS5 produced the opposite effect. Clinically, increased METTL3 and YTHDF2 levels corresponded with decreased GAS5 expression, increased m6A mRNA content and mitochondrial fission, and increased cardiac fibrosis in human heart tissue with atrial fibrillation. We describe a novel mechanism wherein METTL3 boosts mitochondrial fission, cardiac fibroblast proliferation, and fibroblast migration: METTL3 catalyzes m6A methylation of GAS5 methylation in a YTHDF2-dependent manner. Our findings provide insight into the development of preventative measures for cardiac fibrosis.
    Keywords:  Cardiac fibroblast; Collagen; Proliferation; YTHDF2; m(6)A methylation
    DOI:  https://doi.org/10.1016/j.phrs.2023.106840
  13. Virology. 2023 Jun 15. pii: S0042-6822(23)00130-7. [Epub ahead of print]585 186-195
    Cells at early and late stages of infection with Senecavirus A: Comparative analysis of N6-methyladenosine modification on mRNAs.
    Hailan Meng, Ziwei Li, Ling Wang, Liangpeng Lyu, Shuqing Liu, Rong Wei, Bo Ni, Fuxiao Liu.
      Infection with Senecavirus A (SVA) causes differential phenotypes in cells. In this study, cells were inoculated with SVA for culture. At 12 and 72 h post infection, cells were independently harvested for high-throughput RNA sequencing, and further methylated RNA immunoprecipitation sequencing. The resultant data were comprehensively analyzed for mapping N6-methyladenosine (m6A)-modified profiles of SVA-infected cells. More importantly, m6A-modified regions were identified in the SVA genome. A dataset of m6A-modified mRNAs was generated for screening out differentially m6A-modified mRNAs, further subjected to a series of in-depth analyses. This study not only showed statistical differentiation of m6A-modified sites between two SVA-infected groups, but also demonstrated that SVA genome, as a positive-sense, single-stranded mRNA, itself could be modified through the m6A pattern. Out of the six samples of SVA mRNAs, only three were identified to be m6A-modified, implying that the epigenetic effect might not be a crucial driving force for SVA evolution.
    Keywords:  DEMs; DMMs; Enrichment analysis; MeRIP-seq; RNA-Seq; SVA genome; Senecavirus A; m(6)A modification
    DOI:  https://doi.org/10.1016/j.virol.2023.06.006
  14. Int J Mol Sci. 2023 Jun 14. pii: 10111. [Epub ahead of print]24(12):
    An Exploration of the Coherent Effects between METTL3 and NDUFA10 on Alzheimer's Disease.
    Lin Yang, Xinping Pang, Wenbo Guo, Chengjiang Zhu, Lei Yu, Xianghu Song, Kui Wang, Chaoyang Pang.
      Alzheimer's disease (AD) is a neurodegenerative disorder characterized primarily by a decline in cognitive function. However, the etiopathogenesis of AD is unclear. N6-methyladenosine (m6A) is abundant in the brain, and it is interesting to explore the relationship between m6A and AD causes. In this paper, the gene expression of METTL3 and NDUFA10 were found to correlate with the Mini-mental State Examination (MMSE), which is a clinical indicator of the degree of dementia. METTL3 is involved in post-transcriptional methylation and the formation of m6A. NDUFA10 encodes the protein with NADH dehydrogenase activity and oxidoreductase activity in the mitochondrial electron transport chain. The following three characteristics were observed in this paper: 1. The lower the expression level of NDUFA10, the smaller the MMSE, and the higher the degree of dementia. 2. If the expression level of METTL3 dropped below its threshold, the patient would have a risk of AD with a probability close to 100%, suggesting a basic necessity for m6A to protect mRNA. 3. The lower the expression levels of both METTL3 and NDUFA10, the more likely the patient would suffer from AD, implying the coherence between METTL3 and NDUFA10. Regarding the above discovery, the following hypothesis is presented: METTL3 expression level is downregulated, then the m6A modification level of NDUFA10 mRNA is also decreased, thereby reducing the expression level of NDUFA10-encoded protein. Furthermore, the abnormal expression of NDUFA10 contributes to the assembly disorder of mitochondrial complex I and affects the process of the electron respiratory chain, with the consequent development of AD. In addition, to confirm the above conclusions, the AI Ant Colony Algorithm was improved to be more suitable for discovering the characteristics of AD data, and the SVM diagnostic model was applied to mine the coherent effects on AD between METTL3 and NDUFA10. In conclusion, our findings suggest that dysregulated m6A leads to altered expression of its target genes, thereby affecting AD's development.
    Keywords:  Alzheimer’s disease; METTL3; NDUFA10; m6A
    DOI:  https://doi.org/10.3390/ijms241210111
  15. Front Cell Dev Biol. 2023 ;11 1136096
    Comprehensive analysis of differences in N6-methyladenosine RNA methylomes in Helicobacter pylori infection.
    Huan Li, Jiahui Lin, Sha Cheng, Jingshu Chi, Ju Luo, Yu Tang, Wenfang Zhao, Yufeng Shu, Xiaoming Liu, Canxia Xu.
      Background:Helicobacter pylori (H.pylori) infection is an important factor in the occurrence of human gastric diseases, but its pathogenic mechanism is not clear. N6-methyladenosine (m6A) is the most prevalent reversible methylation modification in mammalian RNA and it plays a crucial role in controlling many biological processes. However, there are no studies reported that whether H. pylori infection impacts the m6A methylation of stomach. In this study, we measured the overall level changes of m6A methylation of RNA under H. pylori infection through in vitro and in vivo experiment. Methods: The total quantity of m6A was quantified in gastric tissues of clinical patients and C57 mice with H. pylori infection, as well as acute infection model [H. pylori and GES-1 cells were cocultured for 48 h at a multiplicity of infection (MOI) from of 10:1 to 50:1]. Furthermore, we performed m6A methylation sequencing and RNA-sequencing on the cell model and RNA-sequencing on animal model. Results: Quantitative detection of RNA methylation showed that H. pylori infection group had higher m6A modification level. M6A methylation sequencing identified 2,107 significantly changed m6A methylation peaks, including 1,565 upregulated peaks and 542 downregulated peaks. A total of 2,487 mRNA was upregulated and 1,029 mRNA was downregulated. According to the comprehensive analysis of MeRIP-seq and RNA-seq, we identified 200 hypermethylation and upregulation, 129 hypermethylation but downregulation, 19 hypomethylation and downregulation and 106 hypomethylation but upregulation genes. The GO and KEGG pathway analysis of these differential methylation and regulatory genes revealed a wide range of biological functions. Moreover, combining with mice RNA-seq results, qRT- PCR showed that m6A regulators, METTL3, WTAP, FTO and ALKBH5, has significant difference; Two key genes, PTPN14 and ADAMTS1, had significant difference by qRT- PCR. Conclusion: These findings provide a basis for further investigation of the role of m6A methylation modification in H. pylori-associated gastritis.
    Keywords:  Helicobacter pylori; M6A; MeRIP-seq; N6-methyladenosine; gastritis
    DOI:  https://doi.org/10.3389/fcell.2023.1136096
  16. J Mol Histol. 2023 Jun 26.
    YTHDF2 negatively correlates with tumor immune infiltration in small cell lung cancer.
    Gang Chen, Dandan Ren, Yuanhan Wang, Hui Wang, Junhong Zhang, Song Yang.
      In recent times, RNA modifications have garnered increased attention due to their involvement in the onset and progression of tumors, with N6-methyladenosine (m6A) modification being the most prevalent form. YTHDF2 is an m6A reading protein that can modulate RNA stability, transcription, and translation. This study aimed to explore the role of YTHDF2 in small cell lung cancer (SCLC) by collecting 20 SCLC patients from our hospital (cohort 1) and 48 Chinese SCLC patients from the GEO database (cohort 2). We evaluated the prognostic value of YTHDF2 using Kaplan-Meier survival analysis, Log-rank test, and Cox regression analysis. Additionally, we employed Gene Set Enrichment Analysis (GSEA) to screen different signaling pathways. We also investigated the correlation between the expression of m6A-related genes and SCLC molecular subtype and tumor immune microenvironment (TIME). Furthermore, we utilized multiplex immunofluorescence (mIF) staining to validate the immune infiltration of SCLC patient tissue sections. Our study revealed that YTHDF2 is an independent prognostic factor, which high expression is associated with low overall survival rate in SCLC. Low expression of YTHDF2 in SCLC tumors may enhance the molecular subtype transition from neuroendocrine (NE) to non-neuroendocrine (non-NE) subtype. Low YTHDF2 expression was closely associated with high immune infiltration, immune checkpoints, and other immune-related molecular features. Additionally, mIF detection showed a correlation between the low expression of YTHDF2 and CD4 + T cells and CD8 + T cells. Taken together, YTHDF2 could serve as a potential prognostic biomarker negatively correlated with tumor immune infiltration in SCLC.
    Keywords:  Immune infiltrating; Prognosis; Small cell lung cancer; YTHDF2; m6A
    DOI:  https://doi.org/10.1007/s10735-023-10129-6
  17. Cell Death Dis. 2023 Jun 29. 14(6): 385
    High glucose induces tau hyperphosphorylation in hippocampal neurons via inhibition of ALKBH5-mediated Dgkh m6A demethylation: a potential mechanism for diabetic cognitive dysfunction.
    Minli Qu, Linhui Zuo, Mengru Zhang, Peng Cheng, Zhanjun Guo, Junya Yang, Changjun Li, Jing Wu.
      Tau hyperphosphorylation in hippocampal neurons has an important pathogenetic role in the development of diabetic cognitive dysfunction. N6-methyladenosine (m6A) methylation is the most common modification of eukaryotic mRNA and is involved in regulating diverse biological processes. However, the role of m6A alteration in tau hyperphosphorylation of hippocampus neurons has not been reported. We found lower ALKBH5 expression in the hippocampus of diabetic rats and in HN-h cells with high-glucose intervention, accompanied by tau hyperphosphorylation. ALKBH5 overexpression significantly reversed tau hyperphosphorylation in high-glucose-stimulated HN-h cells. Furthermore, we found and confirmed by m6A-mRNA epitope transcriptome microarray and transcriptome RNA sequencing coupled with methylated RNA immunoprecipitation that ALKBH5 regulates the m6A modification of Dgkh mRNA. High glucose inhibited the demethylation modification of Dgkh by ALKBH5, resulting in decreases in Dgkh mRNA and protein levels. Overexpression of Dgkh reversed tau hyperphosphorylation in HN-h cells after high-glucose stimulation. Overexpression of Dgkh by adenovirus suspension injection into the bilateral hippocampus of diabetic rats significantly ameliorated tau hyperphosphorylation and diabetic cognitive dysfunction. In addition, ALKBH5 targeted Dgkh to activate PKC-α, leading to tau hyperphosphorylation under high-glucose conditions. The results of this study reveal that high glucose suppresses the demethylation modification of Dgkh by ALKBH5, which downregulates Dgkh and leads to tau hyperphosphorylation through activation of PKC-α in hippocampal neurons. These findings may indicate a new mechanism and a novel therapeutic target for diabetic cognitive dysfunction.
    DOI:  https://doi.org/10.1038/s41419-023-05909-7
  18. Cancers (Basel). 2023 Jun 11. pii: 3148. [Epub ahead of print]15(12):
    METTL3-Modulated circUHRF2 Promotes Colorectal Cancer Stemness and Metastasis through Increasing DDX27 mRNA Stability by Recruiting IGF2BP1.
    Miao Chen, Buning Tian, Gui Hu, Yihang Guo.
      Increasing evidence has implicated that circular RNAs (circRNAs) exert important roles in colorectal cancer (CRC) occurrence and progression. However, the role of a novel circRNA, circUHRF2, remains unknown in CRC. Our work aimed at identifying the functional roles of circUHRF2 in CRC and illustrating the potential mechanisms. As assessed by quantitative real-time PCR (qRT-PCR), circUHRF2 and methyltransferase-like 3 (METTL3) were highly expressed in CRC specimens and cells. Sanger sequencing and RNase R assays were performed to verify the ring structure of circUHRF2. Notably, aberrantly increased expression of circUHRF2 was positively correlated with poor prognosis of CRC patients. Functional experiments indicated that CRC stemness, migration, and epithelial-mesenchymal transition (EMT) were suppressed by the knockdown of circUHRF2 or METTL3. Mechanistically, METTL3 enhanced circUHRF2 expression through N6-methyladenine (m6A) modification. Rescue experiments showed that overexpression of circUHRF2 reversed the repressive effect of METTL3 silencing on CRC progression. Moreover, circUHRF2 inhibited the loss of DEAD-box helicase 27 (DDX27) protein via promoting the interaction between insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and DDX27 mRNA. DDX27 knockdown repressed CRC malignant properties, which was counteracted by circUHRF2 overexpression. The in vivo assays in nude mice demonstrated that circUHRF2 or METTL3 silencing exerted a suppressive effect on CRC growth and liver metastasis via repressing DDX27 protein expression. Taken together, METTL3-mediated m6A modification upregulated circUHRF2 and subsequently inhibited loss of DDX27 protein via recruitment of IGF2BP1, which conferred CRC stemness and metastasis. These findings shed light on CRC pathogenesis and suggest circUHRF2 as a novel target for CRC treatment.
    Keywords:  DDX27 signaling; N6-methyladenine (m6A) modification; circUHRF2; colorectal cancer; stemness; tumor metastasis
    DOI:  https://doi.org/10.3390/cancers15123148
  19. BMB Rep. 2023 Jun 26. pii: 5926. [Epub ahead of print]
    Intron retention decreases METTL3 expression by inhibiting mRNA export to the cytoplasm.
    Sangsoo Lee, Haesoo Jung, Sunkyung Choi, Namjoon Cho, Eun-Mi Kim, Kee K Kim.
      Methyltransferase-like 3 (METTL3), a key component of the m6A methyltransferase complex, regulates the splicing, nuclear transport, stability, and translation of its target genes. However, the mechanism underlying the regulation of METTL3 expression by alternative splicing (AS) remains unknown. We analyzed the expression pattern of METTL3 after AS in human tissues and confirmed the expression of an isoform retaining introns 8 and 9 (METTL3-IR). We confirmed the different intracellular localizations of METTL3-IR and METTL3 proteins using immunofluorescence microscopy. Furthermore, the endogenous expression of METTL3-IR at the protein level was different from that at the mRNA level. We found that 3'-UTR generation by intron retention (IR) inhibited the export of METTL3-IR mRNA to the cytoplasm, which in turn suppressed protein expression. To the best of our knowledge, this is the first study to confirm the regulation of METTL3 gene expression by AS, providing evidence that the suppression of METTL3 protein expression by IR is an integral part of the mechanism by which 3'-UTR generation regulates protein expression via inhibition of RNA export to the cytoplasm.
  20. FASEB J. 2023 Aug;37(8): e23071
    Long noncoding RNA AI662270 promotes kidney fibrosis through enhancing METTL3-mediated m6 A modification of CTGF mRNA.
    Yanyan Sun, Jia Ge, Fang Shao, Zhengrong Ren, Zhen Huang, Zhi Ding, Lei Dong, Jiangning Chen, Junfeng Zhang, Yuhui Zang.
      The sustained release of profibrotic cytokines, mainly transforming growth factor-β (TGF-β), leads to the occurrence of kidney fibrosis and chronic kidney disease (CKD). Connective tissue growth factor (CTGF) appears to be an alternative target to TGF-β for antifibrotic therapy in CKD. In this study, we found that long noncoding RNA AI662270 was significantly increased in various renal fibrosis models. In vivo, ectopic expression of AI662270 alone was sufficient to activate interstitial fibroblasts and drive kidney fibrosis, whereas inhibition of AI662270 blocked the activation of interstitial fibroblasts and ameliorated kidney fibrosis in various murine models. Mechanistic studies revealed that overexpression of AI662270 significantly increased CTGF product, which was required for the role of AI662270 in driving kidney fibrosis. Furthermore, AI662270 binds to the CTGF promoter and directly interacts with METTL3, the methyltransferase of RNA N6 -methyladenosine (m6 A) modification. Functionally, AI662270-mediated recruitment of METTL3 increased the m6 A methylation of CTGF mRNA and consequently enhanced CTGF mRNA stability. In conclusion, our results support that AI662270 promotes CTGF expression at the posttranscriptional stage by recruiting METTL3 to the CTGF promoter and depositing m6 A modifications on the nascent mRNA, thereby, uncovering a novel regulatory mechanism of CTGF in the pathogenesis of kidney fibrosis.
    Keywords:  CTGF; METTL3; kidney fibrosis; lncRNA; m6A modification
    DOI:  https://doi.org/10.1096/fj.202202012RRR
  21. J Gastroenterol. 2023 Jun 30.
    VIRMA facilitates intrahepatic cholangiocarcinoma progression through epigenetic augmentation of TMED2 and PARD3B mRNA stabilization.
    Hongfa Xu, Xiaowen Lin, Zhongliang Li, Xu He, Yong Li, Lige Qiu, Ligong Lu, Bing Liu, Meixiao Zhan, Ke He.
       BACKGROUND: N6-methyladenine modification of RNA, a critical component of the regulatory role at the post-transcriptional level, has a crucial effect on tumor development and progression. vir-Like m6A methyltransferase associated (VIRMA) has been recently discovered as an N6-methyladenine methyltransferase; however, its specific role in intrahepatic cholangiocarcinoma (ICC) remains to be investigated in-depth.
    METHODS: VIRMA expression and its association with clinicopathological characteristics were evaluated using The Cancer Genome Atlas (TCGA) dataset and tissue microarrays. In vivo and in vitro assays were performed to determine the role of VIRMA in ICC proliferation and metastasis. The underlying mechanism by which VIRMA influences ICC was clarified by RNA sequencing (RNA-seq), methylated RNA immunoprecipitation sequencing (MeRIP-seq), SLAM sequencing (SLAM-seq), RNA immunoprecipitation, a luciferase reporter assay, and chromatin immunoprecipitation assay.
    RESULTS: VIRMA showed high expression in ICC tissues, and this finding predicted a dismal prognostic outcome. The high expression of VIRMA in ICC was due to the demethylation of H3K27me3 modification in the promoter region. Functionally, VIRMA is required for the endothelial-mesenchymal transition (EMT) process in ICC cells, as shown by multiple ICC models in in vitro and in vivo experiments. Mechanistically, multi-omics analysis using ICC cells demonstrated that TMED2 and PARD3B were the direct downstream target of VIRMA. The methylated TMED2 and PARD3B transcripts were directly recognized by HuR, which exerted stabilizing effects on its bound RNA. VIRMA-induced expression of TMED2 and PARD3B activated the Akt/GSK/β-catenin and MEK/ERK/Slug signaling pathways, thereby promoting ICC proliferation and metastasis.
    CONCLUSIONS: The present study showed that VIRMA plays a critical role in ICC development by stabilizing TMED2 and PARD3B expression through the m6A-HuR-mediated mechanism. Thus, demonstrating VIRMA and its pathway as candidate therapeutic targets for ICC treatment.
    Keywords:  ICC; N6-methyladenosine; PARD3B; TMED2; VIRMA
    DOI:  https://doi.org/10.1007/s00535-023-02015-5
  22. Ecotoxicol Environ Saf. 2023 Jun 22. pii: S0147-6513(23)00678-4. [Epub ahead of print]262 115174
    N6-methyladenosine methylation mediates non-coding RNAs modification in microplastic-induced cardiac injury.
    Min Zhang, Jun Shi, Jun Zhou, Lei Song, Jingjing Ding, Hui Ping Deng, Li Weng, Yiqian Zhu, Zhongqing Xu.
      Owing to their potential adverse health effects, global contamination by microplastics (MPs) has attracted increased scientific and societal concerns. However, in vivo studies on MP toxicity, along with its effects and underlying mechanisms, remain limited. We recently found that non-coding RNA (ncRNAs) contribute to MP-mediated vascular toxicity. Moreover, previous studies have identified N6-methyladenosine (m6A) modifications in ncRNAs as influencing factors in cardiovascular disease. However, whether and how m6A modifications in ncRNAs are affected by MP-induced cardiotoxicity remain unknown. Herein, we profiled differentially expressed ncRNAs and their related m6A modification profiles in MP-exposed myocardial tissue using RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (MeRIP-seq). First, we observed that MPs accumulated in different organs and upregulated apoptosis in the heart, liver, spleen, and kidney cells. Furthermore, total m6A and METTL3 levels increased in the myocardium after exposure to MPs. RNA-seq results revealed that 392 lncRNAs and 302 circRNAs were differentially expressed in MP-treated mouse myocardium compared to the control group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that these altered lncRNAs and circRNAs were closely associated with endocytosis, cellular senescence, and cell cycle signaling pathways, which may cause cardiotoxicity. Furthermore, MeRIP-seq data showed different distributions and abundances of m6A modifications in lncRNAs and circRNAs. Additionally, we identified differentially m6A methylated lncRNAs and circRNAs through conjoint analysis of the two high-throughput sequencing datasets and found that both m6A modifications and the expression of circ-Arfgef2 and lncG3bp2 were upregulated after exposure to MPs. This suggests that MP-induced m6A modifications in ncRNAs are involved in cardiotoxicity. Our findings contribute to a better understanding of MP-induced cardiotoxicity and new molecular targets for treating cardiac injury.
    Keywords:  Cardiotoxicity; Circ-Arfgef2; LncG3bp2; Microplastics; N6-methyladenosine
    DOI:  https://doi.org/10.1016/j.ecoenv.2023.115174
  23. Nat Rev Mol Cell Biol. 2023 Jun 27.
    Regulation and functions of non-m6A mRNA modifications.
    Hanxiao Sun, Kai Li, Cong Liu, Chengqi Yi.
      Nucleobase modifications are prevalent in eukaryotic mRNA and their discovery has resulted in the emergence of epitranscriptomics as a research field. The most abundant internal (non-cap) mRNA modification is N6-methyladenosine (m6A), the study of which has revolutionized our understanding of post-transcriptional gene regulation. In addition, numerous other mRNA modifications are gaining great attention because of their major roles in RNA metabolism, immunity, development and disease. In this Review, we focus on the regulation and function of non-m6A modifications in eukaryotic mRNA, including pseudouridine (Ψ), N6,2'-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), inosine, 5-methylcytidine (m5C), N4-acetylcytidine (ac4C), 2'-O-methylated nucleotide (Nm) and internal N7-methylguanosine (m7G). We highlight their regulation, distribution, stoichiometry and known roles in mRNA metabolism, such as mRNA stability, translation, splicing and export. We also discuss their biological consequences in physiological and pathological processes. In addition, we cover research techniques to further study the non-m6A mRNA modifications and discuss their potential future applications.
    DOI:  https://doi.org/10.1038/s41580-023-00622-x
  24. J Hazard Mater. 2023 Jun 19. pii: S0304-3894(23)01174-3. [Epub ahead of print]458 131891
    Sperm Rhoa m6A modification mediates intergenerational transmission of paternally acquired hippocampal neuronal senescence and cognitive deficits after combined exposure to environmental cadmium and high-fat diet in mice.
    Jin Zhang, Yong-Wei Xiong, Lu-Lu Tan, Xin-Mei Zheng, Yu-Feng Zhang, Qing Ling, Chao Zhang, Hua-Long Zhu, Wei Chang, Hua Wang.
      Little is currently known about the effect and mechanism of combined paternal environmental cadmium (Cd) and high-fat diet (HFD) on offspring cognitive ability. Here, using in vivo model, we found that combined paternal environmental Cd and HFD caused hippocampal neuronal senescence and cognitive deficits in offspring. MeRIP-seq revealed m6A level of Rhoa, a regulatory gene of cellular senescence, was significantly increased in combined environmental Cd and HFD-treated paternal sperm. Interestingly, combined paternal environmental Cd and HFD markedly enhanced Rhoa mRNA, its m6A and reader protein IGF2BP1 in offspring hippocampus. STM2457, the inhibitor of m6A modification, markedly mitigated paternal exposure-caused the elevation of hippocampal Rhoa m6A, neuronal senescence and cognitive deficits in offspring. In vitro experiments, Rhoa siR significantly reversed mouse hippocampal neuronal senescence. Igf2bp1 siR obviously reduced the level and stability of Rhoa in aging mouse hippocampal neuronal cells. In conclusion, combined paternal environmental Cd and HFD induce offspring hippocampal neuronal senescence and cognitive deficits by promoting IGF2BP1-mediated Rhoa stabilization in offspring hippocampus via elevating Rhoa m6A in paternal sperm.
    Keywords:  Cadmium; Cognitive ability; High-fat diet; Paternal; Sperm m6A
    DOI:  https://doi.org/10.1016/j.jhazmat.2023.131891
  25. J Gastroenterol. 2023 Jun 28.
    N6-methyladenosine RNA methylation in liver diseases: from mechanism to treatment.
    Lan Yang, Siyuan Tian, Xiaohong Zheng, Miao Zhang, Xinmin Zhou, Yulong Shang, Ying Han.
      Epigenetic modification occurring in RNA has become the hotspot of the field. N6-methyladenosine (m6A) methylation is the most abundant RNA internal modification mainly occurring at the consensus motif DR (m6A) CH (D = A/G/U, R = A/G, H = A/C/U) in the 3'-UTR particularly the region near stop codons. The life cycle of m6A methylation includes "writers," "erasers," and "readers", which are responsible for the addition, removal, and recognition of m6A, respectively. m6A modification has been reported changing RNA secondary structure or modulating the stability, localization, transport, and translation of mRNAs to play crucial roles in various physiological and pathological conditions. Liver, as the largest metabolic and digestive organ, modulates vital physiological functions, and its dysfunction gives rise to the occurrence of various diseases. Despite the advanced intervening measures, mortality due to liver diseases is continuously high. Recent studies have explored the roles of m6A RNA methylation in the pathogenesis of liver diseases, providing new insights for studying the molecular mechanism of liver diseases. In the review, we extensively summarize the life cycle of m6A methylation, as well as its function and relevant mechanisms in liver fibrosis (LF), nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatitis virus infection, and hepatocellular carcinoma (HCC), and eventually we explore the potential of m6A as a treatment option for these liver diseases.
    Keywords:  Liver diseases; Mechanism; N6-methyladenosine; Treatment
    DOI:  https://doi.org/10.1007/s00535-023-02008-4
  26. Epigenetics. 2023 Dec;18(1): 2230662
    m6A mRNA Methylation Analysis Provides Novel Insights into Pigmentation in Sheep Skin.
    Yuanyuan Zhao, Jinzhu Meng, Xingchao Song, Qingming An.
      N6-methyladenosine (m6A) is the most universal post-transcriptional modification of mRNA which may play important roles in verious species. However, the potential roles of m6A in the pigmentation of skin are not completely understood. To explore the role of m6A modification in pigmentation of sheep skin, we used MeRIP-seq and RNA-seq to profile the skin transcriptome in black and white coat color (n=3). Our results showed that an average of 7701 m6A peaks were obtained for all samples and the average length was 305.89 bp. The GGACUU sequence was the most enrichment motif and shared in black skin and white skin. The m6A peaks were mainly enriched in the CDS, 3'UTR and 5'UTR, especially in CDS region near the stop codon of the transcript. 235 significantly differential peaks were found in black skin vs. white skin. The KEGG signaling pathways of downregulated and upregulated m6A peaks were mainly enriched in AGE-RAGE signaling pathway in diabetic complications, Viral carcinogenesis, Transcriptional misregulation in cancer, ABC transporters, Basal transcription factors and Thyroid hormone synthesis (P value <0.05). For RNA-seq, 71 differently expressed genes (DEGs) were scanned in black skin vs. white skin. DEGs were significantly enriched in tyrosine metabolism, melanogenesis, neuroactive ligand-receptor interaction pathway (P value <0.05). Combined m6A-seq and RNA-seq analysis showed that the hyper-up genes and hypo-up genes were both enriched in ErbB signaling pathway (P value <0.05). In conclusion, it provide a basis for further research into the functions of m6A methylation modifications in pigmentation.
    Keywords:  MeRIP-seq; Pigmentation; Sheep; m6A mRNA methylation
    DOI:  https://doi.org/10.1080/15592294.2023.2230662
  27. Viruses. 2023 06 16. pii: 1381. [Epub ahead of print]15(6):
    m6A Regulates the Stability of Cellular Transcripts Required for Efficient KSHV Lytic Replication.
    Oliver Manners, Belinda Baquero-Perez, Timothy J Mottram, Ivaylo D Yonchev, Christopher J Trevelyan, Katherine L Harper, Sarah Menezes, Molly R Patterson, Andrew Macdonald, Stuart A Wilson, Julie L Aspden, Adrian Whitehouse.
      The epitranscriptomic modification N6-methyladenosine (m6A) is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to the redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m6A in cellular transcripts upregulated during KSHV lytic replication. Our results show that m6A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent-lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NFκB signalling. Overall, this work highlights the central importance of m6A in modulating cellular gene expression to influence viral infection.
    Keywords:  GPCR5A; KSHV; RNA modification; cell signalling; lytic replication; m6A methylation
    DOI:  https://doi.org/10.3390/v15061381
  28. Cell Rep. 2023 Jun 27. pii: S2211-1247(23)00715-5. [Epub ahead of print]42(7): 112704
    Loss of Mettl3 enhances liver tumorigenesis by inducing hepatocyte dedifferentiation and hyperproliferation.
    Tao Wei, Jin Li, Jian Zhang, Qi Zhang, Xiaoyu Liu, Qi Chen, Liang Wen, Ke Ma, Wen Chen, Jianhui Zhao, Cheng Zhang, Jinyan Huang, Yali Xie, Hao Qin, Danfeng Qian, Tingbo Liang.
      While a few works have shown that Mettl3 plays oncogenic roles in hepatocellular carcinoma (HCC), its function in early HCC tumorigenesis remains unclear. In Mettl3flox/flox; Alb-Cre knockout mice, Mettl3 loss leads to aberrant hepatocyte homeostasis and liver damage. Importantly, Mettl3 deletion dramatically accelerates liver tumorigenesis in various HCC mouse models. Depletion of Mettl3 in adult Mettl3flox/flox mice through TBG-Cre administration also enhances liver tumor development, while overexpression of Mettl3 inhibits hepatocarcinogenesis. Mechanistically, aggravated tumorigenesis upon Mettl3 deletion is a consequence of hepatocyte dedifferentiation and hyperproliferation via m6A-mediated modulation on Hnf4α and cell cycle genes. In contrast, by using Mettl3flox/flox; Ubc-Cre mice, depletion of Mettl3 in established HCC ameliorates tumor progression. Additionally, Mettl3 is overexpressed in HCC tumors compared with adjacent non-tumor tissues. The present findings define a tumor-suppressive role of Mettl3 in liver tumorigenesis, indicating its potentially opposite stage-dependent functions in HCC initiation versus progression.
    Keywords:  CP: Cancer; CP: Molecular biology; Hnf4α; Mettl3; context-dependent function; liver cancer; liver damage; m6A modification; tumorigenesis
    DOI:  https://doi.org/10.1016/j.celrep.2023.112704
  29. J Transl Med. 2023 Jun 24. 21(1): 412
    Targeting choroidal vasculopathy via up-regulation of tRNA-derived fragment tRF-22 expression for controlling progression of myopia.
    Chang Liu, Meiyan Li, Yaming Shen, Xiaoyan Han, Ruoyan Wei, Yunzhe Wang, Shanshan Xu, Xingtao Zhou.
       BACKGROUND: Myopia has emerged as a major public health concern globally, which is tightly associated with scleral extracellular matrix (ECM) remodeling and choroidal vasculopathy. Choroidal vasculopathy has gradually been recognized as a critical trigger of myopic pathology. However, the precise mechanism controlling choroidal vasculopathy remains unclear. Transfer RNA-derived fragments (tRFs) are known as a novel class of small non-coding RNAs that plays important roles in several biological and pathological processes. In this study, we investigated the role of tRF-22-8BWS72092 (tRF-22) in choroidal vasculopathy and myopia progression.
    METHODS: The tRF-22 expression pattern under myopia-related stresses was detected by qRT-PCR. MTT assays, EdU incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of tRF-22 in choroidal endothelial cell function in vitro. Isolectin B4 staining and choroidal sprouting assay ex vivo were conducted to detect the role of tRF-22 in choroidal vascular dysfunction in vivo. Immunofluorescent staining, western blot assays and ocular biometric parameters measurement were performed to examine whether altering tRF-22 expression in choroid affects scleral hypoxia and ECM remodeling and myopia progression in vivo. Bioinformatics analysis and luciferase activity assays were conducted to identify the downstream targets of tRF-22. RNA-sequencing combined with m6A-qPCR assays were used to identify the m6A modified targets of METTL3. Gain-of-function and Loss-of-function analysis were performed to reveal the mechanism of tRF-22/METTL3-mediated choroidal vascular dysfunction.
    RESULTS: The results revealed that tRF-22 expression was significantly down-regulated in myopic choroid. tRF-22 overexpression alleviated choroidal vasculopathy and retarded the progression of myopia in vivo. tRF-22 regulated choroidal endothelial cell viability, proliferation, migration, and tube formation ability in vitro. Mechanistically, tRF-22 interacted with METTL3 and blocked m6A methylation of Axin1 and Arid1b mRNA transcripts, which led to increased expression of Axin1 and Arid1b.
    CONCLUSIONS: Our study reveals that the intervention of choroidal vasculopathy via tRF-22-METTL3- Axin1/Arid1b axis is a promising strategy for the treatment of patients with myopic pathology.
    Keywords:  Choroidal vasculopathy; METTL3; Myopia; Transfer RNA-derived fragment; m6A modification
    DOI:  https://doi.org/10.1186/s12967-023-04274-5
  30. J Cancer Res Clin Oncol. 2023 Jun 30.
    The m6A modulator-mediated cytarabine sensitivity and immune cell infiltration signature in acute myeloid leukemia.
    Jincai Yang, Liangliang Li, Juan Cheng, Jianle Lu, Shuling Zhang, Shan Wang, Li Zhao, Lanxia Zhou.
       PURPOSE: The study aims to investigate the impact of m6A modulators on drug resistance and the immune microenvironment in acute myeloid leukemia (AML). The emergence of drug resistance is a significant factor that contributes to relapse and refractory AML, leading to a poor prognosis.
    METHODS: The AML transcriptome data were retrieved from the TCGA database. The "oncoPredict" R package was utilized to assess the sensitivity of each sample to cytarabine (Ara-C) and classify them into distinct groups. Differential expression analysis was performed to identify m6A modulators differentially expressed between the two groups. Select Random Forest (RF) to build a predictive model. Model performance was evaluated using calibration curve, clinical decision curve, and clinical impact curve. The impacts of METTL3 on Ara-C sensitivity and immune microenvironment in AML were examined using GO, KEGG, CIBERSORT, and GSEA analyses.
    RESULTS: Seventeen out of 26 m6A modulators exhibited differential expression between the Ara-C-sensitive and resistant groups, with a high degree of correlation. We selected the 5 genes with the highest scores in the RF model to build a reliable and accurate prediction model. METTL3 plays a vital role in m6A modification, and further analysis shows its impact on the sensitivity of AML cells to Ara-C through its interaction with 7 types of immune-infiltrating cells and autophagy.
    CONCLUSION: This study utilizes m6A modulators to develop a prediction model for the sensitivity of AML patients to Ara-C, which can assist in treating AML drug resistance by targeting mRNA methylation.
    Keywords:  AML; Ara-C; Drug sensitivity; Immune infiltrating cells; METTL3; m6A
    DOI:  https://doi.org/10.1007/s00432-023-05029-x
  31. Cell. 2023 Jun 21. pii: S0092-8674(23)00598-6. [Epub ahead of print]
    QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.
    Zhicong Zhao, Ying Qing, Lei Dong, Li Han, Dong Wu, Yangchan Li, Wei Li, Jianhuang Xue, Keren Zhou, Miao Sun, Brandon Tan, Zhenhua Chen, Chao Shen, Lei Gao, Andrew Small, Kitty Wang, Keith Leung, Zheng Zhang, Xi Qin, Xiaolan Deng, Qiang Xia, Rui Su, Jianjun Chen.
      N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.
    Keywords:  G3BP1; METTL1; N(7)-methylguanosine; QKI; drug resistance; m(7)G; mRNA metabolism; mRNA stability; stress granule; translational regulation
    DOI:  https://doi.org/10.1016/j.cell.2023.05.047
  32. Cancers (Basel). 2023 Jun 14. pii: 3188. [Epub ahead of print]15(12):
    Adenosine Methylation Level of miR-125a-5p Promotes Anti-PD-1 Therapy Escape through the Regulation of IGSF11/VSIG3 Expression.
    Gwenola Bougras-Cartron, Arulraj Nadaradjane, Marie-Pierre Joalland, Lisenn Lalier-Bretaudeau, Judith Raimbourg, Pierre-François Cartron.
       BACKGROUND: Despite encouraging anti-tumour activity in lung cancer, anti-PD-1 therapy has encountered increasing resistance to treatment. Several companion diagnostic assays have been performed to identify patients who may benefit from this immunotherapy and to adapt this therapy in case of acquired resistance.
    METHODS: A large panel of methods was used for the analysis of expression and methylation levels of miRNAs (qPCR, MemiRIP, …), protein/miRNA interactions (CLIP, oligo pull-down, …), and protein-protein interactions (CoIP) in cells and/or blood samples.
    RESULTS: Our work highlights that the saturation of PD-1 by anti-PD1 therapies induces an immune escape phenomenon due to the overexpression of IGSF11 following adenosine methylation of miR-125a-5p. Mechanistically, we identify METTL3/KHDRBS3 and HuR as two crucial players in the methylation and the loss of the repressive function of this miRNA. Finally, our work shows that the adenosine methylation of miR-125a-5p is analyzable from EVs/exosomes from longitudinal blood samples and that such EVs/exosomes modulate the IGSF11/VSIG3 expression in lung cancer cells to promote an immune escape phenomenon.
    CONCLUSIONS: Our data provide a biomarker (m6A-miR-125a-5p level) and two therapeutic solutions (anti-IGSF11 antibody and METTL3 inhibitor) that could potentially address the anti-PD1 therapy failure in the context of precision and personalized medicine.
    Keywords:  PD-1 therapy; adenosine; epitranscriptomics; lung cancer; methylation
    DOI:  https://doi.org/10.3390/cancers15123188
  33. Transl Oncol. 2023 Jun 26. pii: S1936-5233(23)00112-2. [Epub ahead of print]35 101726
    Single-cell and spatial transcriptomics reveal 5-methylcytosine RNA methylation regulators immunologically reprograms tumor microenvironment characterizations, immunotherapy response and precision treatment of clear cell renal cell carcinoma.
    Cheng-Peng Gui, Jin-Huan Wei, Chi Zhang, Yi-Ming Tang, Guan-Nan Shu, Rong-Pei Wu, Jun-Hang Luo.
      Clear cell Renal Cell Carcinoma (ccRCC) is a highly heterogeneous disease, making it challenging to predict prognosis and therapy efficacy. In this study, we aimed to explore the role of 5-methylcytosine (m5C) RNA modification in ccRCC and its potential as a predictor for therapy response and overall survival (OS). We established a novel 5-methylcytosine RNA modification-related gene index (M5CRMRGI) and studied its effect on the tumor microenvironment (TME) using single-cell sequencing data for in-depth analysis, and verified it using spatial sequencing data. Our results showed that M5CRMRGI is an independent predictor of OS in multiple datasets and exhibited outstanding performance in predicting the OS of ccRCC. Distinct mutation profiles, hallmark pathways, and infiltration of immune cells in TME were observed between high- and low-M5CRMRGI groups. Single-cell/spatial transcriptomics revealed that M5CRMRGI could reprogram the distribution of tumor-infiltrating immune cells. Moreover, significant differences in tumor immunogenicity and tumor immune dysfunction and exclusion (TIDE) were observed between the two risk groups, suggesting a better response to immune checkpoint blockade therapy of the high-risk group. We also predicted six potential drugs binding to the core target of the M5CRMRGI signature via molecular docking. Real-world treatment cohort data proved once again that high-risk patients were appropriate for immune checkpoint blockade therapy, while low-risk patients were appropriate for Everolimus. Our study shows that the m5C modification landscape plays a role in TME distribution. The proposed M5CRMRGI-guided strategy for predicting survival and immunotherapy efficacy, we reported here, might also be applied to more cancers other than ccRCC.
    Keywords:  Clear cell renal cell carcinoma; Single-cell transcriptomics; Spatial transcriptomics; Tumor microenvironment; m5C RNA modification
    DOI:  https://doi.org/10.1016/j.tranon.2023.101726
  34. Cell Oncol (Dordr). 2023 Jun 24.
    LncRNA MIR4435-2HG drives cancer progression by modulating cell cycle regulators and mTOR signaling in stroma-enriched subtypes of urothelial carcinoma of the bladder.
    Lu Pei, Dong Yan, Qingqing He, Jianqiu Kong, Meihua Yang, Honglian Ruan, Qiongqiong Lin, Lifang Huang, Jian Huang, Tianxin Lin, Haide Qin.
       BACKGROUND: The risk for recurrence and metastasis after treatment for urothelial carcinoma of the bladder (UCB) is high. Therefore, identifying efficient prognostic markers and novel therapeutic targets is urgently needed. Several long noncoding RNAs (lncRNAs) have been reported to be correlated with UCB progression. In this study, we found that the subtype-specific lncRNA MIR4435-2 host gene (MIR4435-2HG) plays a novel oncogenic role in UCB.
    METHODS: RNA-Seq data of TCGA/BLCA were analyzed. The expression of MIR4435-2HG was measured by qRT-PCR in 16 pairs of bladder cancer tissues and adjacent normal tissues. The clinical relecance of MIR4435-2HG was validated via in situ hybridization performed on an in-house cohort of 116 UCB patient samples. RNA pull-down followed by mass spectrometry was performed to identify MIR4435-2HG-binding proteins. To identify signaling pathways involved in MIR4435-2HG activity, comprehensive in vitro and in vivo studies and RNA-Seq assays were performed using UCB cells in which MIR4435-2HG expression was knocked down or exogenously overexpressed. In addition, we performed RNA immunoprecipitation and Western blot analyses to validate the identified MIR4435-2HG-binding proteins and to determine the molecular mechanisms by which MIR4435-2HG promotes UCB progression.
    RESULTS: We found that MIR4435-2HG was significantly upregulated in the stromal-enriched subtype of UCB. Increased MIR4435-2HG expression was positively correlated with a high histological grade, advanced T stages, larger tumors, lymph node metastasis and a poor prognosis. In vitro experiments revealed that MIR4435-2HG expression silencing suppressed cell proliferation and induced apoptosis. Inhibition of MIR4434-2HG delayed xenograft tumor growth, while MIR4435-2HG overexpression reversed the MIR4435-2HG silencing-induced inhibition of UCB tumor phenotype acquisition. Mechanistically, we found that MIR4435-2HG positively regulated the expression of a variety of cell cycle regulators, including BRCA2 and CCND1. Knocking down MIR4435-2HG increased the sensitivity of tumor cells to the VEGFR inhibitor cediranib. Furthermore, we found that MIR4435-2HG regulated mTOR signaling and epithelial-mesenchymal transition (EMT) signaling pathways by modulating the phosphorylation of mTOR, 70S6K and 4EBP1. Finally, we confirmed that MIR4435-2HG enhances tumor metastasis through regulation of the EMT pathway.
    CONCLUSIONS: Our data indicate that upregulated MIR4435-2HG expression levels are significantly correlated with a poor prognosis of UCB patients. MIR4435-2HG promotes bladder cancer progression, mediates cell cycle (de)regulation and modulates mTOR signaling. MIR4435-2HG is an oncogenic lncRNA in UCB that may serve as a diagnostic and therapeutic target.
    Keywords:  LncRNA; MIR4435-2HG; Molecular subtype; Urothelial carcinoma of the bladder (UCB) ; mTOR
    DOI:  https://doi.org/10.1007/s13402-023-00826-5
  35. Nucleic Acids Res. 2023 Jun 28. pii: gkad534. [Epub ahead of print]
    Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers.
    Giuseppe Nicastro, Giancarlo Abis, Pierre Klein, Sofia Esteban-Serna, Christopher Gallagher, Belen Chaves-Arquero, Yuyang Cai, Angelo Miguel Figueiredo, Stephen R Martin, Rickie Patani, Ian A Taylor, Andres Ramos.
      m6A methylation provides an essential layer of regulation in organismal development, and is aberrant in a range of cancers and neuro-pathologies. The information encoded by m6A methylation is integrated into existing RNA regulatory networks by RNA binding proteins that recognise methylated sites, the m6A readers. m6A readers include a well-characterised class of dedicated proteins, the YTH proteins, as well as a broader group of multi-functional regulators where recognition of m6A is only partially understood. Molecular insight in this recognition is essential to build a mechanistic understanding of global m6A regulation. In this study, we show that the reader IMP1 recognises the m6A using a dedicated hydrophobic platform that assembles on the methyl moiety, creating a stable high-affinity interaction. This recognition is conserved across evolution and independent from the underlying sequence context but is layered upon the strong sequence specificity of IMP1 for GGAC RNA. This leads us to propose a concept for m6A regulation where methylation plays a context-dependent role in the recognition of selected IMP1 targets that is dependent on the cellular concentration of available IMP1, differing from that observed for the YTH proteins.
    DOI:  https://doi.org/10.1093/nar/gkad534
  36. Cancer Lett. 2023 Jun 22. pii: S0304-3835(23)00236-7. [Epub ahead of print]567 216285
    NUSAP1-LDHA-Glycolysis-Lactate feedforward loop promotes Warburg effect and metastasis in pancreatic ductal adenocarcinoma.
    Ming Chen, Kaili Cen, Yinjing Song, Xiaochen Zhang, Yih-Cherng Liou, Pu Liu, Jinyan Huang, Jian Ruan, Jia He, Wanyi Ye, Tianyue Wang, Xing Huang, Jiaqi Yang, Yunlu Jia, Xue Liang, Peng Shen, Qingqing Wang, Tingbo Liang.
      Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxia and hypovascular tumor microenvironment. Nucleolar and spindle associated protein 1 (NUSAP1) is a microtubule-associated protein that is known to be involved in cancer biology. Our study aimed to investigate the role of NUSAP1 in glycolytic metabolism and metastasis in PDAC. Expression and prognostic value of NUSAP1 in PDAC and common gastrointestinal tumors was evaluated. The function of NUSAP1 in PDAC progression was clarified by single-cell RNA-seq and further experiments in vitro, xenograft mouse model, spontaneous PDAC mice model and human tissue microarray. The downstream genes and signaling pathways regulated by NUSAP1 were explored by RNA-Seq. And the regulation of NUSAP1 on Lactate dehydrogenase A (LDHA)-mediated glycolysis and its underlying mechanism was further clarified by CHIP-seq. NUSAP1 was an independent unfavorable predictor of PDAC prognosis that playing a critical role in metastasis of PDAC by regulating LDHA-mediated glycolysis. Mechanically, NUSAP1 could bind to c-Myc and HIF-1α that forming a transcription regulatory complex localized to LDHA promoter region and enhanced its expression. Intriguingly, lactate upregulated NUSAP1 expression by inhibiting NUSAP1 protein degradation through lysine lactylated (Kla) modification, thus forming a NUSAP1-LDHA-glycolysis-lactate feedforward loop. The NUSAP1-LDHA-glycolysis-lactate feedforward loop is one of the underlying mechanisms to explain the metastasis and glycolytic metabolic potential in PDAC, which also provides a novel insights to understand the Warburg effect in cancer. Targeting NUSAP1 would be an attractive paradigm for PDAC treatment.
    Keywords:  Glycolysis; LDHA; Lactylation; NUSAP1; PDAC
    DOI:  https://doi.org/10.1016/j.canlet.2023.216285
  37. Cell Rep. 2023 Jun 23. pii: S2211-1247(23)00695-2. [Epub ahead of print]42(7): 112684
    METTL3-mediated m6A methylation orchestrates mRNA stability and dsRNA contents to equilibrate γδ T1 and γδ T17 cells.
    Zhiqiang Xiao, Shanshan Wang, Yixia Tian, Wenkai Lv, Hao Sheng, Mingjie Zhan, Qiongxiao Huang, Zhanpeng Zhang, Leqing Zhu, Chuyun Zhu, Hui Zhong, Qiong Wen, Zonghua Liu, Jingyi Tan, Yan Xu, Meixiang Yang, Yumei Liu, Richard A Flavell, Quanli Yang, Guangchao Cao, Zhinan Yin.
      γδ T cells make key contributions to tissue physiology and immunosurveillance through two main functionally distinct subsets, γδ T1 and γδ T17. m6A methylation plays critical roles in controlling numerous aspects of mRNA metabolism that govern mRNA turnover, gene expression, and cellular functional specialization; however, its role in γδ T cells remains less well understood. Here, we find that m6A methylation controls the functional specification of γδ T17 vs. γδ T1 cells. Mechanistically, m6A methylation prevents the formation of endogenous double-stranded RNAs and promotes the degradation of Stat1 transcripts, which converge to prevent over-activation of STAT1 signaling and ensuing inhibition of γδ T17. Deleting Mettl3, the key enzyme in the m6A methyltransferases complex, in γδ T cells reduces interleukin-17 (IL-17) production and ameliorates γδ T17-mediated psoriasis. In summary, our work shows that METTL3-mediated m6A methylation orchestrates mRNA stability and double-stranded RNA (dsRNA) contents to equilibrate γδ T1 and γδ T17 cells.
    Keywords:  CP: Immunology; CP: Molecular biology; IL-17; m6A methylation; psoriasis; γδ T cells
    DOI:  https://doi.org/10.1016/j.celrep.2023.112684
  38. Int Immunopharmacol. 2023 Jun 28. pii: S1567-5769(23)00825-1. [Epub ahead of print]122 110502
    Schisandrin treatment suppresses the proliferation, migration, invasion, and inflammatory responses of fibroblast-like synoviocytes from rheumatoid arthritis patients and attenuates synovial inflammation and joint destruction in CIA mice.
    Wei Lin, Yingli Liu, Shuoyang Zhang, Siqi Xu, Qian Qiu, Cuicui Wang, Di Liu, Chuyu Shen, Meilin Xu, Maohua Shi, Youjun Xiao, Guoqiang Chen, Hanshi Xu, Liuqin Liang.
       BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disease causing joint dysfunction. As disease-modifying anti-rheumatic drugs (DMARDs) have poor efficacy in 20% to 25% of RA patients, additional novel RA medications are urgently needed. Schisandrin (SCH) has multiple therapeutic effects. However, whether SCH is effective against RA remains unknown.
    PURPOSE: To investigate how SCH affects the abnormal behaviours of RA fibroblast-like synoviocytes (FLSs) and further elucidate the underlying mechanism of SCH in RA FLSs and collagen-induced arthritis (CIA) mice.
    METHODS: Cell Counting Kit-8 (CCK8) assays were used to characterize cell viability. EdU assays were performed to assess cell proliferation. Annexin V-APC/PI assays were used to determine apoptosis. Transwell chamber assays were used to measure cell migration and invasion in vitro. RT-qPCR was used to assess proinflammatory cytokine and MMP mRNA expression. Western blotting was used to detect protein expression. RNA sequencing was performed to explore the potential downstream targets of SCH. CIA model mice were used to assess the treatment efficacy of SCH in vivo.
    RESULTS: Treatments with SCH (50, 100, and 200 μΜ) inhibited RA FLSs proliferation, migration, invasion, and TNF-α-induced IL-6, IL-8, and CCL2 expression in a dose-dependent manner but did not affect RA FLSs viability or apoptosis. RNA sequencing and Reactome enrichment analysis indicated that SREBF1 might be the downstream target in SCH treatment. Furthermore, knockdown of SREBF1 exerted effects similar to those of SCH in inhibiting RA FLSs proliferation, migration, invasion, and TNF-α-induced expression of IL-6, IL-8, and CCL2. Both SCH treatment and SREBF1 knockdown decreased activation of the PI3K/AKT and NF-κB signalling pathways. Moreover, SCH ameliorated joint inflammation and cartilage and bone destruction in CIA model mice.
    CONCLUSION: SCH controls the pathogenic behaviours of RA FLSs by targeting SREBF1-mediated activation of the PI3K/AKT and NF-κB signalling pathways. Our data suggest that SCH inhibits FLS-mediated synovial inflammation and joint damage and that SCH might have therapeutic potential for RA.
    Keywords:  AKT signalling pathway; Fibroblast-like synoviocytes; NF-κB signalling pathway; Rheumatoid arthritis; SREBF1; Schisandrin
    DOI:  https://doi.org/10.1016/j.intimp.2023.110502
  39. J Transl Med. 2023 06 27. 21(1): 418
    RP11-296E3.2 acts as an important molecular chaperone for YBX1 and promotes colorectal cancer proliferation and metastasis by activating STAT3.
    Qian Shi, Ying He, Shouyu He, Jingjing Li, Ji Xia, Tianwei Chen, Lixia Huo, Yuhang Ling, Qinchen Liu, Wei Zang, Qiang Wang, Chengwu Tang, Xiang Wang.
       BACKGROUND: RP11-296E3.2 is a novel long noncoding RNA (lncRNA) associated with colorectal cancer (CRC) metastasis, that was reported in our previous clinical studies. However, the mechanisms of RP11-296E3.2 in colorectal tumorigenesis remain elusive.
    METHODS: RNA sequencing (RNA-seq), Fluorescence in situ hybridization (FISH), Transwell assays and others, were performed to evaluate the function of RP11-296E3.2 for proliferation and metastasis in vitro. In situ and metastatic tumor models were performed to evaluate the function of RP11-296E3.2 for proliferation and metastasis in vivo. RNA-pulldown, RNA-interacting protein immunoprecipitation (RIP), tissue microarray (TMA) assay, a luciferase reporter assay, chromatin immunoprecipitation (ChIP) and others were performed to explore the mechanisms by which RP11-296E3.2 regulates CRC tumorigenesis.
    RESULTS: RP11-296E3.2 was confirmed to be associated with CRC cell proliferation and metastasis in vitro and in vivo. Mechanistically, RP11-296E3.2 directly bound to recombinant Y-Box Binding Protein 1 (YBX1) and enhanced signal transducer and activator of transcription 3 (STAT3) transcription and phosphorylation. YBX1 promoted the CRC cell proliferation and migration, while knockdown of RP11-296E3.2 attenuated the effects of YBX1 on CRC cell proliferation, and metastasis and the expression of several related downstream genes. We are the first to discover and confirm the existence of the YBX1/STAT3 pathway, a pathway dependent on RP11-296E3.2.
    CONCLUSION: Together, these novel findings show that the RP11-296E3.2/YBX1 pathway promotes colorectal tumorigenesis and progression by activating STAT3 transcription and phosphorylation, and suggest that RP11-296E3.2 is a potential diagnostic biomarker and therapeutic target in CRC.
    Keywords:  CRC; Metastasis; Proliferation; RP11-296E3.2/YBX1; STAT3 transcription
    DOI:  https://doi.org/10.1186/s12967-023-04267-4
  40. Cell Death Discov. 2023 Jun 27. 9(1): 194
    SOX1 acts as a tumor hypnotist rendering nasopharyngeal carcinoma cells refractory to chemotherapy.
    Xin-Xing Lei, Shu-Lan Wang, Ying Xia, Min Yan, Bin He, Bo Wang, Zi-Jie Long, Quentin Liu.
      SOX1, a well-known tumor suppressor, delays malignant progression in most cancer types. However, high expression of SOX1 in late-stage head and neck squamous cell carcinoma leads to poor prognosis. In this study, we show that SOX1 induces nasopharyngeal carcinoma (NPC) cells to enter a quiescent state. Using a model that mimics therapeutic resistance and tumor recurrence, a subpopulation of SOX1-induced NPC cells is refractory to paclitaxel, a cell cycle-specific chemotherapy drug. These cells maintain a quiescent state with decreased translational activity and down-regulated cell growth potential. However, once SOX1 expression is decreased, the NPC cells recover and enter a proliferative state. The chemotherapy resistance induced by SOX1 can not pass to next generation, as the cells that undergo re-proliferation become sensitive to paclitaxel again. Moreover, SOX1 directly binds to the promoter region of the MYC gene, leading to transcriptional suppression. When switching to a paclitaxel-free culture environment, the cells with decreased levels of SOX1 re-express MYC, resulting in increased abundance of proliferative cancer cells. Our study presents an evolutionary trade-off between tumor growth and chemoresistance orchestrated by SOX1-MYC in NPC. Basing on the dynamic role of SOX1 in different stages of cancer development, SOX1 would be regarded as a "tumor hypnotist".
    DOI:  https://doi.org/10.1038/s41420-023-01479-x
  41. J Immunother Cancer. 2023 Jun;pii: e006890. [Epub ahead of print]11(6):
    PRMT5 reduces immunotherapy efficacy in triple-negative breast cancer by methylating KEAP1 and inhibiting ferroptosis.
    Zhe Wang, Ruolei Li, Niuniu Hou, Juliang Zhang, Ting Wang, Pengyu Fan, Cheng Ji, Bo Zhang, Liuyin Liu, Yaping Wang, Jing Kong, Qing Yao, Jie Duan, Ge Zhao, Rui Ling, Jian Zhang.
       BACKGROUND: As an emerging treatment strategy for triple-negative breast cancer (TNBC), immunotherapy acts in part by inducing ferroptosis. Recent studies have shown that protein arginine methyltransferase 5 (PRMT5) has distinct roles in immunotherapy among multiple cancers by modulating the tumor microenvironment. However, the role of PRMT5 during ferroptosis, especially for TNBC immunotherapy, is unclear.
    METHODS: PRMT5 expression in TNBC was measured by IHC (immunohistochemistry) staining. To explore the function of PRMT5 in ferroptosis inducers and immunotherapy, functional experiments were conducted. A panel of biochemical assays was used to discover potential mechanisms.
    RESULTS: PRMT5 promoted ferroptosis resistance in TNBC but impaired ferroptosis resistance in non-TNBC. Mechanistically, PRMT5 selectively methylated KEAP1 and thereby downregulated NRF2 and its downstream targets which can be divided into two groups: pro-ferroptosis and anti-ferroptosis. We found that the cellular ferrous level might be a critical factor in determining cell fate as NRF2 changes. In the context of higher ferrous concentrations in TNBC cells, PRMT5 inhibited the NRF2/HMOX1 pathway and slowed the import of ferrous. In addition, a high PRMT5 protein level indicated strong resistance of TNBC to immunotherapy, and PRMT5 inhibitors potentiated the therapeutic efficacy of immunotherapy.
    CONCLUSIONS: Our results reveal that the activation of PRMT5 can modulate iron metabolism and drive resistance to ferroptosis inducers and immunotherapy. Accordingly, PRMT5 can be used as a target to change the immune resistance of TNBC.
    Keywords:  Breast Neoplasms; Immunotherapy
    DOI:  https://doi.org/10.1136/jitc-2023-006890
  42. Gynecol Oncol. 2023 Jun 23. pii: S0090-8258(23)00324-4. [Epub ahead of print]175 133-141
    The differences in immune features and genomic profiling between squamous cell carcinoma and adenocarcinoma - A multi-center study in Chinese patients with uterine cervical cancer.
    Jin Li, Xiaohong Xue, Yan Zhang, Fengna Ding, Wenyan Wu, Cuicui Liu, Yang Xu, Hanlin Chen, Qiuxiang Ou, Yang Shao, Xinjun Li, Fei Wu, Xiaohua Wu.
       BACKGROUND: Squamous cell carcinoma (SCC) and adenocarcinoma (AC) of the uterine cervix have distinct biological behaviors and different treatment responses. Studies on immune features and genomic profiling of these two pathologic types were limited and mainly focused on small patient cohorts.
    METHODS: From 2014 to 2021, 336 (254 SCC vs. 82 AC) cervical cancer patients who were diagnosed/treated in 7 medical centers in China were enrolled in the study. Next-generation sequencing of 425 cancer-relevant genes was performed on tumor tissues and liquid biopsies. Somatic alterations and immune response-related biomarkers were analyzed. Patient prognosis and immune infiltration were analyzed using data from The Cancer Genome Atlas (TCGA).
    RESULTS: AC tended to have more immunotherapy resistance-related STK11 alterations (P = 0.039), a higher proportion of microsatellite instability (P = 0.21), and more actionable mutations (P = 0.161). In contrast, higher tumor mutational burdens (TMB; P = 0.01), a higher proportion of TMB-high patients (P = 0.016), and more PD-L1-high patients (P = 0.0013) were observed in SCC. Multiple genetic alterations and aberrant signaling pathways were specifically enriched in AC (e.g., TP53, KRAS, ERBB2, and ARID1A alterations) or SCC (e.g., PIK3CA, FBXW7, EP300, and BAP1 mutations). Notably, AC-enriched genetic changes were significantly associated with decreased infiltrations of various B cells, T cells, and dendritic cells, whereas SCC-associated molecular features tended to be associated with increased CD4+ T cell infiltrations.
    CONCLUSIONS: This was the first multi-center study revealing the immunologic and genomic features between SCC and AC in Chinese patients with cervical cancer. Our findings have illustrated the difference in genetic profiles of those two cervical cancer subtypes, which may suggest the possibility of differential treatment regimens, with better immunotherapy efficacy in SCC and targeted therapy options more favorable in AC.
    Keywords:  Adenocarcinoma; Cervical cancer; Genomic profiles; Immune features; Squamous cell carcinoma
    DOI:  https://doi.org/10.1016/j.ygyno.2023.05.071
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