RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

Solution structure ensemble of human obesity-associated protein FTO reveals druggable surface pockets at the interface between the N- and C-terminal domain.

The fat mass and obesity-associated FTO protein catalyzes demethylation of the N6-methyladenosine, an epigenetic mark that controls several metabolic pathways by modulating the transcription, translation, and cellular localization of RNA molecules. Since the discovery that its overexpression links to the development of obesity and cancer, FTO was the target of screening campaigns and structure-based drug design efforts. Although several FTO inhibitors were generated, these often lack potency or selectivity. Herein, we investigate the structure and dynamics of human FTO in solution. We show that the structure of the catalytic N-terminal domain is unstable in the absence of the C-terminal domain, which explains why the isolated N-terminal domain is incompetent for catalysis and suggests that the domain interaction represents a target for the development of specific inhibitors. Then, by using NMR relaxation measurements, we show that the interface between the FTO structural domains, the active site, and several peripheral loops undergo conformational dynamics on both the picosecond-nanosecond and microsecond-millisecond timescales. Consistent with this, we found that the backbone amide residual dipolar couplings measured for FTO in phage pf1 are inconsistent with the static crystal structure of the enzyme. Finally, we generated a conformational ensemble for apo FTO that satisfies the solution NMR data by combining the experimental residual dipolar couplings with accelerated molecular dynamics simulations. Altogether, the structural ensemble reported in this work provides an atomic-resolution model of apo FTO and reveals transient surface pockets at the domain interface that represent potential targets for the design of allosteric inhibitors.

RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation.

N(6)-methyladenosine (m(6)A) is the most prevalent and internal modification that occurs in the messenger RNAs (mRNA) of most eukaryotes, although its functional relevance remained a mystery for decades. This modification is installed by the m(6)A methylation "writers" and can be reversed by demethylases that serve as "erasers." In this review, we mainly summarize recent progress in the study of the m(6)A mRNA methylation machineries across eukaryotes and discuss their newly uncovered biological functions. The broad roles of m(6)A in regulating cell fates and embryonic development highlight the existence of another layer of epigenetic regulation at the RNA level, where mRNA is subjected to chemical modifications that affect protein expression.

Multi-substrate selectivity based on key loops and non-homologous domains: new insight into ALKBH family.

AlkB homologs (ALKBH) are a family of specific demethylases that depend on Fe2+ and α-ketoglutarate to catalyze demethylation on different substrates, including ssDNA, dsDNA, mRNA, tRNA, and proteins. Previous studies have made great progress in determining the sequence, structure, and molecular mechanism of the ALKBH family. Here, we first review the multi-substrate selectivity of the ALKBH demethylase family from the perspective of sequence and structural evolution. The construction of the phylogenetic tree and the comparison of key loops and non-homologous domains indicate that the paralogs with close evolutionary relationship have similar domain compositions. The structures show that the lack and variations of four key loops change the shape of clefts to cause the differences in substrate affinity, and non-homologous domains may be related to the compatibility of multiple substrates. We anticipate that the new insights into selectivity determinants of the ALKBH family are useful for understanding the demethylation mechanisms.

Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates.

FTO demethylates internal N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am; at the cap +1 position) in mRNA, m6A and m6Am in snRNA, and N1-methyladenosine (m1A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N6-methyldeoxyadenosine (6mA), 3-methylthymine (3mT), and 3-methyluracil (m3U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate-in vitro and in vivo-that FTO has extensive demethylation enzymatic activity on both internal m6A and cap m6Am Considering that 6mA, m6A, and m6Am all share the same nucleobase, we present a crystal structure of human FTO bound to 6mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m6A and m6Am in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications.

Regulation of autophagy in leukocytes through RNA N6-adenosine methylation in chronic kidney disease patients.

Patients with chronic kidney diseases have multiple cellular dysfunctions leading to increased atherosclerosis, impaired immunity, and disturbed metabolism. However, it is unclear what is the fundamental signaling served as a marker or as a mediator for the dysregulated function in their leukocytes or tissues. Here we hypothesized that the N6-Methyladenosine (m6A) modification of the RNA in the leukocytes is responsible for the cellular dysfunction in chronic kidney diseases. Patients with chronic kidney diseases had significantly less m6A abundances in leukocytes and elevated RNA demethylase FTO proteins. The uremic toxin, indoxyl sulfate, activated the autophagy flux through modulation of FTO and m6A modifications in RNA. Notably, knockdown of FTO or inhibit the m6A by 3-deazaadenosine blocks the effects of indoxyl sulfate on autophagy activation in cells. These findings provide new insights into the mechanisms underlying chronic kidney disease-associated cellular dysfunction. Targeting RNA m6A modification may be a novel strategy for the treatment of chronic kidney diseases and autophagy.

mRNA Traffic Control Reviewed: N6-Methyladenosine (m6 A) Takes the Driver's Seat.

Messenger RNA is a flexible tool box that plays a key role in the dynamic regulation of gene expression. RNA modifications variegate the message conveyed by the mRNA. Similar to DNA and histone modifications, mRNA modifications are reversible and play a key role in the regulation of molecular events. Our understanding about the landscape of RNA modifications is still rudimentary in contrast to DNA and histone modifications. The major obstacle has been the lack of sensitive detection methods since they are non-editing events. However, with the advent of next-generation sequencing techniques, RNA modifications are being identified precisely at single nucleotide resolution. In recent years, methylation at the N6 position of adenine (m6 A) has gained the attention of RNA biologists. The m6 A modification has a set of writers (methylases), erasers (demethylases), and readers. Here, we provide a summary of interesting facts, conflicting findings, and recent advances in the technical and functional aspects of the m6 A epitranscriptome.

Identification of Flavin Mononucleotide as a Cell-Active Artificial N6 -Methyladenosine RNA Demethylase.

N6 -Methyladenosine (m6 A) represents a common and highly dynamic modification in eukaryotic RNA that affects various cellular pathways. Natural dioxygenases such as FTO and ALKBH5 are enzymes that demethylate m6 A residues in mRNA. Herein, the first identification of a small-molecule modulator that functions as an artificial m6 A demethylase is reported. Flavin mononucleotide (FMN), the metabolite produced by riboflavin kinase, mediates substantial photochemical demethylation of m6 A residues of RNA in live cells. This study provides a new perspective to the understanding of demethylation of m6 A residues in mRNA and sheds light on the development of powerful small molecules as RNA demethylases and new probes for use in RNA biology.

Sprouts of RNA epigenetics: the discovery of mammalian RNA demethylases.

More than 100 structurally distinct RNA modifications have been identified in all kingdoms of life. These post-transcriptional modifications are widely present in various RNAs, including ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), long non-coding RNA (lncRNA), etc. We have shown that the methylation of N(6)-methyladenine (m(6)A) can be reversed through the discovery of the first RNA demethylase, the human fat mass and obesity-associated protein, FTO, in 2011. (Most recently, we have identified a new mammalian RNA demethylase, ALKBH5, which is also able to remove the methyl group of m(6)A from RNA both in vitro and in vivo (Fig. 1A). The ALKBH5 protein colocalizes with nuclear speckles where pre-mRNA processing occurs. This protein is actively involved in mRNA export regulation, in which its demethylation activity seems to play an important role, as well as in RNA synthesis. A knockout of the Alkbh5 gene in mice resulted in impaired male fertility due to compromised spermatogenesis. Importantly, increased m(6)A levels were observed in mRNA isolated from the Alkbh5-knockout mouse organs compared to those from wild-type littermates. RNA-Seq results indicate aberrant gene expression in spermatogenic cells of the seminoferous tubulus of testes from Alkbh5-deficient mice, thereby showing that the loss of the m(6)A demethylase influences gene expression, which, in turn, leads to defects in spermatogenesis and increased apoptosis of meiotic cells. Thus, the discovery of FTO and this new RNA demethylase strongly suggests that the methylation of RNA, like DNA and histone modifications, is dynamically regulated and likely to play broad roles in mammalian cells.

Label-free and sensitive detection of N6-methyladenosine demethylase activity in crude cell extracts and clinical cancer tissues based on demethylation-triggered exponential signal amplification.

The m6A demethylase catalyzes the removal of m6A modification to establish proper RNA methylation patterns, and it has emerged as a promising disease biomarker and a therapeutic target. The reported m6A demethylase assays often suffer from tedious producers, expensive reagents, radioactive risk, limited sensitivity, and poor specificity. Herein, we develop a simple, selective, label-free, and highly sensitive fluorescent biosensor for m6A demethylase assay based on demethylation-triggered exponential signal amplification. In this biosensor, m6A demethylase-catalyzed demethylation can protect the circular DNA from the digestion by DpnI, subsequently triggering hyperbranched rolling circle amplification to achieve exponential signal amplification for producing abundant ssDNA and dsDNA products. The amplified DNA signal can be sensitively and simply detected by SYBR Gold in a label-free manner. This biosensor avoids any antibodies, washing/separation procedures, and fluorophore-/quencher-labeled probes, great simplifying the assay procedures and reducing the assay cost. Moreover, this biosensor achieves good specificity and excellent sensitivity with a detection limit of 1.2 fg/µL, which is superior to conventional ELISA (36.3 pg/µL). Especially, this biosensor enables direct monitoring of m6A demethylase activity in crude cell extracts with high accuracy, and it can be further applied for the screening of m6A demethylase inhibitor, measurement of m6A demethylase activity in different cell lines, and discrimination of m6A demethylase level in clinical cancer and healthy tissues, providing a facile and robust platform for RNA methylation-related biomedical research, disease diagnosis, and drug discovery.

Down-regulated FTO and ALKBH5 co-operatively activates FOXO signaling through m6A methylation modification in HK2 mRNA mediated by IGF2BP2 to enhance glycolysis in colorectal cancer.

BACKGROUND: N6-methyladenosine (m6A) modification is the most abundant reversible methylation modification in eukaryotes, and it is reportedly closely associated with a variety of cancers progression, including colorectal cancer (CRC). This study showed that activated lipid metabolism and glycolysis play vital roles in the occurrence and development of CRC. However, only a few studies have reported the biological mechanisms underlying this connection. METHODS: Protein and mRNA levels of FTO and ALKBH5 were measured using western blot and qRT-PCR. The effects of FTO and ALKBH5 on cell proliferation were examined using CCK-8, colony formation, and EdU assays, and the effects on cell migration and invasion were tested using a transwell assay. m6A RNA immunoprecipitation (MeRIP) and RNA-seq was used to explore downstream target gene. RIP was performed to verify the interaction between m6A and HK2. The function of FTO and ALKBH5 in vivo was determined by xenograft in nude mice. RESULTS: In this study, FTO and ALKBH5 were significantly down-regulated in CRC patients and cells both in vivo and in vitro in a high-fat environment. Moreover, FTO and ALKBH5 over-expression hampered cell proliferation both in vitro and in vivo. Conversely, FTO and ALKBH5 knockdown accelerated the malignant biological behaviors of CRC cells. The mechanism of action of FTO and ALKBH5 involves joint regulation of HK2, a key enzyme in glycolysis, which was identified by RNA sequencing and MeRIP-seq. Furthermore, reduced expression of FTO and ALKBH5 jointly activated the FOXO signaling pathway, which led to enhanced proliferation ability in CRC cells. IGF2BP2, as a m6A reader, positively regulated HK2 mRNA in m6A dependent manner. Additionally, down-regulation of FTO/ALKBH5 increased METTL3 and decreased METTL14 levels, further promoting CRC progression. CONCLUSION: In conclusion, our study revealed the FTO-ALKBH5/IGF2BP2/HK2/FOXO1 axis as a mechanism of aberrant m6A modification and glycolysis regulation in CRC.

A study of RNA m6A demethylases in oral epithelial dysplasia and oral squamous cell carcinoma.

Purpose: N6-Methyladenosine (m6A) modification is involved in the tumorigenesis of various cancers. However, the roles of RNA m6A demethylases, fat mass and obesity-associated protein (FTO), and AlkB homolog 5 (ALKBH5) in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC) remain unclear. This study focuses on FTO and ALKBH5 expression by using immunohistochemistry. Material and methods: Twenty specimens each of OED, OSCC, and normal oral mucosa (NOM) were included. The expression pattern, the number of positive cells, the cell-staining intensity, and the histochemical scoring (H-score) were examined and analyzed. Results: In all the OED and OSCC specimens, FTO and ALKBH5 were mainly expressed with moderate to strong staining intensity in the nuclei of the abnormal epithelial cells, respectively. Regarding the NOM, both RNA demethylases showed mild cell staining intensity and was present in 50-60% of the specimens. Interestingly, the percentage of cell positivity, the cell-staining intensity, and the H-score of the FTO and ALKBH5 in NOM, OED, and OSCC were increased, respectively (p < 0.001). There was also a positive correlation between the FTO and ALKBH5 expressions in OSCC (r = 0.62, p = 0.003), but not in the NOM and OED. Conclusion: These results suggest a possible prognostic role of FTO and ALKBH5 expression in the malignant transformation of OED and tumor progression. Further studies are needed to elucidate the mechanisms underlying the roles of FTO and ALKBH5 in carcinogenesis.

Case report: A novel biallelic FTO variant causing multisystem anomalies with severe epilepsy, widening the spectrum of FTO syndrome.

The fat mass and obesity-associated gene (FTO) codes for a DNA/RNA demethylase. Pathological variants in this gene are rare, with only three reports in the literature, all with mutations in the catalytic domain. We report the first biallelic human variant in fat mass and obesity-associated gene (c.287G>C, p.Arg96Pro/R96P) outside the catalytic site, causing numerous abnormalities across multiple organ systems, affecting respiratory, cardiovascular, and neurological function. Biochemical assays of cells with the patient's variant were performed to further quantify the effect of the variant on function. Loss-of-function resulting from the patient's R96P missense variant was demonstrated with in vitro biochemical characterization of demethylase activity, resulting in a 90% reduction in function of the fat mass and obesity-associated protein compared to wild-type. Our findings demonstrate a novel fat mass and obesity-associated gene non-catalytic site variant with a unique patient phenotype of bilateral multifocal epilepsy and multisystem congenital anomalies.

Maslinic Acid Suppresses High Glucose-induced Inflammation by Epigenetically Inhibiting TXNIP Expression.

OBJECTIVE: Hyperglycemia-induced inflammation and subsequent endothelial injuries ultimately lead to the pathogenesis of cardiovascular diseases associated with high mortality, such as atherosclerosis. Maslinic acid (MA) is a phytochemical with anti-inflammatory activity. However, it remains unknown whether it can inhibit diabetes-associated cardiovascular inflammation. The present study aimed to determine the effect of MA on high glucose-induced endothelial inflammation and apoptosis in human umbilical vein endothelial cells (HUVECs) and to explore the underlying mechanism. METHODS: HUVECs were treated with high glucose to induce inflammation and apoptosis. Apoptosis was determined by flow cytometry. CCK-8 assay was used to examine cell viability. Production levels of cytokines were detected by quantitative realtime PCR (qPCR) and ELISA. Protein expression levels and signaling pathways activation were detected by Western blotting. RNA immunoprecipitation and qPCR were used to determine the N6-methyladenosine (m6A) levels of target mRNAs. RESULTS: MA promoted the recruitment of RNA demethylase ALKBH5 to TXNIP mRNA, and subsequently enhanced its m6A demethylation. By this means, MA decreased the stability of TXNIP mRNA and downregulated its expression level. Subsequently, reactive oxygen species (ROS) and production of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1ß, were inhibited. And high glucose-induced apoptosis in HUVECs was inhibited by MA. CONCLUSION: MA ameliorates high glucose-induced endothelial inflammation and injury, serving as a new potential therapeutic application for protecting against diabetes-associated atherosclerosis and other inflammatory diseases.

Tanshinone IIA alleviates cardiac hypertrophy through m6A modification of galectin-3.

Cardiac hypertrophy results from the adaptive response of the myocardium to pressure overload on the heart. Tanshinone IIA (Tan IIA) is the major active compound extracted from Salvia miltiorrhiza Bunge, which possesses various pharmacological benefits. In the present study, the effect and mechanism of action of Tan IIA on cardiac hypertrophy were studied. Ang II-induced and transverse aortic constriction (TAC)-induced cardiomyocyte hypertrophy models were used to evaluate the effect of Tan IIA. An adenoviral vector system was utilized to overexpress galectin-3. The results revealed that Tan IIA significantly inhibited Ang II-induced hypertrophy in vitro and TAC-induced cardiac hypertrophy in vivo. Furthermore, Tan IIA notably inhibited the expression of galectin-3. Rescue experiments indicated that galectin-3 overexpression reversed the effects of Tan IIA, which further validated the interaction between Tan IIA and galectin-3. Additionally, Tan IIA suppressed alkB homolog 5, RNA demethylase (ALKBH5)-mediated N6-methyladenosine (m6A) modification of galectin-3. In summary, the results of the present study indicated that Tan IIA attenuates cardiac hypertrophy by targeting galectin-3, suggesting that galectin-3 plays a critical role in cardiac hypertrophy and represents a new therapeutic target.

The controversial role and therapeutic development of the m6A demethylase FTO in renal cell carcinoma.

Fat mass and obesity-associated (FTO) protein, the first m6A demethylase identified in 2011, regulates multiple aspects of RNA biology including splicing, localization, stability, and translation. Accumulating data show that FTO is involved in numerous physiological processes and is implicated in multiple cancers including renal cell carcinoma (RCC). However, the exact role of FTO in RCC remains controversial. Some studies demonstrated that decreased FTO expression was associated with aggressive clinical features and shorter overall survival in clear cell RCC (ccRCC) patients, while others found that FTO inhibition selectively reduced the growth and survival of VHL-deficient ccRCC cells in vitro and in vivo. Here, we review the evidence supporting either a promoting or suppressive role of FTO in kidney cancers, the mechanisms of action of FTO, and recent progress in developing FTO inhibitors.

mRNA modifications in cardiovascular biology and disease: with a focus on m6A modification.

Among several known RNA modifications, N6-methyladenosine (m6A) is the most studied RNA epitranscriptomic modification and controls multiple cellular functions during development, differentiation, and disease. Current research advancements have made it possible to examine the regulatory mechanisms associated with RNA methylation and reveal its functional consequences in the pathobiology of many diseases, including heart failure. m6A methylation has been described both on coding (mRNA) and non-coding RNA species including rRNA, tRNA, small nuclear RNA and circular RNAs. The protein components which catalyze the m6A methylation are termed methyltransferase or 'm6A writers'. The family of proteins that recognize this methylation are termed 'm6A readers' and finally the enzymes involved in the removal of a methyl group from RNA are known as demethylases or 'm6A erasers'. At the cellular level, different components of methylation machinery are tightly regulated by many factors to maintain the m6A methylation dynamics. The m6A methylation process impacts different stages of mRNA metabolism and the biogenesis of long non-coding RNA and miRNA. Although, mRNA methylation was initially described in the 1970s, its regulatory roles in various diseases, including cardiovascular diseases are broadly unexplored. Recent investigations suggest the important role of m6A mRNA methylation in both hypertrophic and ischaemic heart diseases. In the present review, we evaluate the significance of m6A methylation in the cardiovascular system, in cardiac homeostasis and disease, all of which may help to improve therapeutic intervention for the treatment of heart failure. RNA methylation in cardiovascular diseases: altered m6A RNA (coding and non-coding RNA) methylation is identified during different cardiovascular diseases. Increased cardiac hypertrophy is observed following METTL3 overexpression. In contrast, reduced FTO level was seen in mice following myocardial infarction. Increased cardiac fibroblasts activation or increased atherosclerotic plaques were also co-related with m6A RNA methylation.

ALKBH5 Regulates SPHK1-Dependent Endothelial Cell Angiogenesis Following Ischemic Stress.

BACKGROUND: Endothelial cells dysfunction has been reported in many heart diseases including acute myocardial infarction, and atherosclerosis. The molecular mechanism for endothelial dysfunction in the heart is still not clearly understood. We aimed to study the role of m6A RNA demethylase alkB homolog 5 (ALKBH5) in ECs angiogenesis during ischemic injury. METHODS AND RESULTS: ECs were treated with ischemic insults (lipopolysaccharide and 1% hypoxia) to determine the role of ALKBH5 in ECs angiogenesis. siRNA mediated ALKBH5 gene silencing was used for examining the loss of function. In this study, we report that ALKBH5 levels are upregulated following ischemia and are associated with maintaining ischemia-induced ECs angiogenesis. To decipher the mechanism of action, we found that ALKBH5 is required to maintain eNOS phosphorylation and SPHK1 protein levels. ALKBH5 silencing alone or with ischemic stress significantly increased SPHK1 m6A mRNA methylation. In contrast, METTL3 (RNA methyltransferase) overexpression resulted in the reduced expression of SPHK1. CONCLUSION: We reported that ALKBH5 helps in the maintenance of angiogenesis in endothelial cells following acute ischemic stress via reduced SPHK1 m6A methylation and downstream eNOS-AKT signaling.

Novel Prognostic Implications of Methylated RNA and Demethylases in Resected HCC and Background Liver Tissue.

BACKGROUND/AIM: N6-Methyladenosine (m6A), the most abundant internal modification of RNA, plays a critical role in cancer development. However, the clinical implications of m6A in hepatocellular carcinoma (HCC) remain unclear. MATERIALS AND METHODS: We analyzed 177 HCC and paired noncancerous liver tissues from patients who underwent hepatectomy according to global m6A quantification and expression of m6A demethylases fat mass and obesity-associated protein (FTO) and alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5). RESULTS: The global m6A quantification revealed no significant difference between HCC and non-cancerous tissue. The expression of m6A demethylases FTO and ALKBH5, was significantly lower in HCC than in non-cancerous tissues (both p<0.001). Furthermore, low ALKBH5 expression in non-cancerous tissues was significantly correlated with worse recurrence-free survival (median of 16.3 vs. 38.9 months, p=0.001). CONCLUSION: m6A in HCC and its demethylase in surrounding non-cancerous liver tissues might be involved in inherent mechanisms for HCC development and affect malignant potential after HCC resection.

Cyclophosphamide Regulates N6-Methyladenosine and m6A RNA Enzyme Levels in Human Granulosa Cells and in Ovaries of a Premature Ovarian Aging Mouse Model.

Cyclophosphamide (CTX) is one of the most frequently used alkylating anticancer drugs. CTX is associated with reproductive failure and premature ovarian insufficiency (POI) or premature ovarian aging. Much less is known about the mechanism by which CTX affects female fertility through N6-methyladenosine (m6A) levels. In this case-controlled study, we employed human ovarian granulosa cells and mice as experimental models in vitro and in vivo. m6A test kit was developed to determine the content in RNA, and qPCR and western blot were used to examine the expression levels of RNA methyltransferases, demethylases, and effectors. Results showed that CTX increased the m6A level in a time- and concentration-dependent manner. The expression levels of RNA methyltransferases were significantly higher in the CTX treatment group than in the control group with time and concentration dependence, except for RBM15 and WTAP. CTX significantly inhibited the expression levels of RNA demethylase FTO in a time- and concentration-dependent manner but not ALKBH5. The expression levels of RNA effectors were reduced by CTX in a time- and concentration-dependent manner. These data suggest that CTX increased the expression levels of m6A and may be responsible for the increase in RNA methyltransferases and decrease in RNA demethylases in a time- and concentration-dependent manner.