m6A mRNA Research Articles

Expression profiling of N6-methyladenosine-modified mRNA in PC12 cells in response to unconjugated bilirubin

BackgroundAbnormal 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.MethodsRat 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).ResultsCompared 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.ConclusionOur study suggests that the modulation of m6A methylation modifications plays a significant role in UCB neurotoxicity.

K235 acetylation couples with PSPC1 to regulate the m6A demethylation activity of ALKBH5 and tumorigenesis

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.

METTL3 boosts mitochondrial fission and induces cardiac fibrosis by enhancing LncRNA GAS5 methylation

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.

METTL3-mediated m6A RNA modification promotes corneal neovascularization by upregulating the canonical Wnt pathway during HSV-1 infection

BackgroundCorneal 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. MethodsA 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. ResultsMETTL3 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. ConclusionOur 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.

O-257 IGF2BP3 induces apoptosis of spermatogenic cells by regulating the stability of Tcf4 mRNA in an m6A-dependent manner, thereby affecting the occurrence of azoospermia

Abstract Study question Does IGF2BP3 regulate Tcf4 mRNA stability in an N6-methyladenosine (m6A)-dependent manner, affect spermatogenesis, and lead to idiopathic non-obstructive azoospermia (iNOA)? Summary answer IGF2BP3 induces apoptosis of spermatogenic cells by regulating the stability of Tcf4 mRNA in an m6A-dependent manner, which in turn affects spermatogenesis leading to iNOA. What is known already Azoospermia is the most serious phenotype of male infertility, affecting approximately 10-15% of male infertility patients. iNOA refers to a class of non-obstructive azoospermia of unknown etiology for which there is no effective treatment. The etiology of iNOA is poorly understood, and its genetic and molecular mechanisms are poorly studied. IGF2BP3 was found to be highly expressed in tumor tissues and to be a post-transcriptional regulator in cancers. The study found that IGF2BP3 is an m6A reading protein that recognizes mRNA m6A modification and enhances mRNA stability and translation. Study design, size, duration We collected testicular tissue from 120 iNOA and 120 patients with obstructive azoospermia through prospective studies to analyze differentially expressed genes by high-throughput RNA sequencing. Participants/materials, setting, methods IGF2BP3 knockout and overexpression mouse models were constructed. Spermatogenesis was assessed by immunostaining, HE staining, spermatogenic specific gene expression, and computer-assisted sperm analysis (CASA). The stability of target mRNA was conducted by ActD. Merip-qRCR was used to detect IGF2BP3 binds to the target gene in an m6A-dependent manner; the m6A modification site of Tcf4 was predicted by SRAMP and verified by double luciferase reporting experiments. Overexpression Tcf4 to analyze its effect on spermatogenesis. Main results and the role of chance RNA-seq showed that IGF2BP3 was lowly expressed in iNOA testicular tissue which was verified by qRT-PCR and immunohistochemistry. After knocking down IGF2BP3, the spermatogenesis process is impaired, HE showed that the arrangement of spermatogenic cells in the seminiferous tubule was disordered and reduced, the spermatogonia and spermatoblasts were significantly reduced, and there were very few sperm in the seminiferous tubule. CASA showed that sperm density was significantly lower than that of the control group (p < 0.05). Western blot and qRT-PCR showed that spermatogenic cells-specific genes Nanos3, Sycp3, Tnp1, proliferation indexes PLZF and PCNA were all reduced compared with the control group. Overexpression of IGF2BP3 can reverse spermatogenesis in azoospermia mice; after knocking down IGF2BP3, the fertility of mice in vivo and in vitro was impaired; apoptosis of spermatogenic cells increased in testicular tissues of mice after knocking down IGF2BP3 and decreased in mouse testicular tissue after knocking down IGF2BP3. IGF2BP3 induces apoptosis of spermatogenic cells by regulating the stability of Tcf4 mRNA in an m6A-dependent manner, resulting in impaired spermatogenesis; overexpression of Tcf4 in GC1 cells knocked down IGF2BP3 reduces apoptosis and increases cell proliferation. Limitations, reasons for caution In this study, we constructed knockdown and overexpression mice models by in situ injection of IGF2BP3 lentivirus in the testes, and the results were more accurate if conditional knockout mice model was used. Wider implications of the findings We found direct clinical and in vivo animal model evidence for the causes of male infertility in patients with idiopathic non-obstructive azoospermia, and for the first time found that IGF2BP3 affects spermatogenesis by regulating the stability of target genes in an m6A-dependent manner. Trial registration number not applicable

83-OR: Defining the Transcriptome and Epitranscriptome of the ß-Cell Compensation to Insulin Resistance

Type 2 diabetes (T2D) is characterized by decreased functional β-cell mass and poor compensation to insulin resistance. Thus, understanding of the mechanism(s) responsible for the physiological adaptation of β-cells to states of high metabolic demand is desirable. We aimed to dissect the transcriptome and m6A mRNA methylation landscape in the β-cell adaptation to insulin resistance. To this end we conducted RNA-sequencing in sorted β-cells and m6A-sequencing in whole islets from three mouse models: a) a dietary model (60% high-fat diet: Control-HF), b) a genetic model (liver-specific insulin receptor KO mouse on chow: LIRKO-C), and c) a superimposed dietary and genetic model of insulin resistance (LIRKO on a high fat diet: LIRKO-HF). These data were compared to littermate controls fed a chow diet (Control-C). Control-HF, LIRKO-C and LIRKO-HF each presented an increase in insulin resistance compared to Control-C. However, LIRKO-C and LIRKO-HF presented a robust insulin secretion capacity and a graded increase in β-cell proliferation and mass compared to Control-C. First, we correlated the transcriptome with surrogates of β-cell adaptation such as insulin secretion, β-cell mass or proliferation. Next, we intersected the genes that positively correlated with β-cell compensation with single-cell RNA seq datasets performed in islets from humans with or without T2D. This bioinformatics analyses identified extracellular matrix (ECM) as the most enriched pathway in the β-cell adaptation to insulin resistance. On the other hand, m6A-sequencing of Control-C, LIRKO-C and LIRKO-HF islets and the intersection of this dataset with the differentially methylated genes in human T2D islets, revealed enrichment in pathways associated with chromatin modifications. Together, these results point to ECM pathways as being important in the transcriptomic remodeling and that m6A acts on chromatin-modifying enzymes in the β-cell compensation to mammalian insulin resistance. Disclosure D.F.De jesus: None. Z.Zhang: Research Support; SinoVac. J.Wei: None. N.K.Brown: None. J.Hu: None. S.Kahraman: Employee; Boehringer Ingelheim Pharmaceuticals Inc. E.Dirice: None. R.Kulkarni: Advisory Panel; Novo Nordisk, Inversago, Biomea Fusion, Inc., REDD Pharma, Research Support; Inversago. Funding American Diabetes Association (7-21-PDF-140 to D.F.DJ.); National Institutes of Health (R01067536)

Transcriptome-wide m6A methylation in natural yellow leaf of Catalpa fargesii.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA, and involved in various biological processes in plants. However, the distribution features and functions of mRNA m6A methylation have been poorly explored in woody perennial plants. In this study, a new natural variety with yellow-green leaves, named Maiyuanjinqiu, was screened from the seedlings of Catalpa fargesii. Based on the preliminary experiment, the m6A methylation levels in the leaves of Maiyuanjinqiu were significantly higher than those in C. fargesii. Furthermore, a parallel analysis of m6A-seq and RNA-seq was carried out in different leaf color sectors. The result showed that m6A modification were mostly identified around the 3'-untranslated regions (3'-UTR), which was slightly negatively correlated with the mRNA abundance. KEGG and GO analyses showed that m6A methylation genes were associated with photosynthesis, pigments biosynthesis and metabolism, oxidation-reduction and response to stress, etc. The overall increase of m6A methylation levels in yellow-green leaves might be associated with the decreased the expression of RNA demethylase gene CfALKBH5. The silencing of CfALKBH5 caused a chlorotic phenotype and increased m6A methylation level, which further confirmed our hypothesis. Our results suggested that mRNA m6A methylation could be considered as a vital epigenomic mark and contribute to the natural variations in plants.

Ythdf2 regulates cardiac remodeling through its mRNA target transcripts

m6A mRNA methylation controls cardiomyocyte function and increased overall m6A levels are a stereotyping finding in heart failure independent of the underlying etiology. However, it is largely unknown how the information is read by m6A reader proteins in heart failure. Here we show that the m6A reader protein Ythdf2 controls cardiac function and identified a novel mechanism how reader proteins control gene expression and cardiac function. Deletion of Ythdf2 in cardiomyocytes in vivo leads to mild cardiac hypertrophy, reduced heart function, and increased fibrosis during pressure overload as well as during aging. Similarly, in vitro the knockdown of Ythdf2 results in cardiomyocyte growth and remodeling. Mechanistically, we identified the eucaryotic elongation factor 2 as post-transcriptionally regulated by Ythdf2 using cell type specific Ribo-seq data. Our study expands our understanding on the regulatory functions of m6A methylation in cardiomyocytes and how cardiac function is controlled by the m6A reader protein Ythdf2.

The plant cytosolic m6A RNA methylome stabilizes photosynthesis in the cold

The sessile lifestyle of plants requires an immediate response to environmental stressors that affect photosynthesis, growth, and crop yield. Here, we showed that three abiotic perturbations—heat, cold, and high light—triggered considerable changes in the expression signatures of 42 epitranscriptomic factors (writers, erasers, and readers) with putative chloroplast-associated functions that formed clusters of commonly expressed genes in Arabidopsis. The expression changes under all conditions were reversible upon deacclimation, identifying epitranscriptomic players as modulators in acclimation processes. Chloroplast dysfunctions, particularly those induced by the oxidative stress-inducing norflurazon in a largely GENOME UNCOUPLED-independent manner, triggered retrograde signals to remodel chloroplast-associated epitranscriptomic expression patterns. N6-methyladenosine (m6A) is known as the most prevalent RNA modification and impacts numerous developmental and physiological functions in living organisms. During cold treatment, expression of components of the primary nuclear m6A methyltransferase complex was upregulated, accompanied by a significant increase in cellular m6A mRNA marks. In the cold, the presence of FIP37, a core component of the writer complex, played an important role in positive regulation of thylakoid structure, photosynthetic functions, and accumulation of photosystem I, the Cytb6f complex, cyclic electron transport proteins, and Curvature Thylakoid1 but not that of photosystem II components and the chloroplast ATP synthase. Downregulation of FIP37 affected abundance, polysomal loading, and translation of cytosolic transcripts related to photosynthesis in the cold, suggesting m6A-dependent translational regulation of chloroplast functions. In summary, we identified multifaceted roles of the cellular m6A RNA methylome in coping with cold; these were predominantly associated with chloroplasts and served to stabilize photosynthesis.

EIF4A3-induced Circ_0001187 facilitates AML suppression through promoting ubiquitin-proteasomal degradation of METTL3 and decreasing m6A modification level mediated by miR-499a-5p/RNF113A pathway

Aberrant expression of circRNAs has been proven to play a crucial role in the progression of acute myeloid leukemia (AML); however, its regulatory mechanism remains unclear. Herein, we identified a novel circRNA, Circ_0001187, which is downregulated in AML patients, and its low level contributes to a poor prognosis. We further validated their expression in large-scale samples and found that only the expression of Circ_0001187 was significantly decreased in newly diagnosed (ND) AML patients and increased in patients with hematological complete remission (HCR) compared with controls. Knockdown of Circ_0001187 significantly promoted proliferation and inhibited apoptosis of AML cells in vitro and in vivo, whereas overexpression of Circ _0001187 exerted the opposite effects. Interestingly, we found that Circ_0001187 decreases mRNA m6A modification in AML cells by enhancing METTL3 protein degradation. Mechanistically, Circ_0001187 sponges miR-499a-5p to enhance the expression of E3 ubiquitin ligase RNF113A, which mediates METTL3 ubiquitin/proteasome-dependent degradation via K48-linked polyubiquitin chains. Moreover, we found that the low expression of Circ _0001187 is regulated by promoter DNA methylation and histone acetylation. Collectively, our findings highlight the potential clinical implications of Circ _0001187 as a key tumor suppressor in AML via the miR-499a-5p/RNF113A/METTL3 pathway.

PRMT1 methylates METTL14 to modulate its oncogenic function

N6-methyladenosine (m6A), the most abundant mRNA modification in mammalian cells, is responsible for mRNA stability and alternative splicing. The METTL3-METTL14-WTAP complex is the only methyltransferase for the m6A modification. Thus, regulation of its enzymatic activity is critical for the homeostasis of mRNA m6A levels in cells. However, relatively little is known about the upstream regulation of the METTL3-METTL14-WTAP complex, especially at the post-translational modification level. The C-terminal RGG repeats of METTL14 are critical for RNA binding. Therefore, modifications on these residues may play a regulatory role in its function. Arginine methylation is a post-translational modification catalyzed by protein arginine methyltransferases (PRMTs), among which PRMT1 preferentially methylates protein substrates with an arginine/glycine-rich motif. In addition, PRMT1 functions as a key regulator of mRNA alternative splicing, which is associated with m6A modification. To this end, we report that PRMT1 promotes the asymmetric methylation of two major arginine residues at the C-terminus of METTL14, and the reader protein SPF30 recognizes this modification. Functionally, PRMT1-mediated arginine methylation on METTL14 is likely essential for its function in catalyzing the m6A modification. Moreover, arginine methylation of METTL14 promotes cell proliferation that is antagonized by the PRMT1 inhibitor MS023. These results indicate that PRMT1 likely regulates m6A modification and promotes tumorigenesis through arginine methylation at the C-terminus of METTL14.

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.

M6A mRNA methylation initiated by METTL3 inhibits KDR translation to increase the efficacy of 131I therapy in papillary thyroid carcinoma

m6A mRNA methylation initiated by METTL3 inhibits KDR translation to increase the efficacy of 131I therapy in papillary thyroid carcinoma

Activation of METTL3 promotes white adipose tissue beiging and combats obesity.

The induction of beige adipocytes in white adipose tissue, also known as WAT beiging, improves glucose and lipid metabolism. However, the regulation of WAT beiging at the posttranscriptional level remains to be studied. Here, we report that METTL3, the methyltransferase of N6-methyladenosine (m6A) mRNA modification, is induced during WAT beiging in mice. Adipose-specific depletion of Mettl3 gene undermines WAT beiging and impairs the metabolic capability of mice fed with a high-fat diet (HFD). Mechanistically, METTL3-catalyzed m6A installation on thermogenic mRNAs, including Krüppel-like factor 9 (Klf9), prevents their degradation. Activation of METTL3 complex by its chemical ligand methyl piperidine-3-carboxylate promotes WAT beiging, reduces body weight, and corrects metabolic disorders in diet-induced obese mice. These findings uncover a novel epitranscriptional mechanism in WAT beiging and identify METTL3 as a potential therapeutic target for obesity-associated diseases.

METTL3-mediated m6A mRNA modification was involved in cadmium-induced liver injury

Cadmium is an environmental pollutant that has extensive deleterious effects. However, the mechanisms underlying the hepatotoxicity induced by long-term exposure to cadmium remained undefined. In the present study, we explored the role of m6A methylation in the development of cadmium-induced liver disease. We showed a dynamic change of RNA methylation in liver tissue from mice administrated with cadmium chloride (CdCl2) for 3, 6 and 9 months, respectively. Particularly, the METTL3 expression was declined in a time-dependent manner, associated with the degree of liver injury, indicating the involvement of METTL3 in hepatotoxicity induced by CdCl2. Moreover, we established a mouse model with liver-specific over-expression of Mettl3 and administrated these mice with CdCl2 for 6 months. Notably, METTL3 highly expressed in hepatocytes attenuated CdCl2-induced steatosis and liver fibrosis in mice. In vitro assay also showed METTL3 overexpression ameliorated the CdCl2-induced cytotoxicity and activation of primary hepatic stellate cells. Furthermore, transcriptome analysis identified 268 differentially expressed genes both in mice liver tissue treated with CdCl2 for 3 months and 9 months. Among them, 115 genes were predicted to be regulated by METTL3 determined by m6A2Target database. Further analysis revealed the perturbation of metabolic pathway, glycerophospholipid metabolism, ErbB signaling pathway, Hippo signaling pathway, and choline metabolism in cancer, and circadian rhythm, led to hepatotoxicity induced by CdCl2. Collectively, our findings reveal new insight into the crucial role of epigenetic modifications in hepatic diseases caused by long-term exposure to cadmium.

Short-chain fatty acid-butyric acid ameliorates granulosa cells inflammation through regulating METTL3-mediated N6-methyladenosine modification of FOSL2 in polycystic ovarian syndrome

Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder characterized by chronic low-grade inflammation. Previous studies have demonstrated that the gut microbiome can affect the host tissue cells’ mRNA N6-methyladenosine (m6A) modifications. This study aimed to understand the role of intestinal flora in ovarian cells inflammation by regulating mRNA m6A modification particularly the inflammatory state in PCOS. The gut microbiome composition of PCOS and Control groups was analyzed by 16S rRNA sequencing, and the short chain fatty acids were detected in patients’ serum by mass spectrometry methods. The level of butyric acid was found to be decreased in the serum of the obese PCOS group (FAT) compared to other groups, and this was correlated with increased Streptococcaceae and decreased Rikenellaceae based on the Spearman’s rank test. Additionally, we identified FOSL2 as a potential METTL3 target using RNA-seq and MeRIP-seq methodologies. Cellular experiments demonstrated that the addition of butyric acid led to a decrease in FOSL2 m6A methylation levels and mRNA expression by suppressing the expression of METTL3, an m6A methyltransferase. Additionally, NLRP3 protein expression and the expression of inflammatory cytokines (IL-6 and TNF-α) were downregulated in KGN cells. Butyric acid supplementation in obese PCOS mice improved ovarian function and decreased the expression of local inflammatory factors in the ovary. Taken together, the correlation between the gut microbiome and PCOS may unveil crucial mechanisms for the role of specific gut microbiota in the pathogenesis of PCOS. Furthermore, butyric acid may present new prospects for future PCOS treatments.

YTHDC2 Retards Cell Proliferation and Triggers Apoptosis in Papillary Thyroid Cancer by Regulating CYLD-Mediated Inactivation of Akt Signaling.

N6-Methyladenosine (m6A) mRNA methylation modification is regarded as an important mechanism involved in diverse physiological processes. YT521-B homology (YTH) domain family members are associated with the tumorigenesis of several cancers. However, the role of YTHDC2 in papillary thyroid cancer (PTC) progression remains unknown. Results showed that YTHDC1, YTHDF1, YTHDF2, and YTHDF3 showed no observable difference in thyroid cancer samples. YTHDC2 was significantly downregulated in thyroid cancer samples and cells. YTHDC2 inhibited cell proliferation in PTC cells. YTHDC2 elicited apoptosis in PTC cells, as demonstrated by the elevated expression of pro-apoptotic factors cl-caspase-3/caspase-3 and Bcl-2-associated (Bax), and the reduced anti-apoptotic B cell lymphoma-2 (Bcl-2) expression. There was a positive correlation between YTHDC2 and cylindromatosis (CYLD) expression based on GEPIA database. YTHDC2 increased CYLD expression in PTC cells. CYLD knockdown abolished the effects of YTHDC2 on PTC cell proliferation and apoptosis. Additionally, YTHDC2 inactivated the protein kinase B (Akt) pathway by increasing CYLD in PTC cells. Overall, YTHDC2 inhibited cell proliferation and induced apoptosis in PTC cells by regulating CYLD-mediated inactivation of Akt pathway.

M6A methylation regulates hypoxia-induced pancreatic cancer glycolytic metabolism through ALKBH5-HDAC4-HIF1α positive feedback loop

Pancreatic cancer (PC) is the most hypoxic cancer type among solid tumors. The dynamic changes of RNA N6-methyl-adenosine (m6A) contribute to tumor cells adaption to hypoxic microenvironmental. However, the regulatory mechanisms of hypoxia response in PC remains elusive. Here, we reported that the m6A demethylase ALKBH5 mediated a decrease of total mRNA m6A modification during hypoxia. Subsequently, methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA sequencing (RNA-seq) revealed transcriptome-wide gene expression alteration and identified histone deacetylase type 4 (HDAC4) as a key target gene of m6A modification under hypoxic conditionds. Mechanistically, m6A methylation recognized by m6A reader-YTHDF2 enhanced the stability of HDAC4, and then promoted glycolytic metabolism and migration of PC cells. Our assays also demonstrated that hypoxia-induced HDAC4 enhanced HIF1a protein stability, and overexpressed HIF1a promoted transcription of ALKBH5 in hypoxic pancreatic cancer cells. Together, these results found a ALKBH5/HDAC4/HIF1α positive feedback loop for cellular response to hypoxia in pancreatic cancer. Our studies uncover the crosstalk between histone acetylation and RNA methylation modification on layer of epigenetic regulation.

METTL3 silenced inhibited the ferroptosis development via regulating the TFRC levels in the Intracerebral hemorrhage progression

Intracerebral hemorrhage (ICH) refers to the hemorrhage caused by the increase and rupture of vascular brittleness in non traumatic brain parenchyma, which has been demonstrated to be closely related to ferroptosis. This study aimed to examine the effects of methyltransferase like 3 (METTL3) on the ferroptosis in the ICH progression. The PC12 cells was stimulated by hemin to establish a ICH model. The cell viability was tested by CCK8 assay. The Fe2+, reactive oxygen species (ROS), and malondialdehyde (MDA) levels were determined by the corresponding commercial kits. The cell death was analyzed by propidium Iodide (PI) staining. The lactylation levels were detected by western blot. M6A dot blot assay was performed to detected the total m6A levels and MeRIP assay was conducted to determine the m6A levels of transferrin receptor (TFRC). We found that the METTL3 and m6A levels were increased in the hemin treated PC12 cells. METTL3 knockdown increased the cell viability and decreased Fe2+, ROS and MDA levels in the hemin treated PC12 cells. The role of METTL3 knockdown in the hemin treated PC12 cells was reversed after TFRC overexpression. Mechanistically, the METTL3 lactylation was increased in the hemin treated PC12 cells, which further enhanced the protein stability and expression of METTL3. The up-regulated METTL3 increased the m6A levels and mRNA expressions of TFRC, which further induced the ferroptosis of the PC12 cells. In conclusion, the up-regulation of METTL3 lactylation enhanced the METTL3 protein stability and expression levels in hemin treated PC12 cells. METTL3 silenced suppressed the ferroptosis development through regulating the m6A levels of TFRC mRNA.

O-GlcNAcylation promotes the cytosolic localization of the m6A reader YTHDF1 and colorectal cancer tumorigenesis

O-linked GlcNAc (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalk with phosphorylation and ubiquitination to orchestrate various biological processes. The mechanisms underlying the involvement of O-GlcNAc modifications in N6-methyladenosine (m6A) regulation are not fully characterized. Herein, we show that O-GlcNAc modifies the m6A mRNA reader YTH domain family 1 (YTHDF1) and fine-tunes its nuclear translocation by the exportin protein Crm1. First, we present evidence that YTHDF1 interacts with the sole O-GlcNAc transferase (OGT). Second, we verified Ser196/Ser197/Ser198 as the YTHDF1 O-GlcNAcylation sites, as described in numerous chemoproteomic studies. Then we constructed the O-GlcNAc-deficient YTHDF1-S196A/S197F/S198A (AFA) mutant, which significantly attenuated O-GlcNAc signals. Moreover, we revealed that YTHDF1 is a nucleocytoplasmic protein, whose nuclear export is mediated by Crm1. Furthermore, O-GlcNAcylation increases the cytosolic portion of YTHDF1 by enhancing binding with Crm1, thus upregulating downstream target (e.g. c-Myc) expression. Molecular dynamics simulations suggest that O-GlcNAcylation at S197 promotes the binding between the nuclear export signal motif and Crm1 through increasing hydrogen bonding. Mouse xenograft assays further demonstrate that YTHDF1-AFA mutants decreased the colon cancer mass and size via decreasing c-Myc expression. In sum, we found that YTHDF1 is a nucleocytoplasmic protein, whose cytosolic localization is dependent on O-GlcNAc modification. We propose that the OGT-YTHDF1-c-Myc axis underlies colorectal cancer tumorigenesis.