RNA m6 A modification enzymes shape innate responses to DNA by regulating interferon ß.

Modification of mRNA by N6-adenosine methylation (m6A) on internal bases influences gene expression in eukaryotes. How the dynamic genome-wide landscape of m6A-modified mRNAs impacts virus infection and host immune responses remains poorly understood. Here, we show that type I interferon (IFN) production triggered by dsDNA or human cytomegalovirus (HCMV) is controlled by the cellular m6A methyltrasferase subunit METTL14 and ALKBH5 demethylase. While METTL14 depletion reduced virus reproduction and stimulated dsDNA- or HCMV-induced IFNB1 mRNA accumulation, ALKBH5 depletion had the opposite effect. Depleting METTL14 increased both nascent IFNB1 mRNA production and stability in response to dsDNA. In contrast, ALKBH5 depletion reduced nascent IFNB1 mRNA production without detectably influencing IFN1B mRNA decay. Genome-wide transcriptome profiling following ALKBH5 depletion identified differentially expressed genes regulating antiviral immune responses, while METTL14 depletion altered pathways impacting metabolic reprogramming, stress responses, and aging. Finally, we determined that IFNB1 mRNA was m6A-modified within both the coding sequence and the 3' untranslated region (UTR). This establishes that the host m6A modification machinery controls IFNß production triggered by HCMV or dsDNA. Moreover, it demonstrates that responses to nonmicrobial dsDNA in uninfected cells, which shape host immunity and contribute to autoimmune disease, are regulated by enzymes controlling m6A epitranscriptomic changes.

RNA m6A demethylase ALKBH5 regulates the development of γδ T cells.

γδ T cells are an abundant T cell population at the mucosa and are important in providing immune surveillance as well as maintaining tissue homeostasis. However, despite γδ T cells' origin in the thymus, detailed mechanisms regulating γδ T cell development remain poorly understood. N6-methyladenosine (m6A) represents one of the most common posttranscriptional modifications of messenger RNA (mRNA) in mammalian cells, but whether it plays a role in γδ T cell biology is still unclear. Here, we show that depletion of the m6A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells, which confers enhanced protection against gastrointestinal Salmonella typhimurium infection. Mechanistically, loss of ALKBH5 favors the development of γδ T cell precursors by increasing the abundance of m6A RNA modification in thymocytes, which further reduces the expression of several target genes including Notch signaling components Jagged1 and Notch2. As a result, impairment of Jagged1/Notch2 signaling contributes to enhanced proliferation and differentiation of γδ T cell precursors, leading to an expanded mature γδ T cell repertoire. Taken together, our results indicate a checkpoint role of ALKBH5 and m6A modification in the regulation of γδ T cell early development.

Bisphenol C Induces Cardiac Developmental Defects by Disrupting m6A Homeostasis.

Bisphenol A (BPA) is a commonly used plastic additive. Since BPA has been banned in maternal and infant food containers in many countries, BPA substitutes have been widely introduced to replace it. By systematically assessing the potential developmental toxicity of BPA substitutes, we observed that the 41-150 nM in vivo BPC exposure (around the reported concentration detected in infant urine: 6-186 nM) induced cardiac defects in zebrafish. Mechanistically, BPC disrupted m6A homeostasis by downregulation of the key m6A methyltransferase, Mettl3, thereby causing the m6A reader, Igf2bp2b, to fail in recognizing and stabilizing the inefficiently m6A-modified acox1 and tnnt2d mRNA. Then, downregulation of Acox1 (a regulator in cardiac fatty acid metabolism) and Tnnt2d (a component of cardiac troponin for muscle contraction) led to cardiac defects. Indeed, the dual cardiac functional axes regulated by the same m6A reader in response to BPC provided new insight into the regulatory mechanisms of epitranscriptomics and cardiac development. Collectively, our study not only presented evidence showing that the internal exposure levels of BPC in humans could lead to cardiac developmental defects but also demonstrated the underlying mechanism of BPC-mediated defects by disrupting the Mettl3-m6A-Igf2bp2b-Acox1/Tnnt2d pathways, which provided potential molecular markers associated with BPC exposure.

The METHYLTRANSFERASE B-SERRATE interaction mediates the reciprocal regulation of microRNA biogenesis and RNA m6A modification.

In eukaryotes, RNA N6-methyladenosine (m6A) modification and microRNA (miRNA)-mediated RNA silencing represent two critical epigenetic regulatory mechanisms. The m6A methyltransferase complex (MTC) and the microprocessor complex both undergo liquid-liquid phase separation to form nuclear membraneless organelles. Although m6A methyltransferase has been shown to positively regulate miRNA biogenesis, a mechanism of reciprocal regulation between the MTC and the microprocessor complex has remained elusive. Here, we demonstrate that the MTC and the microprocessor complex associate with each other through the METHYLTRANSFERASE B (MTB)-SERRATE (SE) interacting module. Knockdown of MTB impaired miRNA biogenesis by diminishing microprocessor complex binding to primary miRNAs (pri-miRNAs) and their respective MIRNA loci. Additionally, loss of SE function led to disruptions in transcriptome-wide m6A modification. Further biochemical assays and fluorescence recovery after photobleaching (FRAP) assay indicated that SE enhances the liquid-liquid phase separation and solubility of the MTC. Moreover, the MTC exhibited enhanced retention on chromatin and diminished binding to its RNA substrates in the se mutant background. Collectively, our results reveal the substantial regulatory interplay between RNA m6A modification and miRNA biogenesis.

Up-regulation of RNA m6A methyltransferase like-3 expression contributes to arsenic and benzo[a]pyrene co-exposure-induced cancer stem cell-like property and tumorigenesis.

While arsenic or BaP alone exposure can cause lung cancer, studies showed that arsenic plus BaP co-exposure displays a significantly stronger lung tumorigenic effect. However, the underlying mechanism has not been well understood. Studies showed that RNA molecules are chemically modified. The most frequently occurring RNA modification in eukaryotic messenger RNAs is the N6-methyladenosine (m6A) methylation. This study aimed to determine whether arsenic plus BaP exposure alters RNA m6A methylation and its role in lung tumorigenic effect of arsenic plus BaP exposure. Human bronchial epithelial cells transformed by exposure to arsenic or BaP alone, and arsenic plus BaP and mouse xenograft tumorigenesis models were used in this study. It was found that arsenic plus BaP exposure-transformed cells have significantly higher levels of RNA m6A methylation than arsenic or BaP alone exposure-transformed human bronchial epithelial cells. Western blot analysis showed that arsenic plus BaP exposure greatly up-regulates the m6A writer methyltransferase like-3 (METTL3) expression levels in cultured cells and mouse lung tissues. METTL3 knockdown in cells transformed by arsenic plus BaP exposure drastically reduced their RNA m6A methylation levels. Functional studies revealed that METTL3 knockdown in cells transformed by arsenic plus BaP exposure greatly reduces their anchorage-dependent and -independent growth, cancer stem cell characters and tumorigenesis. The findings from this study suggest that arsenic plus BaP co-exposure causes epitranscriptomic dysregulation, which may contribute significantly to arsenic plus BaP co-exposure-caused synergistic lung tumorigenic effect.

Dysregulated N6-methyladenosine (m6A) processing in hepatocellular carcinoma.

N6-methyladenosine (m6A) is the most thoroughly studied type of internal RNA modification, as this epigenetic modification is the most abundant in eukaryotic RNAs to date. This modification occurs in various types of RNAs and plays significant roles in dominant RNA-related processes, such as translation, splicing, export and degradation. These processes are catalyzed by three types of prominent enzymes: writers, erasers and readers. Increasing evidence has shown that m6A modification is vital for the regulation of gene expression, carcinogenesis, tumor progression and other abnormal changes, and recent studies have shown that m6A is important in the development of hepatocellular carcinoma (HCC). Herein, we summarize the nature and regulatory mechanisms of m6A modification, including its role in the pathogenesis of HCC and related chronic liver diseases. We also highlight the clinical significance and future strategies involving RNA m6A modifications in HCC.

The m6A methyltransferase METTL14 inhibits the proliferation, migration, and invasion of gastric cancer by regulating the PI3K/AKT/mTOR signaling pathway.

BACKGROUND: N6-methyladenosine (m6A) modification may participate in the regulation of occurrence and development of tumors. However, the m6A level and the potential regulatory mechanism of m6A in gastric cancer (GC) remain uncertain. METHODS: RNA m6A quantification assay was conducted to detect the m6A level in GC tissues and cell lines. Methyltransferase-like 14 (METTL14) expression in GC tissues was explored by bioinformatics and immunohistochemistry. Then, the function of METTL14 in GC cells was examined by CCK-8, colony formation assay, wound healing assay, and Transwell assay. Besides, Western blotting was conducted to probe the PI3K/AKT/mTOR pathway and the epithelial-mesenchymal transformation (EMT) pathway-related gene expression. RESULTS: The m6A modification level was decreased in GC and METTL14 was a key regulator resulting in m6A disorder in GC. METTL14 was downregulated in GC by analyzing both clinical samples and bioinformatics. METTL14 overexpression suppressed GC cell proliferation and aggression by deactivating the PI3K/AKT/mTOR pathway and the EMT pathway, respectively. CONCLUSIONS: Our findings indicate that METTL14 partakes in the biological process of GC as a tumor suppressor and may be an emerging biomarker in GC.

miR-338-5p inhibits cell growth and migration via inhibition of the METTL3/m6A/c-Myc pathway in lung cancer.

Lung cancer is a common type of cancer that causes a very large public health burden worldwide. Achieving a better understanding of the molecular mechanism underlying the progression of lung cancer is of benefit for the diagnosis, prognosis, and treatment of lung cancer. Here, we first identified dramatically decreased expression of miR-338-5p in lung cancer tissues and cells using quantitative polymerase chain reaction (qPCR) analysis. We then revealed that miR-338-5p inhibited the cell growth and migration of lung cancer cells using cell counting kit 8 (CCK8), EdU, and Transwell analysis. Furthermore, we demonstrated that miR-338-5p inhibited METTL3 expression by qPCR, western blot analysis, and luciferase reporter assay, while upregulation of METTL3 alleviated the role of miR-338-5p in lung cancer cells. We also showed that METTL3 promoted c-Myc expression by increasing the m6A modification of c-Myc, and overexpression of c-Myc restored the inhibition of cell growth and migration of lung cancer cells induced by METTL3 silencing. Ultimately, this research illustrated that modification of the miR-338-5p/METTL3/c-Myc pathway affected cellular progression in lung cancer cells. Collectively, our study revealed the underlying mechanism of miR-338-5p in lung cancer, providing a novel regulatory pathway in lung cancer. There is potential for this pathway to serve as a diagnostic, prognostic, and therapeutic biomarker for lung cancer.

m6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress.

Translocated in LipoSarcoma/Fused in Sarcoma (TLS/FUS) is a nuclear RNA binding protein whose mutations cause amyotrophic lateral sclerosis. TLS/FUS undergoes LLPS and forms membraneless particles with other proteins and nucleic acids. Interaction with RNA alters conformation of TLS/FUS, which affects binding with proteins, but the effect of m6A RNA modification on the TLS/FUS-RNA interaction remains elusive. Here, we investigated the binding specificity of TLS/FUS to m6A RNA fragments by RNA pull down assay, and elucidated that both wild type and ALS-related TLS/FUS mutants strongly bound to m6A modified RNAs. TLS/FUS formed cytoplasmic foci by treating hyperosmotic stress, but the cells transfected with m6A-modified RNAs had a smaller number of foci. Moreover, m6A-modified RNA transfection resulted in the cells obtaining higher resistance to the stress. In summary, we propose TLS/FUS as a novel candidate of m6A recognition protein, and m6A-modified RNA fragments diffuse cytoplasmic TLS/FUS foci and thereby enhance cell viability.

N6-Methyladenosine-binding proteins suppress HIV-1 infectivity and viral production.

The internal N6-methyladenosine (m6A) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1-3 or Y1-3) bind to m6A-modified cellular mRNAs and modulate their metabolism and processing, thereby affecting cellular protein translation. We previously reported that HIV-1 RNA contains the m6A modification and that Y1-3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription activity. Here, we investigated the mechanisms of Y1-3-mediated inhibition of HIV-1 infection in target cells and the effect of Y1-3 on viral production levels in virus-producing cells. We found that Y1-3 protein overexpression in HIV-1 target cells decreases viral genomic RNA (gRNA) levels and inhibits both early and late reverse transcription. Purified recombinant Y1-3 proteins preferentially bound to the m6A-modified 5' leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1-3 proteins to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered m6A modification sites in the 5' leader sequence of gRNA exhibited significantly lower infectivity than WT, replication-competent HIV-1, confirming that these sites alter viral infection. HIV-1 produced from cells in which endogenous Y1, Y3, or Y1-3 proteins were knocked down singly or together had increased viral infectivity compared with HIV-1 produced in control cells. Interestingly, we found that Y1-3 proteins and HIV-1 Gag protein formed a complex with RNA in HIV-1-producing cells. Overall, these results indicate that Y1-3 proteins inhibit HIV-1 infection and provide new insights into the mechanisms by which the m6A modification of HIV-1 RNA affects viral replication.

Transcriptome-wide N6-methyladenosine profiling of rice callus and leaf reveals the presence of tissue-specific competitors involved in selective mRNA modification.

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification present in mRNAs of all higher eukaryotes. With the development of MeRIP-seq technique, in-depth identification of mRNAs with m(6)A modification becomes feasible. Here we present a transcriptome-wide m(6)A modification profiling effort for rice transcriptomes of differentiated callus and leaf, which yields 8,138 and 14,253 m(6)A-modified genes, respectively. The m(6)A peak (m(6)A-modified nucleotide position on mRNAs) distribution exhibits preference toward both translation termination and initiation sites. The m(6)A peak enrichment is negatively correlated with gene expression and weakly positively correlated with certain gene features, such as exon length and number. By comparing m(6)A-modified genes between the 2 samples, we define 1,792 and 6,508 tissue-specific m(6)A-modified genes (TSMGs) in callus and leaf, respectively. Among which, 626 and 5,509 TSMGs are actively expressed in both tissues but are selectively m(6)A-modified (SMGs) only in one of the 2 tissues. Further analyses reveal characteristics of SMGs: (1) Most SMGs are differentially expressed between callus and leaf. (2) Two conserved RNA-binding motifs, predicted to be recognized by PUM and RNP4F, are significantly over-represented in SMGs. (3) GO enrichment analysis shows that SMGs in callus mainly participate in transcription regulator/factor activity whereas SMGs in leaf are mainly involved in plastid and thylakoid. Our results suggest the presence of tissue-specific competitors involved in SMGs. These findings provide a resource for plant RNA epitranscriptomic studies and further enlarge our knowledge on the function of RNA m(6)A modification.

Arginase 1 does not affect RNA m6A methylation in mouse fetal lung.

BACKGROUND: Arginase 1 (Arg1) encodes a key enzyme that catalyzes the metabolism of arginine to ornithine and urea. In our recent study, we found that knockdown of Arg1 in the lungs of fetal mice induces apoptosis of epithelial cells and dramatically delays initiation of labor. As the most abundant internal mRNA modification, N6 -methyladenosine (m6 A) has been found to play important roles in lung development and cellular differentiation. However, if the knockdown of Arg1 affects the RNA m6A modification in fetal lungs remains unknown. METHODS: In the current study, the RNA m6A levels and the expression of RNA m6A related enzymes were validated in 13.0 dpc fetal lungs that Arg1 was knocked down by adeno-associated virus carrying Arg1-shRNA, using western blot, immunofluorescence, and RT-qPCR. RESULTS: No statistical differences were found in the expression of methyltransferase, demethylases, and binding proteins in the fetal lungs between AAV-shArg1-injected mice and AAV-2/9-injected mice. Besides, there is no significant change of overall RNA m6A level in fetal lungs from AAV-shArg1-injected mice, compared with that from AAV-2/9-injected mice. CONCLUSIONS: These results indicate that arginase 1 does not affect RNA m6A methylation in mouse fetal lung, and the mechanisms other than RNA m6A modification underlying the effects of Arg1 knockdown on the fetal lung development and their interaction with labor initiation need to be further explored.

The landscape of epigenetic regulation and therapeutic application of N6-methyladenosine modifications in non-coding RNAs.

RNA N6-methyladenosine (m6A) methylation is the most abundant and conserved RNA modification in eukaryotes. It participates in the regulation of RNA metabolism and various pathophysiological processes. Non-coding RNAs (ncRNAs) are defined as small or long transcripts which do not encode proteins and display numerous biological regulatory functions. Similar to mRNAs, m6A deposition is observed in ncRNAs. Studying RNA m6A modifications on ncRNAs is of great importance specifically to deepen our understanding of their biological roles and clinical implications. In this review, we summarized the recent research findings regarding the mutual regulation between RNA m6A modification and ncRNAs (with a specific focus on microRNAs, long non-coding RNAs, and circular RNAs) and their functions. We also discussed the challenges of m6A-containing ncRNAs and RNA m6A as therapeutic targets in human diseases and their future perspective in translational roles.

Landscape of transcriptome-wide m6A modification in diabetic liver reveals rewiring of PI3K-Akt signaling after physical exercise.

AIM: Type 2 diabetes mellitus (T2DM) is one of the most common diseases, and epigenetic modification N6-methyladenosine (m6A) is essential for transcriptional modulation involved in its development. However, the precise role and landscape of transcriptome-wide m6A alterations in molecular adaptations after physical exercise have yet to be fully elucidated. METHODS: Four-week-old male C57BL/6J mice received a high-fat diet (HFD) for 12 weeks to establish a diabetic state, and HFD mice were simultaneously subjected to physical exercise (HFD + EX). The hepatic RNA m6A methylome was examined, the conjoint MeRIP-seq and RNA-seq was performed, and the exercise-modulated genes were confirmed. RESULTS: Physical exercise significantly ameliorates liver metabolic disorder and triggers a dynamic change in hepatic RNA m6A. By analyzing the distribution of m6A in transcriptomes, an abundance of m6A throughout mRNA transcripts and a pattern of conserved m6A after physical exercise was identified. It is noteworthy that conjoint MeRIP-seq and RNA-seq data revealed that both differentially methylated genes and differentially expressed genes were enriched in all stages of the PI3K-Akt signaling pathway, in particular the upstream nodes of this pathway, which are considered a valuable therapeutic target for T2DM. Moreover, in vivo and in vitro analyses showed that exercise-mediated methyltransferase Rbm15 positively regulated the expression of two upstream genes (Itga3 and Fgf21) in an m6A-dependent manner. CONCLUSION: These findings highlight the pivotal role of the exercise-induced m6A epigenetic network and contribute insights into the intricate epigenetic mechanism underlying insulin signaling.

Reprogramming of RNA m6A Modification Is Required for Acute Myeloid Leukemia Development.

Hematopoietic homeostasis is maintained by hematopoietic stem cells (HSCs), and it is tightly controlled at multiple levels to sustain the self-renewal capacity and differentiation potential of HSCs. Dysregulation of self-renewal and differentiation of HSCs leads to the development of hematologic diseases, including acute myeloid leukemia (AML). Thus, understanding the underlying mechanisms of HSC maintenance and the development of hematologic malignancies is one of the fundamental scientific endeavors in stem cell biology. N  6-methyladenosine (m6A) is a common modification in mammalian messenger RNAs (mRNAs) and plays important roles in various biological processes. In this study, we performed a comparative analysis of the dynamics of the RNA m6A methylome of hematopoietic stem and progenitor cells (HSPCs) and leukemia-initiating cells (LICs) in AML. We found that RNA m6A modification regulates the transformation of long-term HSCs into short-term HSCs and determines the lineage commitment of HSCs. Interestingly, m6A modification leads to reprogramming that promotes cellular transformation during AML development, and LIC-specific m6A targets are recognized by different m6A readers. Moreover, the very long chain fatty acid transporter ATP-binding cassette subfamily D member 2 (ABCD2) is a key factor that promotes AML development, and deletion of ABCD2 damages clonogenic ability, inhibits proliferation, and promotes apoptosis of human leukemia cells. This study provides a comprehensive understanding of the role of m6A in regulating cell state transition in normal hematopoiesis and leukemogenesis, and identifies ABCD2 as a key factor in AML development.

Epitranscriptomic m6A modifications during reactivation of HIV-1 latency in CD4+ T cells.

Despite effective antiretroviral therapy reducing HIV-1 viral loads to undetectable levels, the presence of latently infected CD4+ T cells poses a major barrier to HIV-1 cure. N6-methyladenosine (m6A) modification of viral and cellular RNA has a functional role in regulating HIV-1 infection. m6A modification of HIV-1 RNA can affect its stability, translation, and splicing in cells and suppresses type-I interferon induction in macrophages. However, the function of m6A modification in regulating HIV-1 latency reactivation remains unknown. We used the Jurkat T cell line-derived HIV-1 latency model (J-Lat cells) to investigate changes in m6A levels of cellular RNA in response to latency reversal. We observed a significant increase in m6A levels of total cellular RNA upon reactivation of latent HIV-1 in J-Lat cells. This increase in m6A levels was transient and returned to steady-state levels despite continued high levels of viral gene expression in reactivated cells compared to control cells. Upregulation of m6A levels occurred without significant changes in the protein expression of m6A writers or erasers that add or remove m6A, respectively. Knockdown of m6A writers in J-Lat cells significantly reduced HIV-1 reactivation. Treatment with an m6A writer inhibitor reduced cellular RNA m6A levels, along with a reduction in HIV-1 reactivation. Furthermore, using m6A-specific sequencing, we identified cellular RNAs that are differentially m6A-modified during HIV-1 reactivation in J-Lat cells. Knockdown of identified m6A-modified RNA validates these results with an established primary CD4+ T cell model of HIV-1 latency. These results show the importance of m6A RNA modification in HIV-1 latency reversal. IMPORTANCE: RNA m6A modification is important for regulating gene expression and innate immune responses to HIV-1 infection. However, the functional significance of m6A modification during HIV-1 latency reactivation is unknown. To address this important question, in this study, we used established cellular models of HIV-1 latency, m6A-specific sequencing at single-base resolution, and functional assays. We demonstrate that HIV-1 latency reversal leads to increased levels of cellular m6A modification, correlates with cellular m6A levels, and is dependent on the catalytic activity of the m6A methyltransferase enzyme. We also identified cellular genes that are differentially m6A-modified during HIV-1 reactivation, as well as the sites of m6A within HIV-1 RNA. Our novel findings point toward a significant role for m6A modification in HIV-1 latency reversal.

Suppression of the METTL3-m6A-integrin ß1 axis by extracellular acidification impairs T cell infiltration and antitumor activity.

The acidic metabolic byproducts within the tumor microenvironment (TME) hinder T cell effector functions. However, their effects on T cell infiltration remain largely unexplored. Leveraging the comprehensive The Cancer Genome Atlas dataset, we pinpoint 16 genes that correlate with extracellular acidification and establish a metric known as the "tumor acidity (TuAci) score" for individual patients. We consistently observe a negative association between the TuAci score and T lymphocyte score (T score) across various human cancer types. Mechanistically, extracellular acidification significantly impedes T cell motility by suppressing podosome formation. This phenomenon can be attributed to the reduced expression of methyltransferase-like 3 (METTL3) and the modification of RNA N6-methyladenosine (m6A), resulting in a subsequent decrease in the expression of integrin ß1 (ITGB1). Importantly, enforced ITGB1 expression leads to enhanced T cell infiltration and improved antitumor activity. Our study suggests that modulating METTL3 activity or boosting ITGB1 expression could augment T cell infiltration within the acidic TME, thereby improving the efficacy of cell therapy.

Celastrol suppresses human pancreatic cancer via m6A-YTHDF3-mediated downregulation of Claspin and Bcl-2.

BACKGROUND: Celastrol has been revealed to exhibit anticancer pharmacological activity, however, the molecular mechanisms of celastrol involved in pancreatic cancer remain to be further elucidated. The present study was to illustrate whether celastrol suppresses pancreatic cancer through modulating RNA m6A modification. METHODS: Effect of celastrol treatment on the malignant phenotypes of pancreatic cancer cells was evaluated by CCK-8 assay, EdU assay, colony formation assay, flow cytometry analysis and subcutaneous xenograft experiments. RNA sequencing (RNA-seq) analysis was carried out to analyze the genes differentially expressed in celastrol-treated pancreatic cancer cells. RT-qPCR, Western blotting and immunohistochemistry were employed to evaluate the expression of the indicated genes. RNA dot blot and quantification of total RNA m6A modification assays, MeRIP-qPCR assay, RIP-qPCR assay, RNA stability and protein stability assays were applied to evaluate the regulatory mechanism of celastrol treatment in pancreatic cancer cells. RESULTS: We demonstrated that celastrol suppressed cell proliferation and induced cell cycle arrest and apoptosis of pancreatic cancer cells in vitro, and decreased tumor growth in vivo. Specifically, Bcl-2, Claspin, METTL3 and YTHDF3 were identified as the potential targets of celastrol treatment in pancreatic cancer cells. Moreover, our results indicated that celastrol treatment downregulated METTL3 and decreased m6A levels of Claspin and Bcl-2 mRNA, leading to the degradation of Claspin and Bcl-2 mRNA in pancreatic cancer cells. Furthermore, we revealed that celastrol treatment downregulated Claspin and Bcl-2, at least in part, in an m6A-YTHDF3-mediated manner in pancreatic cancer cells. CONCLUSION: Our study highlighted a novel mechanism underlying celastrol-induced cellular proliferation inhibition and apoptosis in pancreatic cancer cells via m6A-YTHDF3-mediated downregulation of Claspin and Bcl-2.

Manipulation of IME4 expression, a global regulation strategy for metabolic engineering in Saccharomyces cerevisiae.

Metabolic engineering has been widely used for production of natural medicinal molecules. However, engineering high-yield platforms is hindered in large part by limited knowledge of complex regulatory machinery of metabolic network. N6-Methyladenosine (m6A) modification of RNA plays critical roles in regulation of gene expression. Herein, we identify 1470 putatively m6A peaks within 1151 genes from the haploid Saccharomyces cerevisiae strain. Among them, the transcript levels of 94 genes falling into the pathways which are frequently optimized for chemical production, are remarkably altered upon overexpression of IME4 (the yeast m6A methyltransferase). In particular, IME4 overexpression elevates the mRNA levels of the methylated genes in the glycolysis, acetyl-CoA synthesis and shikimate/aromatic amino acid synthesis modules. Furthermore, ACS1 and ADH2, two key genes responsible for acetyl-CoA synthesis, are induced by IME4 overexpression in a transcription factor-mediated manner. Finally, we show IME4 overexpression can significantly increase the titers of isoprenoids and aromatic compounds. Manipulation of m6A therefore adds a new layer of metabolic regulatory machinery and may be broadly used in bioproduction of various medicinal molecules of terpenoid and phenol classes.

Studies on the fat mass and obesity-associated (FTO) gene and its impact on obesity-associated diseases.

Obesity has become a major health crisis in the past ∼50 years. The fat mass and obesity-associated (FTO) gene, identified by genome-wide association studies (GWAS), was first reported to be positively associated with obesity in humans. Mice with more copies of the FTO gene were observed to be obese, while loss of the gene in mice was found to protect from obesity. Later, FTO was found to encode an m6A RNA demethylase and has a profound effect on many biological and metabolic processes. In this review, we first summarize recent studies that demonstrate the critical roles and regulatory mechanisms of FTO in obesity and metabolic disease. Second, we discuss the ongoing debates concerning the association between FTO polymorphisms and obesity. Third, since several small molecule drugs and micronutrients have been found to regulate metabolic homeostasis through controlling the expression or activity of FTO, we highlight the broad potential of targeting FTO for obesity treatment. Improving our understanding of FTO and the underlying mechanisms may provide new approaches for treating obesity and metabolic diseases.