Engineering cascade biocatalysis in whole cells for syringic acid bioproduction.

BACKGROUND: Syringic acid (SA) is a high-value natural compound with diverse biological activities and wide applications, commonly found in fruits, vegetables, and herbs. SA is primarily produced through chemical synthesis, nonetheless, these chemical methods have many drawbacks, such as considerable equipment requirements, harsh reaction conditions, expensive catalysts, and numerous by-products. Therefore, in this study, a novel biotransformation route for SA production was designed and developed by using engineered whole cells. RESULTS: An O-methyltransferase from Desulfuromonas acetoxidans (DesAOMT), which preferentially catalyzes a methyl transfer reaction on the meta-hydroxyl group of catechol analogues, was identified. The whole cells expressing DesAOMT can transform gallic acid (GA) into SA when S-adenosyl methionine (SAM) is used as a methyl donor. We constructed a multi-enzyme cascade reaction in Escherichia coli, containing an endogenous shikimate kinase (AroL) and a chorismate lyase (UbiC), along with a p-hydroxybenzoate hydroxylase mutant (PobA**) from Pseudomonas fluorescens, and DesAOMT; SA was biosynthesized from shikimic acid (SHA) by using whole cells catalysis. The metabolic system of chassis cells also affected the efficiency of SA biosynthesis, blocking the chorismate metabolism pathway improved SA production. When the supply of the cofactor NADPH was optimized, the titer of SA reached 133 µM (26.2 mg/L). CONCLUSION: Overall, we designed a multi-enzyme cascade in E. coli for SA biosynthesis by using resting or growing whole cells. This work identified an O-methyltransferase (DesAOMT), which can catalyze the methylation of GA to produce SA. The multi-enzyme cascade containing four enzymes expressed in an engineered E. coli for synthesizing of SA from SHA. The metabolic system of the strain and biotransformation conditions influenced catalytic efficiency. This study provides a new green route for SA biosynthesis.

5-Fluorouracil dose escalation generated desensitized colorectal cancer cells with reduced expression of protein methyltransferases and no epithelial-to-mesenchymal transition potential.

Background: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers. In many cases, the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil (5-FU). The epithelial-to-mesenchymal transition (EMT) and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers. This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC. Materials and Methods: HCT-116, Caco-2, and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU. The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays. This was followed by a Western blot which analyzed the protein expressions of the epithelial marker E-cadherin, mesenchymal marker vimentin, and the EMT transcription factor (EMT-TF), the snail family transcriptional repressor 1 (Snail) in the parental and desensitized cells. Western blotting was also conducted to study the protein expressions of the protein methyltransferases (PMTs), Euchromatic histone lysine methyltransferase 2 (EHMT2/G9A), protein arginine methyltransferase (PRMT5), and SET domain containing 7/9 (SETD7/9) along with the global lysine and arginine methylation profiles. Results: The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU. The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells. This was reflected in the observed reduction in E-cadherin, vimentin, and Snail in the desensitized cell lines. Additionally, the protein expressions of EHMT2/G9A, PRMT5, and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment. Conclusion: This study showed that continuous, dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.

Two genes encoding caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1) are candidate genes for physical seed dormancy in cowpea (Vigna unguiculata (L.) Walp.).

KEY MESSAGE: The major QTL Sdp1.1+ controlling seed dormancy in cowpea was finely mapped, and two CCoAOMT1 genes were identified as candidate genes for the dormancy. Seed dormancy in wild cowpea may be useful in breeding cultivated cowpea with pre-harvest sprouting resistance. A previous study identified a major quantitative trait locus (QTL) for seed dormancy, Sdp1.1+ , using the population of the cross between cultivated cowpea 'JP81610' and wild cowpea 'JP89083.' However, the molecular basis of seed dormancy in cowpea is not yet known. In this study, we aimed to finely map the locus Sdp1.1+ and identify candidate gene(s) for it. Germination tests demonstrated that the seed coat is the major factor controlling seed dormancy in the wild cowpea JP89083. Microscopic observations revealed that wild cowpea seeds, unlike cultivated cowpea seeds, possessed a palisade cuticle layer. Fine mapping using a large F2 population of the cross JP81610 × JP89083 grown in Thailand revealed a single QTL, Sdp1.1+ , controlling seed dormancy. The Sdp1.1+ was confirmed using a small F2 population of the same cross grown in Japan. The Sdp1.1+ was mapped to a 37.34-Kb region containing three genes. Two closely linked genes, Vigun03g278900 (VuCCoAOMT1a) and Vigun03g290000 (VuCCoAOMT1b), located 4.844 Kb apart were considered as candidate genes for seed dormancy. The two genes encoded caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1). DNA sequencing and alignment of VuCCoAOMT1a and VuCCoAOMT1b between JP89083 and JP81610 revealed a single nucleotide polymorphism (SNP) causing an amino acid change in VuCCoAOMT1a and several SNPs leading to six amino acid changes in VuCCoAOMT1b. Altogether, these results indicate that VuCCoAOMT1a and VuCCoAOMT1b are candidate genes controlling physical seed dormancy in the wild cowpea JP89083.

Marrow mesenchymal stem cell mediates diabetic nephropathy progression via modulation of Smad2/3/WTAP/m6A/ENO1 axis.

Diabetic nephropathy (DN) is one of the common microvascular complications in diabetic patients. Marrow mesenchymal stem cells (MSCs) have attracted attention in DN therapy but the underlying mechanism remains unclear. Here, we show that MSC administration alleviates high glucose (HG)-induced human kidney tubular epithelial cell (HK-2 cell) injury and ameliorates renal injury in DN mice. We identify that Smad2/3 is responsible for MSCs-regulated DN progression. The activity of Smad2/3 was predominantly upregulated in HG-induced HK-2 cell and DN mice and suppressed with MSC administration. Activation of Smad2/3 via transforming growth factor-ß1 (TGF-ß1) administration abrogates the protective effect of MSCs on HG-induced HK-2 cell injury and renal injury of DN mice. Smad2/3 has been reported to interact with methyltransferase of N6-methyladenosine (m6A) complex and we found a methyltransferase, Wilms' tumor 1-associating protein (WTAP), is involved in MSCs-Smad2/3-regulated DN development. Moreover, WTAP overexpression abrogates the improvement of MSCs on HG-induced HK-2 cell injury and renal injury of DN mice. Subsequently, α-enolase (ENO1) is the downstream target of WTAP-mediated m6A modification and contributes to the MSCs-mediated regulation. Collectively, these findings reveal a molecular mechanism in DN progression and indicate that Smad2/3/WTAP/ENO1 may present a target for MSCs-mediated DN therapy.

METTL1-mediated tRNA m7G methylation and translational dysfunction restricts breast cancer tumorigenesis by fueling cell cycle blockade.

BACKGROUND: RNA modifications of transfer RNAs (tRNAs) are critical for tRNA function. Growing evidence has revealed that tRNA modifications are related to various disease processes, including malignant tumors. However, the biological functions of methyltransferase-like 1 (METTL1)-regulated m7G tRNA modifications in breast cancer (BC) remain largely obscure. METHODS: The biological role of METTL1 in BC progression were examined by cellular loss- and gain-of-function tests and xenograft models both in vitro and in vivo. To investigate the change of m7G tRNA modification and mRNA translation efficiency in BC, m7G-methylated tRNA immunoprecipitation sequencing (m7G tRNA MeRIP-seq), Ribosome profiling sequencing (Ribo-seq), and polysome-associated mRNA sequencing were performed. Rescue assays were conducted to decipher the underlying molecular mechanisms. RESULTS: The tRNA m7G methyltransferase complex components METTL1 and WD repeat domain 4 (WDR4) were down-regulated in BC tissues at both the mRNA and protein levels. Functionally, METTL1 inhibited BC cell proliferation, and cell cycle progression, relying on its enzymatic activity. Mechanistically, METTL1 increased m7G levels of 19 tRNAs to modulate the translation of growth arrest and DNA damage 45 alpha (GADD45A) and retinoblastoma protein 1 (RB1) in a codon-dependent manner associated with m7G. Furthermore, in vivo experiments showed that overexpression of METTL1 enhanced the anti-tumor effectiveness of abemaciclib, a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor. CONCLUSION: Our study uncovered the crucial tumor-suppressive role of METTL1-mediated tRNA m7G modification in BC by promoting the translation of GADD45A and RB1 mRNAs, selectively blocking the G2/M phase of the cell cycle. These findings also provided a promising strategy for improving the therapeutic benefits of CDK4/6 inhibitors in the treatment of BC patients.

METTL3 promotes the progression of osteosarcoma through the N6-methyladenosine modification of MCAM via IGF2BP1.

BACKGROUND: The molecular mechanisms of osteosarcoma (OS) are complex. In this study, we focused on the functions of melanoma cell adhesion molecule (MCAM), methyltransferase 3 (METTL3) and insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) in OS development. METHODS: qRT-PCR assay and western blot assay were performed to determine mRNA and protein expression of MCAM, METTL3, IGF2BP1 and YY1. MTT assay and colony formation assay were conducted to assess cell proliferation. Cell apoptosis, invasion and migration were evaluated by flow cytometry analysis, transwell assay and wound-healing assay, respectively. Methylated RNA Immunoprecipitation (MeRIP), dual-luciferase reporter, Co-IP, RIP and ChIP assays were performed to analyze the relationships of MCAM, METTL3, IGF2BP1 and YY1. The functions of METTL3 and MCAM in tumor growth were explored through in vivo experiments. RESULTS: MCAM was upregulated in OS, and MCAM overexpression promoted OS cell growth, invasion and migration and inhibited apoptosis. METTL3 and IGF2BP1 were demonstrated to mediate the m6A methylation of MCAM. Functionally, METTL3 or IGF2BP1 silencing inhibited OS cell progression, while MCAM overexpression ameliorated the effects. Transcription factor YY1 promoted the transcription level of METTL3 and regulated METTL3 expression in OS cells. Additionally, METTL3 deficiency suppressed tumor growth in vivo, while MCAM overexpression abated the effect. CONCLUSION: YY1/METTL3/IGF2BP1/MCAM axis aggravated OS development, which might provide novel therapy targets for OS.

KIAA1429 Induces m6A Modification of LINC01106 to Enhance the Malignancy of Lung Adenocarcinoma Cells via the JAK/STAT3 Pathway.

BACKGROUND: Sustained expression of the long noncoding RNA (lncRNA) LINC01106 in tumors is crucial for the malignant phenotype of tumor cells. Nevertheless, the mechanisms and clinical effects of LINC01106 in lung adenocarcinoma (LUAD) are limited. This study shows the effect of vir-like m6A methyltransferase-associated (KIAA1429)-mediated N6-methyladenosine (m6A) modification on steady LINC01106 expression on LUAD progression. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine LINC01106 and KIAA1429 levels in LUAD tissues. Transwell, 5-ethynyl-2'-deoxyuridine (EdU), and cell counting kit-8 (CCK-8) assays were used to analyze the functional roles of LINC01106. A xenograft was constructed to verify the function of silencing LINC01106 in tumor growth. The regulatory role of LINC01106 was investigated using methylated RNA immunoprecipitation (MeRIP), qRT-PCR, and the actinomycin D assay. Western blotting was used to identify key proteins in the JAK/STAT3 (JAK2, STAT3) pathway. RESULTS: LINC01106 and KIAA1429 were highly expressed in LUAD, and LINC01106 was interconnected with high tumor grade, stage, and poor prognosis. Data revealed that LINC01106 inhibition reduced LUAD cell proliferation, invasion, and migration and restrained LUAD cell tumorigenicity. In addition, LINC01106 silencing reduced phosphorylated JAK2 and STAT3 levels. KIAA1429-mediated LINC01106 enhances its m6A modification and expression in LUAD cells. Moreover, KIAA1429 promotion eliminated the malignant phenotypic suppression induced by low expression in LUAD cells. CONCLUSION: This study showed that KIAA1429 enhanced LINC01106 m6A modification to promote LUAD development. These results may lead to a better understanding of the mechanism of KIAA1429-m6A-LINC01106 in LUAD and offer a valuable therapeutic target for LUAD.

NSUN2/YBX1 promotes the progression of breast cancer by enhancing HGH1 mRNA stability through m5C methylation.

BACKGROUND: RNA m5C methylation has been extensively implicated in the occurrence and development of tumors. As the main methyltransferase, NSUN2 plays a crucial regulatory role across diverse tumor types. However, the precise impact of NSUN2-mediated m5C modification on breast cancer (BC) remains unclear. Our study aims to elucidate the molecular mechanism underlying how NSUN2 regulates the target gene HGH1 (also known as FAM203) through m5C modification, thereby promoting BC progression. Additionally, this study targets at preliminarily clarifying the biological roles of NSUN2 and HGH1 in BC. METHODS: Tumor and adjacent tissues from 5 BC patients were collected, and the m5C modification target HGH1 in BC was screened through RNA sequencing (RNA-seq) and single-base resolution m5C methylation sequencing (RNA-BisSeq). Methylation RNA immunoprecipitation-qPCR (MeRIP-qPCR) and RNA-binding protein immunoprecipitation-qPCR (RIP-qPCR) confirmed that the methylation molecules NSUN2 and YBX1 specifically recognized and bound to HGH1 through m5C modification. In addition, proteomics, co-immunoprecipitation (co-IP), and Ribosome sequencing (Ribo-Seq) were used to explore the biological role of HGH1 in BC. RESULTS: As the main m5C methylation molecule, NSUN2 is abnormally overexpressed in BC and increases the overall level of RNA m5C. Knocking down NSUN2 can inhibit BC progression in vitro or in vivo. Combined RNA-seq and RNA-BisSeq analysis identified HGH1 as a potential target of abnormal m5C modifications. We clarified the mechanism by which NSUN2 regulates HGH1 expression through m5C modification, a process that involves interactions with the YBX1 protein, which collectively impacts mRNA stability and protein synthesis. Furthermore, this study is the first to reveal the binding interaction between HGH1 and the translation elongation factor EEF2, providing a comprehensive understanding of its ability to regulate transcript translation efficiency and protein synthesis in BC cells. CONCLUSIONS: This study preliminarily clarifies the regulatory role of the NSUN2-YBX1-m5C-HGH1 axis from post-transcriptional modification to protein translation, revealing the key role of abnormal RNA m5C modification in BC and suggesting that HGH1 may be a new epigenetic biomarker and potential therapeutic target for BC.

The Effect Mechanism of N6-adenosine Methylation (m6A) in Melatonin Regulated LPS-induced Colon Inflammation.

Colon inflammation is characterized by disturbances in the intestinal microbiota and inflammation. Melatonin (Mel) can improve colon inflammation. However, the underlying mechanism remains unclear. Recent studies suggest that m6A methylation modification may play an important role in inflammatory responses. This study aimed to explore the effects of melatonin and LPS-mediated m6A methylation on colon inflammation. Our study found that melatonin inhibits M1 macrophages, activates M2 macrophages, inhibit the secretion of pro-inflammatory factors, maintain colon homeostasis and improves colon inflammation through MTNR1B. In addition, the increased methylation level of m6A is associated with the occurrence of colon inflammation, and melatonin can also reduce the level of colon methylation to improve colon inflammation. Among them, the main methylated protein METTL3 can be inhibited by melatonin through MTNR1B. In a word, melatonin regulates m6A methylation by improving abnormal METTL3 protein level to reshape the microflora and activate macrophages to improve colon inflammation, mainly through MTNR1B.

N6-methyladenosine modification of KLF2 may contribute to endothelial-to-mesenchymal transition in pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a progressive disease characterized by pulmonary vascular remodeling. Increasing evidence indicates that endothelial-to-mesenchymal transition (EndMT) in pulmonary artery endothelial cells (PAECs) is a pivotal trigger initiating this remodeling. However, the regulatory mechanisms underlying EndMT in PH are still not fully understood. METHODS: Cytokine-induced hPAECs were assessed using RNA methylation quantification, qRT-PCR, and western blotting to determine the involvement of N6-methyladenosine (m6A) methylation in EndMT. Lentivirus-mediated silencing, overexpression, tube formation, and wound healing assays were utilized to investigate the function of METTL3 in EndMT. Endothelial-specific gene knockout, hemodynamic measurement, and immunostaining were performed to explore the roles of METTL3 in pulmonary vascular remodeling and PH. RNA-seq, RNA Immunoprecipitation-based qPCR, mRNA stability assay, m6A mutation, and dual-luciferase assays were employed to elucidate the mechanisms of RNA methylation in EndMT. RESULTS: The global levels of m6A and METTL3 expression were found to decrease in TNF-α- and TGF-ß1-induced EndMT in human PAECs (hPAECs). METTL3 inhibition led to reduced endothelial markers (CD31 and VE-cadherin) and increased mesenchymal markers (SM22 and N-cadherin) as well as EndMT-related transcription factors (Snail, Zeb1, Zeb2, and Slug). The endothelial-specific knockout of Mettl3 promoted EndMT and exacerbated pulmonary vascular remodeling and hypoxia-induced PH (HPH) in mice. Mechanistically, METTL3-mediated m6A modification of kruppel-like factor 2 (KLF2) plays a crucial role in the EndMT process. KLF2 overexpression increased CD31 and VE-cadherin levels while decreasing SM22, N-cadherin, and EndMT-related transcription factors, thereby mitigating EndMT in PH. Mutations in the m6A site of KLF2 mRNA compromise KLF2 expression, subsequently diminishing its protective effect against EndMT. Furthermore, KLF2 modulates SM22 expression through direct binding to its promoter. CONCLUSIONS: Our findings unveil a novel METTL3/KLF2 pathway critical for protecting hPAECs against EndMT, highlighting a promising avenue for therapeutic investigation in PH.

M6A-induced transcription factor IRF5 contributes to the progression of cervical cancer by upregulating PPP6C.

Cervical cancer (CC) is a gynaecological malignancy tumour that seriously threatens women's health. Recent evidence has identified that interferon regulatory factor 5 (IRF5), a nucleoplasm shuttling protein, is a pivotal transcription factor regulating the growth and metastasis of various human tumours. This study aimed to investigate the function and molecular basis of IRF5 in CC development. IRF5, protein phosphatase 6 catalytic subunit (PPP6C) and methyltransferase-like 3 (METTL3) mRNA levels were evaluated by quantitative real-time (qRT)-polymerase chain reaction (PCR). IRF5, PPP6C, METTL3, B-cell lymphoma 2 and Bax protein levels were detected using western blot. Cell proliferation, migration, invasion, angiogenesis and apoptosis were determined by using colony formation, 5-ethynyl-2'-deoxyuridine (EdU), transwell, tube formation assay and flow cytometry assay, respectively. Glucose uptake and lactate production were measured using commercial kits. Xenograft tumour assay in vivo was used to explore the role of IRF5. After JASPAR predication, binding between IRF5 and PPP6C promoter was verified using chromatin immunoprecipitation and dual-luciferase reporter assays. Moreover, the interaction between METTL3 and IRF5 was verified using methylated RNA immunoprecipitation (MeRIP). IRF5, PPP6C and METTL3 were highly expressed in CC tissues and cells. IRF5 silencing significantly inhibited cell proliferation, migration, invasion, angiogenesis and glycolytic metabolism in CC cells, while induced cell apoptosis. Furthermore, the absence of IRF5 hindered tumour growth in vivo. At the molecular level, IRF5 might bind with PPP6C to positively regulate the expression of PPP6C mRNA. Meanwhile, IRF5 was identified as a downstream target of METTL3-mediated m6A modification. METTL3-mediated m6A modification of mRNA might promote CC malignant progression by regulating PPP6C, which might provide a promising therapeutic target for CC treatment.

An integrated multi-omics approach reveals polymethoxylated flavonoid biosynthesis in Citrus reticulata cv. Chachiensis.

Citrus reticulata cv. Chachiensis (CRC) is an important medicinal plant, its dried mature peels named "Guangchenpi", has been used as a traditional Chinese medicine to treat cough, indigestion, and lung diseases for several hundred years. However, the biosynthesis of the crucial natural products polymethoxylated flavonoids (PMFs) in CRC remains unclear. Here, we report a chromosome-scale genome assembly of CRC with the size of 314.96 Mb and a contig N50 of 16.22 Mb. Using multi-omics resources, we discover a putative caffeic acid O-methyltransferase (CcOMT1) that can transfer a methyl group to the 3-hydroxyl of natsudaidain to form 3,5,6,7,8,3',4'-heptamethoxyflavone (HPMF). Based on transient overexpression and virus-induced gene silencing experiments, we propose that CcOMT1 is a candidate enzyme in HPMF biosynthesis. In addition, a potential gene regulatory network associated with PMF biosynthesis is identified. This study provides insights into PMF biosynthesis and may assist future research on mining genes for the biosynthesis of plant-based medicines.

METTL3-mediated m6A methylation of SLC38A1 stimulates cervical cancer growth.

The objective of this study was to better characterize the role of the glutamine transporter SLC38A1 in cervical cancer and explore the underlying mechanisms. Data from public databases and clinical cervical cancer tissue samples were used to assess the expression of SLC38A1 and its prognostic significance. Immunohistochemical staining, qRT-PCR, and Western blotting were used to evaluate the expression of relevant genes and proteins. Cell viability, cell cycle, apoptosis, and intracellular glutamine content were measured using CCK-8, flow cytometry, and biochemical assays. Additionally, the RNA immunoprecipitation (RIP) assay was used to examine the impact of METTL3/IGF2BP3 on the m6A modification of the SLC38A1 3'UTR. Both cervical cancer specimens and cells showed significantly increased expression of SLC38A1 and its expression correlated with an unfavorable prognosis. Knockdown of SLC38A1 inhibited cell viability and cell cycle progression, induced apoptosis, and suppressed tumor growth in vivo. Glutaminase-1 inhibitor CB-839 reversed the effects of SLC38A1 overexpression. METTL3 promoted m6A modification of SLC38A1 and enhanced its mRNA stability through IGF2BP3 recruitment. Moreover, METTL3 silencing inhibited cell viability, cell cycle progression, intracellular glutamine content, and induced apoptosis, but these effects were reversed by SLC38A1 overexpression. In conclusion, METTL3-mediated m6A methylation of SLC38A1 stimulates cervical cancer progression. SLC38A1 inhibition is a potential therapeutic strategy for cervical cancer.

METTL14 decreases FTH1 mRNA stability via m6A methylation to promote sorafenib-induced ferroptosis of cervical cancer.

Cervical cancer (CC) is a prevalent malignancy among women worldwide. This study was designed to investigate the role of METTL14 in sorafenib-induced ferroptosis in CC. METTL14 expression and m6A methylation were determined in CC tissues, followed by analyzes correlating these factors with clinical features. Subsequently, METTL14 was knocked down in CC cell lines, and the effects on cell proliferation, mitochondrial morphology and ferroptosis were assessed using CCK-8, microscopy, and markers associated with ferroptosis, respectively. The regulatory relationship between METTL14 and FTH1 was verified using qRT-PCR and luciferase reporter assays. The functional significance of this interaction was further investigated both in vitro and in vivo by co-transfecting cells with overexpression vectors or shRNAs targeting METTL14 and FTH1 after sorafenib treatment. METTL14 expression and m6A methylation were significantly reduced in CC tissues, and lower METTL14 expression levels were associated with a poorer CC patients' prognosis. Notably, METTL14 expression increased during sorafenib-induced ferroptosis, and METTL14 knockdown attenuated the ferroptotic response induced by sorafenib in CC cells. FTH1 was identified as a direct target of METTL14, with METTL14 overexpression leading to increased m6A methylation of FTH1 mRNA, resulting in reduced stability and expression of FTH1 in CC. Furthermore, FTH1 overexpression or treatment with LY294002 partially counteracted the promotion of sorafenib-induced ferroptosis by METTL14. In vivo xenograft experiments demonstrated that inhibiting METTL14 reduced the anticancer effects of sorafenib, whereas suppression of FTH1 significantly enhanced sorafenib-induced ferroptosis and increased its anticancer efficacy. METTL14 reduces FTH1 mRNA stability through m6A methylation, thereby enhancing sorafenib-induced ferroptosis, which contributes to suppressing CC progression via the PI3K/Akt signaling pathway.

Size-Optimized Layered Double Hydroxide Nanoparticles Promote Neural Progenitor Cells Differentiation of Embryonic Stem Cells Through the Regulation of M6A Methylation.

Purpose: The committed differentiation fate regulation has been a difficult problem in the fields of stem cell research, evidence showed that nanomaterials could promote the differentiation of stem cells into specific cell types. Layered double hydroxide (LDH) nanoparticles possess the regulation function of stem cell fate, while the underlying mechanism needs to be investigated. In this study, the process of embryonic stem cells (ESCs) differentiate to neural progenitor cells (NPCs) by magnesium aluminum LDH (MgAl-LDH) was investigated. Methods: MgAl-LDH with diameters of 30, 50, and 100 nm were synthesized and characterized, and their effects on the cytotoxicity and differentiation of NPCs were detected in vitro. Dot blot and MeRIP-qPCR were performed to detect the level of m6A RNA methylation in nanoparticles-treated cells. Results: Our work displayed that LDH nanoparticles of three different sizes were biocompatible with NPCs, and the addition of MgAl-LDH could significantly promote the process of ESCs differentiate to NPCs. 100 nm LDH has a stronger effect on promoting NPCs differentiation compared to 30 nm and 50 nm LDH. In addition, dot blot results indicated that the enhanced NPCs differentiation by MgAl-LDH was closely related to m6A RNA methylation process, and the major modification enzyme in LDH controlled NPCs differentiation may be the m6A RNA methyltransferase METTL3. The upregulated METTL3 by LDH increased the m6A level of Sox1 mRNA, enhancing its stability. Conclusion: This work reveals that MgAl-LDH nanoparticles can regulate the differentiation of ESCs into NPCs by increasing m6A RNA methylation modification of Sox1.

Functional characterisation of Artemisia annua jasmonic acid carboxyl methyltransferase: a key enzyme enhancing artemisinin biosynthesis.

MAIN CONCLUSION: Overexpression of Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT) leads to enhanced artemisinin content in Artemisia annua. Artemisinin-based combination therapies remain the sole deterrent against deadly disease malaria and Artemisia annua remains the only natural producer of artemisinin. In this study, the 1101 bp gene S-adenosyl-L-methionine (SAM): Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT), was characterised from A. annua, which converts jasmonic acid (JA) to methyl jasmonate (MeJA). From phylogenetic analysis, we confirmed that AaJMT shares a common ancestor with Arabidopsis thaliana, Eutrema japonica and has a close homology with JMT of Camellia sinensis. Further, the Clustal Omega depicted that the conserved motif I, motif III and motif SSSS (serine) required to bind SAM and JA, respectively, are present in AaJMT. The relative expression of AaJMT was induced by wounding, MeJA and salicylic acid (SA) treatments. Additionally, we found that the recombinant AaJMT protein catalyses the synthesis of MeJA from JA with a Km value of 37.16 µM. Moreover, site-directed mutagenesis of serine-151 in motif SSSS to tyrosine, asparagine-10 to threonine and glutamine-25 to histidine abolished the enzyme activity of AaJMT, thus indicating their determining role in JA substrate binding. The GC-MS analysis validated that mutant proteins of AaJMT were unable to convert JA into MeJA. Finally, the artemisinin biosynthetic and trichome developmental genes were upregulated in AaJMT overexpression transgenic lines, which in turn increased the artemisinin content.

METTL3's role in cervical cancer development through m6A modification.

N6-methylated adenosine (m6A) is a crucial RNA modification in eukaryotes, particularly in cancer. However, its role in cervical cancer (CC) is unclear. We aimed to elucidate the part of m6A in CC by analyzing methyltransferase-like 3 (METTL3) expression, identifying downstream targets, and exploring the underlying mechanism. We assessed METTL3 expression in CC using western blotting, quantitative polymerase chain reaction (qPCR), and immunohistochemistry. In vitro and in vivo experiments examined METTL3's role in CC. We employed RNA sequencing, methylated RNA immunoprecipitation sequencing, qPCR, and RNA immunoprecipitation qPCR to explore METTL3's mechanism in CC. METTL3 expression was upregulated in CC, promoting cell proliferation and metastasis. METTL3 knockdown inhibited human cervical cancer by inactivating AKT/mTOR signaling pathway. METTL3-mediated m6A modification was observed in CC cells, targeting phosphodiesterase 3A (PDE3A). METTL3 catalyzed m6A modification on PDE3A mRNA through YTH domain family protein 3 (YTHDF3). Our study indicated the mechanism of m6A modification in CC and suggested the METTL3/YTHDF3/PDE3A axis as a potential clinical target for CC treatment.

[Isolation and functional characterization of (iso)eugenol O-methyltransferase (IEMT) gene in Asarum sieboldii].

Methyleugenol is one of the main active constituents in the volatile oil of the traditional Chinese medicine Asari Radix et Rhizoma. It possesses various pharmacological effects such as analgesic, anesthetic, and anti-inflammatory properties. In biosynthesis, the initial precursor phenylalanine is finally converted into methyleugenol through a series of intermediate compounds including coniferyl acid, courmaryl acid, caffeic acid, ferulic acid/ferulic-CoA, coniferyl aldehyde, conferyl alcohol, cnfiferyl acetate, and eugenol/isoeugenol, which are produced through catalysis of a large number of enzymes. Eugenol O-methyltransferase(EOMT) is one of the key enzymes in the biosynthesis pathway, capable of methylating eugenol on the para-site hydroxyl group of the benzene ring, thereby generating methyleugenol. Here, an(iso)eugenol O-methyltransferase(IEMT) gene was cloned for the first time from Asarum siebo-ldii, holding an open reading frame that consisted of 1 113 bp and encoded a protein containing 370 amino acid residues. Bioinformatics analysis results showed that this protein was equipped with the characteristic structural domains of methyltransferases such as S-adenosylmethionine(SAM) binding sites and dimerization domains. The prokaryotic expression recombinant plasmid pET28a(+)-AsIEMT was constructed, and the candidate protein was induced and purified. In vitro enzyme assays confirmed that AsIEMT had dual functions. The enzyme could catalyze the production either of methyleugenol from eugenol or of methylisoeugenol from isoeugenol, although the latter was more prevalent. When isoeugenol was used as the substrate, the kinetics parameters K_m and V_(max) of catalytic reaction were(0.90±0.06) mmol·L~(-1) and(1.32±0.04)nmol·s~(-1)·mg~(-1), respectively. This study expanded our understandings of critical enzyme genes involved in phenylpropanoid metabolic pathways, and would facilitate the elucidation of quality formation mechanisms of the TCM Asari Radix et Rhizoma.

Identification of a methyltransferase-related long noncoding RNA signature as a novel prognosis biomarker for lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) accounts for a high proportion of tumor deaths globally, while methyltransferase-related lncRNAs in LUAD were poorly studied. METHODS: In our study, we focused on two distinct cohorts, TCGA-LUAD and GSE3021, to establish a signature of methyltransferase-related long non-coding RNAs (MeRlncRNAs) in LUAD. We employed univariate Cox and LASSO regression analyses as our main analytical tools. The GSE30219 cohort served as the validation cohort for our findings. Furthermore, to explore the differential pathway enrichments between groups stratified by risk, we utilized Gene Set Enrichment Analysis (GSEA). Additionally, single-sample GSEA (ssGSEA) was conducted to assess the immune infiltration landscape within each sample. Reverse transcription quantitative PCR (RT-qPCR) was also performed to verify the expression of prognostic lncRNAs in both clinically normal and LUAD samples. RESULTS: In LUAD, we identified a set of 32 MeRlncRNAs. We further narrowed our focus to six prognostic lncRNAs to develop gene signatures. The TCGA-LUAD cohort and GSE30219 were utilized to validate the risk score model derived from these signatures. Our analysis showed that the risk score served as an independent prognostic factor, linked to immune-related pathways. Additionally, the analysis of immune infiltration revealed that the immune landscape in high-risk groups was suppressed, which could contribute to poorer prognoses. We also constructed a regulatory network comprising 6 prognostic lncRNAs, 19 miRNAs, and 21 mRNAs. Confirmatory RT-qPCR results aligned with public database findings, verifying the expression of these prognostic lncRNAs in the samples. CONCLUSION: The prognostic gene signature of LUAD associated with MeRlncRNAs that we provided, may offer us a comprehensive picture of the prognosis prediction for LUAD patients.