N6-methyladenosine Modification Research Articles

Multi-omics integration analysis reveals the role of N6-methyladenosine in lncRNA translation during glioma stem cell differentiation.

Glioblastoma is one of the most lethal brain diseases in humans. Although recent studies have shown reciprocal interactions between N6-methyladenosine (m6A) modifications and long noncoding RNAs (lncRNAs) in gliomagenesis and malignant progression, the mechanism of m6A-mediated lncRNA translational regulation in glioblastoma remains unclear. Herein, we profiled the transcriptomes, translatomes, and epitranscriptomics of glioma stem cells and differentiated glioma cells to investigate the role of m6A in lncRNA translation comprehensively. We found that lncRNAs with numerous m6A peaks exhibit reduced translation efficiency. Transcript-level expression analysis demonstrates an enrichment of m6A around short open reading frames (sORFs) of translatable lncRNA transcripts. Further comparison analysis of m6A modifications in different RNA regions indicates that m6A peaks downstream of sORFs inhibit lncRNA translation more than those upstream. Observations in glioma-associated lncRNAs H19, LINC00467, and GAS5 further confirm the negative effect of m6A methylation on lncRNA translation. Overall, these findings elucidate the dynamic profiles of the m6A methylome and enhance the understanding of the complexity of lncRNA translational regulation.

ALKBH5 regulates etoposide-induced cellular senescence and osteogenic differentiation in osteoporosis through mediating the m6A modification of VDAC3.

Osteoporosis, a common bone disease in older individuals, involves the progression influenced by N6-methyladenosine (m6A) modification. This study aimed to elucidate the effects of VDAC3 m6A modification on human bone mesenchymal stromal cell (BMSC) senescence and osteogenic differentiation. BMSCs were treated with etoposide to induce senescence. Senescence was assessed by β-galactosidase staining and quantitative real-time PCR (qPCR), and osteogenic differentiation was evaluated using Western blot, alkaline phosphatase, and alizarin red S staining. VDAC3 and ALKBH5 expression were quantified by qPCR, and their interaction was assessed by RNA immunoprecipitation (RIP) and luciferase reporter assay. m6A methylation was analyzed using the Me-RIP assay. VDAC3 expression was significantly decreased in etoposide-treated BMSCs (1.00 ± 0.13 vs. 0.26 ± 0.06). VDAC3 overexpression reduced etoposide-induced senescence and promoted osteogenic differentiation. ALKBH5 overexpression inhibited VDAC3 m6A modification (1.00 ± 0.095 vs. 0.233 ± 0.177) and its stability. ALKBH5 knockdown decreased etoposide-induced senescence and promoted osteogenic differentiation, effects that were reversed by VDAC3 knockdown. YTHDF1 was identified as the m6A methylation reader, and its overexpression inhibited VDAC3 stability. We demonstrated that ALKBH5 inhibited osteogenic differentiation of etoposide-induced senescent cells through the inhibition of VDAC3 m6A modification, and YTHDF1 acted as the m6A methylation reader. These findings provide a novel theoretical basis for the treatment of osteoporosis.

HNRNPC mediates lymphatic metastasis of cervical cancer through m6A-dependent alternative splicing of FOXM1.

Cervical cancer (CCa) patients with lymph node (LN) metastasis face poor prognoses and have limited treatment options. Aberrant N6-methyladenosine (m6A) modification of RNAs are known to promote tumor metastasis, but their role in CCa remains unclear. Our study reveals that HNRNPC, an alternative splicing (AS) factor and m6A reader, increases tumor-related variants through m6A-dependent manner, thereby promoting lymphatic metastasis in CCa. We found that HNRNPC overexpression correlates with lymphatic metastasis and poorer prognoses in CCa patients. Functionally, knocking down HNRNPC markedly inhibited the migration and invasion of several CCa cell lines, while supplementing HNRNPC restored the malignant phenotypes of these cells. Mechanistically, HNRNPC regulates exon skipping of FOXM1 by binding to its m6A-modified motif. Mutating the m6A site on FOXM1 weakened the interaction between HNRNPC and FOXM1 pre-RNA, leading to a reduction in the metastasis-related FOXM1-S variant. In conclusion, our findings demonstrate that m6A-dependent alternative splicing mediated by HNRNPC is essential for lymphatic metastasis in CCa, potentially providing novel clinical markers and therapeutic strategies for patients with advanced CCa.

Intracellular C5aR1 inhibits ferroptosis in glioblastoma through METTL3-dependent m6A methylation of GPX4

Glioblastoma (GBM) is the most common primary intracranial malignant tumor. Recent literature suggests that induction of programmed death has become a mainstream cancer treatment strategy, with ferroptosis being the most widely studied mode. Complement C5a receptor 1 (C5aR1) is associated with both tumorigenesis and tumor-related immunity. However, knowledge regarding the role of C5aR1 in GBM progression is limited. In the present study, we observed significant upregulation of C5aR1 in glioma tissue. In addition, C5aR1 expression was found to be closely associated with patient prognosis and survival. Subsequent experimental verification demonstrated that C5aR1 promoted the progression of GBM mainly by suppressing ferroptosis induction, inhibiting the accumulation of lipid peroxides, and stabilizing the expression of the core antiferroptotic factor glutathione peroxidase 4 (GPX4). Aberrant N6-methyladenosine (m6A) modification of GPX4 mRNA contributes significantly to epigenetic tumorigenesis, and here, we report that selective methyltransferase-like 3 (METTL3)-dependent m6A methylation of GPX4 plays a key role in C5AR1 knockdown-induced ferroptosis induction. Mechanistically, ERK1/2 signaling pathway activation increases the METTL3 protein abundance in GBM cells. This activation then increases the stability of METTL3-mediated m6A modifications on GPX4, enabling it to fulfill its transcriptional function. More importantly, in an intracranial xenograft mouse model, PMX205, a C5aR1 inhibitor, promoted alterations in ferroptosis in GBM cells and inhibited GBM progression. In conclusion, our findings suggest that C5aR1 inhibits ferroptosis in GBM cells and promotes MettL3-dependent GPX4 expression through ERK1/2, thereby promoting glioma progression. Our study reveals a novel mechanism by which the intracellular complement receptor C5aR1 suppresses ferroptosis induction and promotes GBM progression. These findings may facilitate the identification of a potential therapeutic target for glioma.

N6-methyladenosine modification-tuned lipid metabolism controls skin immune homeostasis via regulating neutrophil chemotaxis.

Disrupted N6-methyladenosine (m6A) modification modulates various inflammatory disorders. However, the role of m6A in regulating cutaneous inflammation remains elusive. Here, we reveal that the m6A and its methyltransferase METTL3 are down-regulated in keratinocytes in inflammatory skin diseases. Inducible deletion of Mettl3 in murine keratinocytes results in spontaneous skin inflammation and increases susceptibility to cutaneous inflammation with activation of neutrophil recruitment. Therapeutically, restoration of m6A alleviates the disease phenotypes in mice and suppresses inflammation in human biopsy specimens. We support a model in which m6A modification stabilizes the mRNA of the lipid-metabolizing enzyme ELOVL6 via the m6A reader IGF2BP3, leading to a rewiring of fatty acid metabolism with a reduction in palmitic acid accumulation and, consequently, suppressing neutrophil chemotaxis in cutaneous inflammation. Our findings highlight a previously unrecognized epithelial-intrinsic m6A modification-lipid metabolism pathway that is essential for maintaining epidermal and immune homeostasis and lay the basis for potential therapeutic targeting of m6A modulators to attenuate inflammatory skin diseases.

FTO-mediated SMAD2 m6A modification protects cartilage against Osteoarthritis.

N6-methyladenosine (m6A) modification is one of the most prevalent forms of epigenetic modification and plays an important role in the development of degenerative diseases such as osteoarthritis (OA). However, the evidence concerning the role of m6A modification in OA is insufficient. Here, m6A modification was increased in human OA cartilage and degenerated chondrocytes. Among all of the m6A enzymes, the expression of the demethylase fat mass and obesity-associated protein (FTO) decreased dramatically. Conditional knockout of FTO in chondrocytes accelerates OA progression. FTO transcription is regulated by runt-related transcription factor-1 (RUNX1). Reduced FTO elevates m6A modification at the adenosine N6 position in SMAD family member 2 (SMAD2) mRNA, whose stability is subsequently modulated by the recruited m6A reader protein YTH N6-methyladenosine RNA binding protein F2 (YTHDF2). Collectively, these findings reveal the function and mechanism of the m6A family member FTO in OA progression. Therefore, reducing m6A modification to increase SMAD2 stability by activating FTO might be a potential therapeutic strategy for OA treatment.

Notch-Driven Cholangiocarcinogenesis Involves the Hippo Pathway Effector TAZ via METTL3-m6A-YTHDF1

Background and AimsNotch and TAZ are implicated in cholangiocarcinogenesis, but whether and how these oncogenic molecules interact remain unknown. MethodsThe development of CCA was induced by hydrodynamic tail vein (HDTV) injection of oncogenes (NICD/AKT) to the FVB/NJ mice. CCA xenograft was developed by inoculation of human CCA cells into the livers of SCID mice. Tissues and cells were analyzed using qRT-PCR, Western blotting analyses, Immunohistochemistry, ChIP-qPCR and WST-1 cell proliferation Assay. ResultsOur experimental findings show that TAZ is indispensable in NICD-driven cholangiocarcinogenesis. Notch activation induces the expression of METTL3 (Methyltransferase like-3) which catalyzes N6-methyladenosine (m6A) modification of TAZ mRNA and that this mechanism plays a central role in the crosstalk between Notch and TAZ in CCA cells. Mechanistically, Notch regulates the expression of METTL3 through the binding of NICD to its downstream transcription factor CSL in the promoter region of METTL3. METTL3 in turn mediates m6A modification of TAZ mRNA which is recognized by the m6A reader YTHDF1 to enhance TAZ protein translation. We observed that inhibition of Notch signaling decreased the protein levels of both MELLT3 and TAZ. Depletion of METTL3 by shRNAs or by the next generation GapmeR antisense oligonucleotides (ASOs) decreased the level of TAZ protein and inhibited the growth of human CCA cells in vitro and in mice. ConclusionThis study describes a novel Notch-METTL3-TAZ signaling cascade which is important in CCA development and progression. Our experimental results provide new insight into how the Notch pathway cooperates with TAZ signaling in CCA, and the findings may have important therapeutic implications.

Upregulation of LY6K induced by FTO-mediated demethylation promotes the tumorigenesis and metastasis of oral squamous cell carcinoma via CAV-1-mediated ERK1/2 signaling activation.

Lymphocyte antigen 6 complex locus K (LY6K) has been demonstrated to play a significant role in cancers and identified as a therapeutic biomarker for head and neck squamous cell carcinoma. However, the role of LY6K in oral squamous cell carcinoma (OSCC) has not been explored. The current study discovered that LY6K was aberrantly upregulated in OSCC cell lines and tissues and that high LY6K expression significantly correlated with poorer survival of OSCC patients. Through stable knockdown of LY6K, we found that the growth, colony formation, migration, and invasion of OSCC cells were substantially suppressed. In addition, tumor growth and lung metastasis in vivo were effectively inhibited by LY6K depletion. Mechanically, LY6K binds with CAV-1 and activates CAV-1-mediated MAPK/ERK signaling to exert its oncogenic effects on OSCC. In addition, LY6K expression in OSCC was discovered to be regulated by FTO-mediated RNA N6-methyladenosine (m6A) modification in an IGF2BP1-dependent manner. Generally, LY6K expression was upregulated by FTO-mediated demethylation in OSCC, which promoted the tumorigenesis and metastasis of OSCC via activating the CAV-1-mediated ERK1/2 signaling pathway.

Methyltransferase-Like 3-Mediated N6-Methyladenosine RNA Methylation Regulates Hypoxia-Induced Pulmonary Arterial Smooth Muscle Cell Pyroptosis by Targeting PTEN.

Pulmonary hypertension is a rare, progressive disorder that can lead to right ventricular hypertrophy, right heart failure, and even sudden death. N6-methyladenosine modification and the main methyltransferase that mediates it, methyltransferase-like (METTL) 3, exert important effects on many biological and pathophysiological processes. However, the role of METTL3 in pyroptosis remains unclear. Here, we characterized the role of METTL3 and the underlying cellular and molecular mechanisms of pyroptosis, which is involved in pulmonary hypertension. METTL3 was downregulated in a pulmonary hypertension mouse model and in hypoxia-exposed pulmonary artery smooth muscle cell. The small interfering RNA-induced silencing of METTL3 decreased the m6A methylation levels and promoted pulmonary artery smooth muscle cell pyroptosis, mimicking the effects of hypoxia. In contrast, overexpression of METTL3 suppressed hypoxia-induced pulmonary artery smooth muscle cell pyroptosis. Mechanistically, we identified the phosphate and tension homology deleted on chromosome 10 (PTEN) gene as a target of METTL3-mediated m6A modification, and methylated phosphate and tension homology deleted on chromosome 10 mRNA was subsequently recognized by the m6A "reader" protein insulin-like growth factor 2 mRNA-binding protein 2, which directly bound to the m6A site on phosphate and tension homology deleted on chromosome 10 mRNA and enhanced its stability. These results identify a new signaling pathway, the METTL3/phosphate and tension homology deleted on chromosome 10/insulin-like growth factor 2 mRNA-binding protein 2 axis, that participates in the regulation of hypoxia-induced pyroptosis.

Evidence for the potential role of m6A modification in regulating autophagy in models of amyotrophic lateral sclerosis

Objective: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. Research indicates that N6-methyladenosine (m6A) modification plays a crucial role in cellular autophagy during ALS development. This study investigates the role of autophagy in ALS, with a focus on the effect of messenger ribonucleic acid m6A methylation modification on disease progression. Material and Methods: We compared m6A levels and regulatory molecule expressions in transgenic superoxide dismutase (SOD1)-G93A and non-transgenic mice, categorized into end-stage and control groups, using quantitative polymerase chain reaction and Western blotting. The NSC-34 cell line, which was modified to model ALS, enabled the investigation of apoptosis, autophagy, and autophagy disruption through terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assays, Western blotting, and fluorescent staining. Results: Our findings indicate significantly elevated m6A methylation levels in ALS mice (0.262 ± 0.005) compared with the controls (0.231 ± 0.003) and in the ALS model cells (0.242±0.005) relative to those belonging to the wild-type control group (0.183 ± 0.007). Furthermore, the proteins involved in m6A RNA modification differed between groups, which suggest impaired autophagy flux in the ALS models. Conclusion: These results suggest that m6A methylation may accelerate ALS progression through the disruption of autophagic processes. Our study underscores the role of m6A methylation in the pathology of ALS and proposes the targeting of m6A methylation as a potential therapeutic strategy for disease treatment. Although this study primarily used transgenic SOD1-G93A mice and NSC-34 cell models to investigate ALS pathology, potential differences in disease mechanisms between animal models and humans must be considered. Although a correlation was detected between m6A methylation levels and autophagy disruption in ALS, the study primarily established an association rather than provided detailed mechanistic insights.

STM2457 Inhibits METTL3-Mediated m6A Modification of miR-30c to Alleviate Spinal Cord Injury by Inducing the ATG5-Mediated Autophagy.

The study aimed to investigate the role of N6-methyladenosine (m6A) modification in spinal cord injury (SCI) and its underlying mechanism, focusing on the interplay between m6A methyltransferase-like 3 (METTL3), miR-30c, and autophagy-related proteins. An SCI model was established in rats, and changes in autophagy-related proteins, m6A methylation levels, and miR-30c levels were analyzed. Hydrogen peroxide (H2O2)-stimulated spinal cord neuron cells (SCNCs) were used to assess the impact of METTL3 overexpression. The effects of STM2457, an antagonist of METTL3, were evaluated on cell viability, apoptosis, and autophagy markers in H2O2-stimulated SCNCs. In the SCI model, decreased levels of autophagy markers and increased m6A methylation, miR-30c levels, and METTL3 were observed. Overexpression of METTL3 in SCNCs led to reduced cell viability, increased apoptosis, and suppressed autophagy. Conversely, co-overexpression of autophagy-related protein 5 (ATG5) or miR-30c inhibition reversed these effects. Knocking out METTL3 yielded opposite results. STM2457 treatment improved cell viability, reduced apoptosis, and upregulated autophagy markers in SCNCs, which also enhanced functional recovery in rats as measured by the Basso-Beattie-Bresnahan score and inclined plate test. STM2457 alleviated SCI by suppressing METTL3-mediated m6A modification of miR-30c, which in turn induces ATG5-mediated autophagy. This study provides insights into the role of m6A modification in SCI and suggests a potential therapeutic approach through targeting METTL3.

N6-Methyladenosine-mediated phase separation suppresses NOTCH1 expression and promotes mitochondrial fission in diabetic cardiac fibrosis.

N6-methyladenosine (m6A) modification of messenger RNA (mRNA) is crucial for liquid-liquid phase separation in mammals. Increasing evidence indicates that liquid-liquid phase separation in proteins and RNAs affects diabetic cardiomyopathy. However, the molecular mechanism by which m6A-mediated phase separation regulates diabetic cardiac fibrosis remains elusive. Leptin receptor-deficient mice (db/db), cardiac fibroblast-specific Notch1 conditional knockout (POSTN-Cre × Notch1flox/flox) mice, and Cre mice were used to induce diabetic cardiac fibrosis. Adeno-associated virus 9 carrying cardiac fibroblast-specific periostin (Postn) promoter-driven small hairpin RNA targeting Alkbh5, Ythdf2, or Notch1, and the phase separation inhibitor 1,6-hexanediol were administered to investigate their roles in diabetic cardiac fibrosis. Histological and biochemical analyses were performed to determine how Alkbh5 and Ythdf2 regulate Notch1 expression in diabetic cardiac fibrosis. NOTCH1 was reconstituted in ALKBH5- and YTHDF2-deficient cardiac fibroblasts and mouse hearts to study its effects on mitochondrial fission and diabetic cardiac fibrosis. Heart tissue samples from patients with diabetic cardiomyopathy were used to validate our findings. In mice with diabetic cardiac fibrosis, decreased Notch1 expression was accompanied by high m6A mRNA levels and mitochondrial fission. Fibroblast-specific deletion of Notch1 enhanced mitochondrial fission and cardiac fibroblast proliferation and induced diabetic cardiac fibrosis in mice. Notch1 downregulation was associated with Alkbh5-mediated m6A demethylation in the 3'UTR of Notch1 mRNA and elevated m6A mRNA levels. These elevated m6A levels in Notch1 mRNA markedly enhanced YTHDF2 phase separation, increased the recognition of m6A residues in Notch1 mRNA by YTHDF2, and induced Notch1 degradation. Conversely, epitranscriptomic downregulation rescues Notch1 expression, resulting in the opposite effects. Human heart tissues from patients with diabetic cardiomyopathy were used to validate the findings in mice with diabetic cardiac fibrosis. We identified a novel epitranscriptomic mechanism by which m6A-mediated phase separation suppresses Notch1 expression, thereby promoting mitochondrial fission in diabetic cardiac fibrosis. Our findings provide new insights for the development of novel treatment approaches for patients with diabetic cardiac fibrosis.

Hypoxic stabilization of RIPOR3 mRNA via METTL3-mediated m6A methylation drives breast cancer progression and metastasis.

Dysregulated N6-methyladenosine (m6A) modification has been associated with breast cancer pathogenesis. Hypoxia which characterizes solid tumors is known to reprogram the m6A epitranscriptome, but the underlying mechanisms of how this process contributes to breast cancer progression remain poorly understood. Through integrative analyses of m6A-RIP sequencing and RNA sequencing databases, we reveal a cluster of mRNAs with upregulated m6A methylation and expression under hypoxia, that are enriched by many oncogenic pathways, including PI3K-Akt signaling. Furthermore, we identify the mRNA, RIPOR3, as a target of METTL3-mediated m6A methylation in response to hypoxia. We find that m6A methylation stabilizes RIPOR3, increasing its protein expression in a METTL3 catalytic activity-dependent manner, and consequently driving breast tumor growth and metastasis. RIPOR3 is found to be overexpressed in breast cancer cell lines and tumor tissues from breast cancer patients, in whom elevated RIPOR3 is associated with a worse prognosis. Mechanistically, we show that RIPOR3 interacts with EGFR and is essential for the PI3K-Akt pathway activation. In conclusion, we identify RIPOR3 as a hypoxia-stabilized oncogenic driver via METTL3-mediated m6A methylation, thus provide a potential therapeutic target for breast cancer.

Selective Effect of DNA N6-Methyladenosine Modification on Transcriptional Genetic Variations in East Asian Samples

Selective Effect of DNA N6-Methyladenosine Modification on Transcriptional Genetic Variations in East Asian Samples

Propofol attenuates prostate cancer progression by upregulating TRHDE-AS1 expression, and METTL14 could mediate its m6A modification.

Propofol has become a microtubule-stabilizing drug for prostate cancer (PC) therapy, but propofol resistance impairs the therapeutic effect. This study aimed to explore the regulatory mechanism of propofol in the pathogenesis of PC through mechanisms involving N6-methyladenosine (m6A) modification. The changes in PC cell malignancy were evaluated by means of transwell, cell counting kit 8 (CCK-8), western blotting and tumour xenograft model assays. Long noncoding RNA TRHDE-AS1 and m6A methyltransferase METTL14 expression levels were determined via reverse transcription quantitative polymerase chain reaction (RT-qPCR). The m6A modification of TRHDE-AS1 which was mediated by METTL14 was confirmed by conducting methylated RNA immunoprecipitation (MeRIP) assay. We observed that propofol (200 μM) inhibited PC cell malignancy in vivo and in vitro, elucidating that it impaired cell proliferation, migration and tumour growth but induced apoptosis. TRHDE-AS1 expression was observed to be lower in PC cells and tissues, and propofol induced TRHDE-AS1 upregulation in PC cells. Propofol was capable of reversing the tumour-promoting effect of TRHDE-AS1 knockdown in PC cells. Additionally, METTL14 was upstream of TRHDE-AS1 to induce m6A modification of TRHDE-AS1 in PC cells. Collectively, our results show that propofol prevents PC progression by upregulating TRHDE-AS1 expression and METTL14 is involved in the m6A modification of TRHDE-AS1. These findings suggest that TRHDE-AS1 may be a potential therapeutic target for the improvement of propofol's therapeutic effect.

Regulatory role and molecular mechanism of METTL14 in vascular endothelial cell injury in preeclampsia.

Preeclampsia (PE) is a pregnancy-related disease characterized by vascular endothelial cell injury. This study aimed to investigate the role of methyltransferase-like protein 14 (METTL14) in vascular endothelial cell injury in PE. The PE cell model was established by treating human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor-alpha (TNF-α) in vitro. METTL14 and forkhead box protein 1 (FOXP1) were silenced, and miR-34a-5p was overexpressed in HUVECs to evaluate their effects. HUVEC viability, apoptosis, and the levels of intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and endothelin-1 were measured. The N6-methyladenosine (m6A) modification of pri-miR-34a-5p was quantified. The interactions between miR-34a-5p, DiGeorge syndrome critical region 8, and m6A enrichment in miR-34a-5p were analyzed. The relationship between miR-34a-5p and FOXP1 was also verified. The results showed that the expressions of METTL14, FOXP1, and miR-34a-5p were determined. METTL14 expression was elevated in the TNF-α-induced HUVEC injury model. Silencing METTL14 improved HUVEC viability, inhibited apoptosis, and reduced endothelial inflammation. METTL14 promoted miR-34a-5p expression through m6A modification. Overexpression of miR-34a-5p or silencing of FOXP1 reversed the protective effects of METTL14 silencing on cell injury in the PE model. In conclusion, METTL14 mediated m6A modification to promote miR-34a-5p expression, leading to the inhibition of FOXP1 expression, which aggravated endothelial cell damage in the PE cell model.

ALKBH5 Regulates Osteogenic Differentiation via the lncRNA/mRNA Complex

Human adipose-derived stem cells (hASCs) are commonly used in bone tissue regeneration. The N6-methyladenosine (m6A) modification has emerged as a novel regulatory mechanism for gene expression, playing a critical role in osteogenic differentiation of stem cells. However, the precise role and mechanism of alkylation repair homolog 5 (ALKBH5) in hASC osteogenesis remain incompletely elucidated and warrant further investigation. Herein, we employed methylated RNA immunoprecipitation sequencing, RNA sequencing, and weighted gene coexpression network analysis to identify a key long noncoding RNA (lncRNA) in hASCs: lncRNA AK311120. Functional experiments demonstrated that lnc-AK311120 promoted the osteogenic differentiation of hASCs, while a mutation at the m6A central site A of lnc-AK311120 was found to decrease the level of m6A modification. The osteogenic effect of ALKBH5 was confirmed both in vitro and in vivo using a mandibular defect model in nude mice. Subsequent investigations revealed that knockdown of ALKBH5 resulted in a significant increase in the m6A modification level of lnc-AK311120, accompanied by a downregulation in the expression level of lnc-AK311120. Additional rescue experiments demonstrated that overexpression of lnc-AK311120 could restore the phenotype after ALKBH5 knockdown. We observed that AK311120 interacted with the RNA-binding proteins DExH-Box helicase 9 (DHX9) and YTH domain containing 2 (YTHDC2) to form a ternary complex, while mitogen-activated protein kinase kinase 7 (MAP2K7) served as the shared downstream target gene of DHX9 and YTHDC2. Knockdown of AK311120 led to a reduction in the binding affinity between DHX9/YTHDC2 and the target gene MAP2K7. Furthermore, ALKBH5 facilitated the translation of MAP2K7 and activated the downstream JNK signaling pathway through the AK311120–DHX9–YTHDC2 complex, without affecting its messenger RNA level. Collectively, we have investigated the regulatory effect and mechanism of ALKBH5-mediated demethylation of lncRNA in hASC osteogenesis for the first time, offering a promising approach for bone tissue engineering.

DDX5 promotes esophageal squamous cell carcinoma growth through sustaining VAV3 mRNA stability.

Novel therapeutic targets and their inhibitors for esophageal squamous cell carcinoma (ESCC) prevention and therapy are urgently needed. This study aimed to investigate the function of DEAD-box helicase 5 (DDX5) in ESCC progression and to identify a promising inhibitor of DDX5. We verified that DDX5 was highly expressed in ESCC and played an oncogenic role, binding with vav guanine nucleotide exchange factor 3 (VAV3) mRNA and facilitating VAV3 mRNA N6-methyladenosine (m6A) modification by interacting with the m6A methyltransferase 3 (METTL3). M6A-modified VAV3 mRNA was identified by insulin-like growth factor 1 (IGF2BP1), increasing mRNA stability. Methylnissolin-3-β-D-O-glucoside (MD) inhibited ESCC progression through the DDX5-VAV3 axis. Our findings suggest that DDX5 promotes ESCC progression. MD inhibits ESCC progression by targeting DDX5.

Enhanced detection of RNA modifications and read mapping with high-accuracy nanopore RNA basecalling models.

In recent years, nanopore direct RNA sequencing (DRS) became a valuable tool for studying the epitranscriptome, due to its ability to detect multiple modifications within the same full-length native RNA molecules. While RNA modifications can be identified in the form of systematic basecalling 'errors' in DRS datasets, N6-methyladenosine (m6A) modifications produce relatively low 'errors' compared to other RNA modifications, limiting the applicability of this approach to m6A sites that are modified at high stoichiometries. Here, we demonstrate that the use of alternative RNA basecalling models, trained with fully unmodified sequences, increases the 'error'signal of m6A, leading to enhanced detection and improved sensitivity even at low stoichiometries. Moreover, we find that high-accuracy alternative RNA basecalling models can show up to 97% median basecalling accuracy, outperforming currently available RNA basecalling models, which show 91% median basecalling accuracy. Notably, the use of high-accuracy basecalling models is accompanied by a significant increase in the number of mapped reads -especially in shorter RNA fractions- and increased basecalling error signatures at pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ) modified sites. Overall, our work demonstrates that alternative RNA basecalling models can be used to improve the detection of RNA modifications, read mappability, and basecalling accuracy in nanopore DRS datasets.

Long-read RNA sequencing reveals allele-specific N6-methyladenosine modifications.

Long-read sequencing technology enables highly accurate detection of allele-specific RNA expression, providing insights into the effects of genetic variation on splicing and RNA abundance. Furthermore, the ability to directly sequence RNA promises the detection of RNA modifications in tandem with ascertaining the allelic origin of each molecule. Here, we leverage these advantages to determine allele-biased patterns of N6-methyladenosine (m6A) modifications in native mRNA. We utilized human and mouse cells with known genetic variants to assign allelic origin of each mRNA molecule combined with a supervised machine learning model to detect read-level m6A modification ratios. Our analyses revealed the importance of sequences adjacent to the DRACH-motif in determining m6A deposition, in addition to allelic differences that directly alter the motif. Moreover, we discovered allele-specific m6A modification (ASM) events with no genetic variants in close proximity to the differentially modified nucleotide, demonstrating the unique advantage of using long reads and surpassing the capabilities of antibody-based short-read approaches. This technological advancement promises to advance our understanding of the role of genetics in determining mRNA modifications.