m6A Reader Research Articles

METTL16 inhibits pancreatic cancer proliferation and metastasis by promoting MROH8 RNA stability and inhibiting CAPN2 expression.

N6-methyladenosine (m6A) modification plays a crucial role in the progression of various cancers, including pancreatic cancer, by regulating gene expression. However, the specific mechanisms by which m6A affects pancreatic cancer metastasis remain unclear. This study aims to elucidate the role of METTL16, an m6A writer gene, in regulating core genes such as CAPN2 and MROH8, influencing tumor growth and metastasis. Transcriptomic data from pancreatic cancer patients in The Cancer Genome Atlas (TCGA) were analyzed to identify m6A-related genes. We performed correlation and survival analyses to uncover core genes influenced by m6A expression. Functional assays, including METTL16 knockdown and overexpression experiments, were conducted in pancreatic cancer cell lines, patient-derived organoids, and animal models. Immunofluorescence, co-immunoprecipitation (Co-IP), and m6A-specific quantitative PCR were used to validate protein interactions and m6A modifications. Chromatin immunoprecipitation (ChIP) analysis was utilized to investigate transcription factor binding at gene promoter regions. METTL16 and METTL3 were identified as key m6A regulators associated with improved prognosis in pancreatic cancer patients (P<0.05). CAPN2, CHMP2B, ITGA3, ITGA6, ITPR1, and RAC1 were identified as core genes linked to m6A expression, all significantly correlated with patient prognosis (P<0.05). METTL16 overexpression significantly inhibited tumor growth and metastasis (P<0.001) by downregulating CAPN2 through an indirect mechanism involving the transcription factor TBP and the gene MROH8. MROH8 negatively regulated CAPN2 by promoting TBP degradation, with METTL16 enhancing MROH8 mRNA stability through m6A modifications (P<0.01). Functional assays demonstrated that METTL16 and YTHDC2 (an m6A reader) collaboratively enhanced MROH8 mRNA stability, thereby inhibiting CAPN2 expression and reducing tumor proliferation and metastasis (P<0.001). This study reveals a novel regulatory axis involving METTL16, MROH8, and TBP that modulates CAPN2 expression, contributing to the suppression of pancreatic cancer progression. The METTL16-MROH8-TBP-CAPN2 pathway offers potential therapeutic targets for pancreatic cancer treatment, highlighting the significance of m6A modifications in tumor regulation. Further clinical validation is needed to confirm these findings in human patients.

M6A reader YTHDF2 governs the onset of atrial fibrillation by modulating Cacna1c translation.

Atrial fibrillation (AF) is the most common arrhythmia, which is tightly associated with the abnormal expression and function of ion channels in the atrial cardiomyocytes. N6-methyladenosine (m6A), a widespread chemical modification in eukaryotic mRNA, is known to play a significant regulatory role in the pathogenesis of heart disease. However, the significance of m6A regulatory proteins in the onset of AF remains unclear. Here, we demonstrate that the m6A reader protein YTHDF2 regulates atrial electrical remodeling and AF onset by modulating the Cav1.2 expression. Firstly, YTHDF2 expression was selectively upregulated in rat atrial cardiomyocytes with AF. Secondly, YTHDF2 knockout reduced AF susceptibility in mice. Thirdly, the knockout of YTHDF2 increased Cav1.2 protein levels in an m6A-in-dependent manner, ultimately prolonging the atrial myocardial refractory period, a critical electrophysiological substrate for the onset of AF. Fourthly, the N-terminal domain of YTHDF2 was identified as critical for Cacna1c mRNA translation regulation. Overall, our findings unveil that YTHDF2 can alter Cav1.2 protein expression in an m6A-independent manner, thereby facilitating the onset of AF. Our study suggests that YTHDF2 may be a potential intervention target for AF.

DYSREGULATION OF M6A RNA METHYLATION, R-LOOPS AND THE DNA DAMAGE RESPONSE IN PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract AIMS Paediatric high-grade gliomas (pHGG) are virtually incurable malignant brain tumours. The regulation of R- loops is necessary for ensuring genomic stability. The formation of R-loops at DNA damage sites promotes DNA repair via m6A methylation of the RNA strand. We aimed to investigate whether m6A methylation, R-loop homeostasis and the DNA damage response is dysregulated in pHGG and whether inhibitors, such as ATM inhibitors, could be potential therapeutics for pHGG. METHOD To assess the global levels of m6A, R-loops, yH2AX, total ATM and phosphorylated ATM in paediatric HGG cell lines compared to control human astrocytes, immunocytochemistry was initially performed. Immunocyto- chemistry was also performed to assess localisation between m6A and R-loops, R-loops and yH2AX, R-loops and total/phosphorylated ATM, m6A and total/phosphorylated ATM, and total/phosphorylated ATM and yH2AX in pHGG cell lines and human astrocytes. We are also exploring two ATM inhibitors, AZD1390 and AZD0156, and are currently investigating their effects on m6A methylation, R-loops and the DNA damage response in pHGG. As a molecular mechanism involving the tumour suppressor ARID1A was recently discovered that is crucial for maintaining genomic stability, we are also exploring the role of this protein in pHGG. We are also investigating the potential involvement of m6A readers in the pathogenesis of pHGG. RESULTS Immunocytochemistry revealed that dysregulation of global m6A, R-loop, yH2AX and ATM occurs in pHGG cell lines as higher levels of expression was generally observed compared to human astrocytes. Moreover, m6A, R-loops, yH2AX and ATM appear to colocalise as high Pearson’s correlation coefficient values were obtained. Experiments exploring ATM inhibitors, ARID1A and m6A readers in pHGG are currently in progress, however the results will be presented. CONCLUSION It appears that dysregulation of m6A methylation, R-loop homeostasis and the DNA damage response occurs in pHGG and that molecular mechanisms involving m6A methylation and R-loops could be impaired.

The m6A reader IGF2BP1 contributes to the activation of hepatic stellate cells through facilitating TUBB4B mRNA stabilization.

The m6A reader insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) is involved in multiple pathophysiological processes through enhanced expression of the proteins encoded by their target mRNAs. However, the functional role of IGF2BP1-mediated m6A in liver fibrosis remains elusive. Here, we report that IGF2BP1 is highly expressed in activated hepatic stellate cells (HSCs), the major driver of fibrogenesis, and TUBB4B is identified as a potential target of IGF2BP1 by re-analysis of the RNA-seq, RIP-seq, and m6A-seq data. The relevant findings were subsequently demonstrated by a series of molecular and cellular evidences. The knockdown of IGF2BP1 or TUBB4B and pharmacological inhibition of TUBB4B by mebendazole treatments significantly suppress the proliferation, migration, and activation of HSCs. Mechanistically, IGF2BP1 upregulates TUBB4B expression through stabilizing TUBB4B in an m6A-dependent manner, and TUBB4B induces liver fibrosis by activating the FAK signaling pathway. Collectively, our results indicate that targeting IGF2BP1/TUBB4B/FAK axis in HSCs could be a promising therapeutic approach for liver fibrosis.

Data-augmented machine learning scoring functions for virtual screening of YTHDF1 m6A reader protein

Machine learning is rapidly advancing the drug discovery process, significantly enhancing speed and efficiency. Innovation in computer-aided drug design is primarily driven by structure- and ligand-based approaches. When the number of known inhibitors for a target is limited, data augmentation strategies are often preferred to enhance model performance. In this study, we developed predictive machine learning models for structure-based drug discovery leveraging multiple traditional machine learning algorithms trained with target and ligand dynamics-aware datasets.To illustrate our approach, we present a composite model that combines classification and regression to predict YTHDF1 inhibitors, utilizing PLEC features. YTHDF1, a key m6A reader protein involved in mRNA translation, is implicated in various cancers, making it a promising therapeutic target. Traditional structure-based virtual screening (SBVS) using generic scoring functions has struggled to identify potent YTHDF1 inhibitors due to the protein's unique binding characteristics. To overcome this, we developed YTHDF1-specific machine learning scoring functions (MLSFs) to enhance SBVS efficacy.We employed various data augmentation techniques to generate a comprehensive dataset, incorporating multiple conformations of ligands and the YTHDF1 protein. We have trained 64 YTHDF1-specific MLSFs using four machine learning algorithms and evaluated them on ten test sets, focusing on their predictive and ranking power. Our results demonstrate that the artificial neural network with protein-ligand extended connectivity fingerprints (ANN-PLEC) outperforms other MLSFs, consistently achieving high area under the precision-recall curve (PR-AUC) of 0.87. This method shows promise for targets with limited quantities of active molecules, providing a viable path forward for drug discovery research. The ANN-PLEC scoring function is made freely available on GitHub for other researchers to access and utilize https://github.com/JuniML/SBVS-YTHDF1/.

M6A RNA methyltransferase METTL16 induces Cr(VI) carcinogenesis and lung cancer development through glutamine biosynthesis and GLUL expression

Hexavalent chromium [Cr(VI)] exposure increases the risk of cancer occurrence. This study found that the levels of an atypical methyltransferase, METTL16 were greatly upregulated in the cells, and mouse tissues with Cr(VI) exposure, and played a critical role in cell proliferation and tumor growth induced by Cr(VI). Similarly, we found METTL16 was upregulated in various human cancer tissues. To understand mechanism of METTL16 in inducing carcinogenesis and cancer development, we identified that glutamate-ammonia ligase (GLUL) as the METTL16 functional target for regulating glutamine metabolism and tumorigenesis induced by Cr(VI) exposure. We demonstrated that METTL16 promoted GLUL expression in a m6A-dependent manner. Furthermore, METTL16 methylated the specific stem-loop structure of GLUL transcript, thereby increased the recognition and splicing of pre-GLUL RNA modified site by m6A reader YTHDC1, which ultimately accelerated the production of mature GLUL mRNA. Animal model of Cr(VI) exposure further confirmed that the expression levels of METTL16 and GLUL were both significantly induced in vivo, and there had a significant positive correlation between METTL16 and GLUL levels. Furthermore, we found that YTHDC1 was also important in inducing GLUL expression, and MYC was the upstream mediator of METTL16 to increase its transcriptional activation. Our study revealed new mechanism of metal carcinogenesis and cancer development.

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.

SMAD4 enhances the cytotoxic efficacy of human NK cells against colorectal cancer cells via the m6A reader YTHDF2.

Colorectal cancer (CRC) ranks as the third most prevalent malignant neoplasm in terms of both morbidity and mortality. Within the tumor microenvironment (TME) of CRC, the diminished presence and diminished cytotoxic function of natural killer (NK) cells serve as important factors driving the advancement of CRC; however, the precise regulatory mechanisms governing this phenomenon remain incompletely understood. Consequently, the identification of novel, potential anti-CRC targets associated with NK cells emerges as a pressing and paramount concern warranting immediate attention. We examined the regulatory mechanism of SMAD4-mediated NK cell cytotoxicity on CRC by utilizing various experimental techniques, such as qRT-PCR, flow cytometry. Our findings revealed that the expression of SMAD4 is decreased in NK cells within the TME of human CRC. Furthermore, we observed that enforced upregulation of SMAD4 resulted in enhanced cytotoxicity of NK cells towards CRC cells. Furthermore, our research has revealed that YTHDF2 functions as a downstream effector of SMAD4, playing a crucial role in the control of transcription and translation of m6A-modified RNA. Moreover, our investigation demonstrated that increased expression of SMAD4 promoted the activating receptor NKG2D by elevating levels of YTHDF2. Ultimately, the SMAD4-YTHDF2 regulatory axis significantly enhanced the cytotoxicity of NK cells against human CRC cells. Our study unveils a novel mechanism through which SMAD4 modulates the cytotoxicity of NK cells towards CRC cells, suggesting that SMAD4 may hold promise as a potential therapeutic target for NK cell therapy in CRC.

Reading m6A marks in mRNA: A potent mechanism of gene regulation in plants.

Modifications to RNA have recently been recognized as a pivotal regulator of gene expression in living organisms. More than 170 chemical modifications have been identified in RNAs, with N6-methyladenosine (m6A) being the most abundant modification in eukaryotic mRNAs. The addition and removal of m6A marks are catalyzed by methyltransferases (referred to as "writers") and demethylases (referred to as "erasers"), respectively. In addition, the m6A marks in mRNAs are recognized and interpreted by m6A-binding proteins (referred to as "readers"), which regulate the fate of mRNAs, including stability, splicing, transport, and translation. Therefore, exploring the mechanism underlying the m6A reader-mediated modulation of RNA metabolism is essential for a much deeper understanding of the epigenetic role of RNA modification in plants. Recent discoveries have improved our understanding of the functions of m6A readers in plant growth and development, stress response, and disease resistance. This review highlights the latest developments in m6A reader research, emphasizing the diverse RNA-binding domains crucial for m6A reader function and the biological and cellular roles of m6A readers in the plant response to developmental and environmental signals. Moreover, we propose and discuss the potential future research directions and challenges in identifying novel m6A readers and elucidating the cellular and mechanistic role of m6A readers in plants.

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.

Multi-omics analysis unveils the predictive value of IGF2BP3/SPHK1 signaling in cancer stem cells for prognosis and immunotherapeutic response in muscle-invasive bladder cancer

BackgroundMuscle invasive bladder cancer (MIBC) is a life-threatening malignant tumor characterized by high metastasis rates, poor prognosis, and limited treatment options. Immune checkpoint inhibitors (ICIs) targeting PD-1 and PD-L1 represent an emerging treatment for MIBC immunotherapy. However, the characteristics of patients likely to benefit from immunotherapy remain unclear.MethodsWe performed single-cell mass cytometry (CyTOF) analysis of 179,483 single cells to characterize potential immunotherapy-related cancer stem cells (CSCs)-like populations in the tumor microenvironment of 38 MIBC tissues. The upregulated expression of IGF2BP3 in CD274 + ALDH + CSC-like cells, which was associated with poor clinical prognosis, was analyzed by bulk RNA-sequencing data from an in-house cohort. The functional role of IGF2BP3 was determined through cell proliferation, colony formation, cell apoptosis and sphere formation assays. The regulation of SPHK1 expression by IGF2BP3 was investigated using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and bulk RNA-sequencing (bulk RNA-seq). We further utilized single-nucleus RNA sequencing (snRNA-seq) data from 67,988 cells of 25 MIBC tissues and single-cell RNA sequencing (scRNA-seq) data from MIBC patient-derived organoids to characterize the molecular features of bladder cancer cells co-expressing IGF2BP3 and SPHK1. Spatial transcriptomics (ST) and co-detection by indexing (CODEX) analysis were used to describe the spatial distribution and interactions of IGF2BP3 + SPHK1 + bladder cancer cells and immune cells.ResultsA subset of CD274 + ALDH + CSC-like cells was identified, associating with immunosuppression and low survival rates in MIBC patients. IGF2BP3, an m6A reader gene, was found to be upregulated in the CD274 + ALDH + CSC-like cell population and linked to poor clinical prognosis in MIBC. Knockout of IGF2BP3 dramatically promoted cell apoptosis and reduced cell proliferation in T24 cells. By integrating MeRIP-seq and bulk RNA-seq analyses, we identified SPHK1 served as a substrate for IGF2BP3 in an m6A-dependent manner. Further snRNA-seq, scRNA-seq, ST, and CODEX analysis revealed a closer topographical distance between IGF2BP3 + SPHK1 + bladder cancer cells and exhausted CD8 + T cells, providing one explanation for the superior response to immunotherapy in IGF2BP3 + SPHK1 + bladder cancer cells-enriched patients. Finally, an ICI-associated signature was developed based on the enriched genes of IGF2BP3 + SPHK1 + bladder cancer cells, and its potential ability to predict the response to immunotherapy was validated in two independent immunotherapy cohort.ConclusionsOur study highlighted the critical involvement of the IGF2BP3/SPHK1 signaling in maintaining the stemness of CSCs and promoting MIBC progression. Additionally, these findings suggested that the IGF2BP3/SPHK1 signaling might serve as a biomarker for prognosis and immunotherapy response in MIBC.

Advanced paternal age exacerbates neuroinflammation in offspring via m6A modification-mediated intergenerational inheritance

BackgroundThe trend of postponing childbearing age is prevalent worldwide. Advanced paternal age (APA) is associated with adverse pregnancy outcomes and offspring health. However, the underlying mechanism by which paternal aging affects the risk of offspring neuropsychiatric disorders is unclear. Our study aims to explore the behavioral phenotypes and the pathologic epigenetic alterations of APA offspring inherited from aging sperm.MethodsBehavioral tests, ELISA assay, immunofluorescence and western blotting were performed on offspring mice. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA immunoprecipitation sequencing (RIP-seq) were used to investigate the modified N6-methyladenosine (m6A) profiles of paternal sperm and offspring hippocampus. Intervention of gene expression by lentivirus and adeno-associated virus in both vivo and vitro examined the potential therapeutic targets of intergenerational inherited neuroinflammation.ResultsIn our study, APA offspring exhibit cognitive impairment and autism-like behavior. An increase in neuroinflammation in APA offspring is associated with microglial overactivation, which manifests as abnormal morphology and augmented engulfment. MeRIP-seq of F0 sperm and F1 hippocampus reveal that Nr4a2 is hypermethylated with decreased expression in APA offspring involving in synaptic plasticity and microglial function. In addition, Ythdc1, an m6A reader protein, is markedly elevated in aging sperm and remains elevated in adult hippocampus of APA group. Enhanced Ythdc1 recognizes and suppresses the hypermethylated Nr4a2, thereby contributing to the abnormal phenotype in offspring. The overexpression of Ythdc1 triggers microglial activation in vitro and its suppression in the hippocampus of APA progeny alleviates behavioral aberrations and attenuates neuroinflammation.ConclusionOur study provides additional evidence of the abnormal behavioral phenotypes of APA offspring and reveals potential epigenetic inheritance signatures and targeted genes for future research.

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.

FMRP protects breast cancer cells from ferroptosis by promoting SLC7A11 alternative splicing through interacting with hnRNPM

Ferroptosis is a unique modality of regulated cell death that is driven by iron-dependent phospholipid peroxidation. N6-methyladenosine (m6A) RNA modification participates in varieties of cellular processes. However, it remains elusive whether m6A reader Fragile X Mental Retardation Protein (FMRP) are involved in the modulation of ferroptosis in breast cancer (BC). In this study, we found that FMRP expression was elevated and associated with poor prognosis and pathological stage in BC patients. Overexpression of FMRP induced ferroptosis resistance and exerted oncogenic roles by positively regulating a critical ferroptosis defense gene SLC7A11. Mechanistically, upregulated FMRP catalyzes m6A modification of SLC7A11 mRNA and further influences the SLC7A11 translation through METTL3-dependent manner. Further studies revealed that FMRP interacts with splicing factor hnRNPM to recognize the splice site and then modulated the exon skip splicing event of SLC7A11 transcript. Interestingly, SLC7A11-S splicing variant can effectively promote FMRP overexpression-induced ferroptosis resistance in BC cells. Moreover, our clinical data suggested that FMRP/hnRNPM/SLC7A11 expression were significantly increased in the tumor tissues, and this signal axis was important evaluation factors closely related to the worse survival and prognosis of BC patients. Overall, our results uncovered a novel regulatory mechanism by which high FMRP expression protects BC cells from undergoing ferroptosis. Targeting the FMRP–SLC7A11 axis has a dual effect of inhibiting ferroptosis resistance and tumor growth, which could be a promising therapeutic target for treating BC.

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.

NAT10 Phase Separation Regulates YTHDF1 Splicing to Promote Gastric Cancer Progression.

Gastric cancer is an aggressive malignancy with poor patient outcomes. N-Acetyltransferase 10 (NAT10) is an acetyltransferase that has been reported to contribute to gastric cancer progression. In-depth investigation into the underlying molecular mechanisms driven by NAT10 could help identify therapeutic targets to improve gastric cancer treatment. In this study, we found that NAT10 forms condensates to regulate RNA dynamics and promote gastric cancer progression. In samples of patients with gastric cancer, elevated NAT10 expression correlated with an unfavorable prognosis, advanced disease stage, and metastasis. NAT10 enhanced the proliferation, migration, and invasion of gastric cancer cells; supported the growth of patient-derived organoids; and accelerated tumor development. A C-terminal intrinsically disordered region-mediated liquid-liquid phase separation of NAT10 and was essential for its tumor-promoting function in gastric cancer. Moreover, NAT10 interacted with the splicing factor serine/arginine-rich splicing factor 2 (SRSF2), leading to its acetylation and increased stability. Acetylated SRSF2 directly bound to the pre-mRNA of the m6A reader YTHDF1, resulting in enhanced YTHDF1 exon 4 skipping and upregulation of a short YTHDF1 transcript that could stimulate gastric cancer cell proliferation and migration. Furthermore, YTHDF1 exon 4 skipping correlated with NAT10 and SRSF2 expression and was associated with a more aggressive phenotype in samples of patients with gastric cancer. Together, this study uncovers the role of NAT10 liquid-liquid phase separation in modulating YTHDF1 splicing through SRSF2 acetylation to drive gastric cancer progression, providing insights into the oncogenic mechanism of NAT10. Significance: Phase separation of NAT10 enables acetylation of SRSF2 that enhances YTHDF1 exon 4 skipping, which is a tumor-promoting axis in gastric cancer that represents potential therapeutic targets and prognostic biomarkers.

A novel protein SPECC1-415aa encoded by N6-methyladenosine modified circSPECC1 regulates the sensitivity of glioblastoma to TMZ

BackgroundCircular RNAs (circRNAs) can influence a variety of biological functions and act as a significant role in the progression and recurrence of glioblastoma (GBM). However, few coding circRNAs have been discovered in cancer, and their role in GBM is still unknown. The aim of this study was to identify coding circRNAs and explore their potential roles in the progression and recurrence of GBM.MethodsCircSPECC1 was screened via circRNAs microarray of primary and recurrent GBM samples. To ascertain the characteristics and coding ability of circSPECC1, we conducted a number of experiments. Afterward, through in vivo and in vitro experiments, we investigated the biological functions of circSPECC1 and its encoded novel protein (SPECC1-415aa) in GBM, as well as their effects on TMZ sensitivity.ResultsBy analyzing primary and recurrent GBM samples via circRNAs microarray, circSPECC1 was found to be a downregulated circRNA with coding potential in recurrent GBM compared with primary GBM. CircSPECC1 suppressed the proliferation, migration, invasion, and colony formation abilities of GBM cells by encoding a new protein known as SPECC1-415aa. CircSPECC1 restored TMZ sensitivity in TMZ-resistant GBM cells by encoding the new protein SPECC1-415aa. The m6A reader protein IGF2BP1 can bind to circSPECC1 to promote its expression and stability. Mechanistically, SPECC1-415aa can bind to ANXA2 and competitively inhibit the binding of ANXA2 to EGFR, thus resulting in the inhibition of the phosphorylation of EGFR (Tyr845) and its downstream pathway protein AKT (Ser473). In vivo experiments showed that the overexpression of circSPECC1 could combine with TMZ to treat TMZ-resistant GBM, thereby restoring the sensitivity of TMZ-resistant GBM to TMZ.ConclusionsCircSPECC1 was downregulated in recurrent GBM compared with primary GBM. The m6A reader protein IGF2BP1 could promote the expression and stability of circSPECC1. The sequence of SPECC1-415aa, which is encoded by circSPECC1, can inhibit the binding of ANXA2 to EGFR by competitively binding to ANXA2 and inhibiting the phosphorylation of EGFR and AKT, thereby restoring the sensitivity of TMZ-resistant GBM cells to TMZ.

YTHDF1 loss in dendritic cells potentiates radiation-induced antitumor immunity via STING-dependent type I IFN production.

RNA N6-methyladenosine (m6A) reader YTHDF1 is implicated in cancer etiology and progression. We discovered that radiotherapy (RT) increased YTHDF1 expression in dendritic cells (DCs) of PBMCs from cancer patients, but not in other immune cells tested. Elevated YTHDF1 expression of DCs was associated with poor outcomes in patients receiving RT. We found that loss of Ythdf1 in DCs enhanced the antitumor effects of ionizing radiation (IR) via increasing the cross-priming capacity of DCs across multiple murine cancer models. Mechanistically, IR upregulated YTHDF1 expression in DCs through STING-IFN-I signaling. YTHDF1 in turn triggered STING degradation by increasing lysosomal cathepsins, thereby reducing IFN-I production. We created a YTHDF1 deletion/inhibition prototype DC vaccine, significantly improving the therapeutic effect of RT and radio-immunotherapy in a murine melanoma model. Our findings reveal a new layer of regulation between YTHDF1/m6A and STING in response to IR, which opens new paths for the development of YTHDF1-targeting therapies.

Knockdown of IGF2BP3 Down-Regulates PDCD4 Levels to Attenuate Hypoxic-Ischemic Brain Damage

Background: Hypoxic-ischemic brain damage (HIBD) is a prevalent brain injury with high mortality and morbidity. It results from hypoxia and ischemia of the brain due to various perinatal factors. A previous study showed that knockdown of programmed cell death factor 4 (PDCD4) could reduce infarction injury resulting from ischemia/reperfusion injury. However, exact mechanism by which PDCD4 acts in HIBD is not yet understood. Our aim in present investigation was to investigate the function and mechanism of PDCD4 in alleviating HIBD. Methods: An HIBD model was developed using neonatal rats. After 48 h of modeling, short-term neurological function was evaluated and the brain tissue removed for assessment of cerebral infarct volume and brain water content (BWC). A cell model of oxygen glucose deprivation/reoxygenation (OGD/R) was also constructed. Overexpression or knockdown of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) or PDCD4 was performed in pretreated cells. Results: The geotaxis reflex time, cerebral infarct volume, and BWC all increased after HIBD in this neonatal rat model. Additionally, the levels of PDCD4 and of the N6-Methyladenosine (m6A) reader protein IGF2BP3 were increased in HIBD rats and OGD/R-stimulated pheochromocytoma (PC12) cells relative to controls. Moreover, OGD/R-stimulated pheochromocytoma PC12 cells showed decreased cell viability, increased apoptosis, and elevated Interleukin 6 (IL-6), Interleukin 1 β (IL-1β), and tumor necrosis factor-α (TNF-α) contents. These features were reversed after knocking down IGF2BP3. The interaction between IGF2BP3 protein and PDCD4 mRNA was confirmed by RNA immunoprecipitation and RNA pull-down assays. Furthermore, knockdown of IGF2BP3 in OGD/R-stimulated PC12 cells reduced cell damage via down-regulation of PDCD4. Finally, the IGF2BP3/PDCD4 axis alleviated OGD/R-induced cell injury in primary cortical neurons (PCNs). Conclusions: PDCD4 and m6A reader protein IGF2BP3 were up-regulated in an HIBD neonatal rat model. Knockdown of IGF2BP3 in OGD/R-stimulated PC12 cells or PCNs alleviated cell damage through reducing PDCD4.

M6A-modified cenRNA stabilizes CENPA to ensure centromere integrity in cancer cells

m6A modification is best known for its critical role in controlling multiple post-transcriptional processes of the mRNAs. Here, we discovered elevated levels of m6A modification on centromeric RNA (cenRNA) in cancerous cells compared with non-cancerous cells. We then identified CENPA, an H3 variant, as an m6A reader of cenRNA. CENPA is localized at centromeres and is essential in preserving centromere integrity and function during mitosis. The m6A-modified cenRNA stabilizes centromeric localization of CENPA in cancer cells during the S phase of the cell cycle. Mutations of CENPA at the Leu61 and the Arg63 or removal of cenRNA m6A modification lead to loss of centromere-bound CENPA during S phase. This in turn results in compromised centromere integrity and abnormal chromosome separation and hinders cancer cell proliferation and tumor growth. Our findings unveil an m6A reading mechanism by CENPA that epigenetically governs centromere integrity in cancer cells, providing potential targets for cancer therapy.