YTH N6-methyladenosine RNA Binding Protein Research Articles

Unraveling Key m6A Modification Regulators Signatures in Postmenopausal Osteoporosis through Bioinformatics and Experimental Verification.

Bone marrow mesenchymal stem cells (BMSCs) show significant potential for osteogenic differentiation. However, the underlying mechanisms of osteogenic capability in osteoporosis-derived BMSCs (OP-BMSCs) remain unclear. This study aims to explore the impact of YTHDF3 (YTH N6-methyladenosine RNA binding protein 3) on the osteogenic traits of OP-BMSCs and identify potential therapeutic targets to boost their bone formation ability. We examined microarray datasets (GSE35956 and GSE35958) from the Gene Expression Omnibus (GEO) to identify potential m6A regulators in osteoporosis (OP). Employing differential, protein interaction, and machine learning analyses, we pinpointed critical hub genes linked to OP. We further probed the relationship between these genes and OP using single-cell analysis, immune infiltration assessment, and Mendelian randomization. Our in vivo and in vitro experiments validated the expression and functionality of the key hub gene. Differential analysis revealed seven key hub genes related to OP, with YTHDF3 as a central player, supported by protein interaction analysis and machine learning methodologies. Subsequent single-cell, immune infiltration, and Mendelian randomization studies consistently validated YTHDF3's significant link to osteoporosis. YTHDF3 levels are significantly reduced in femoral head tissue from postmenopausal osteoporosis (PMOP) patients and femoral bone tissue from PMOP mice. Additionally, silencing YTHDF3 in OP-BMSCs substantially impedes their proliferation and differentiation. YTHDF3 may be implicated in the pathogenesis of OP by regulating the proliferation and osteogenic differentiation of OP-BMSCs.

METTL3-mediated m6A modification increases Hspa1a stability to inhibit osteoblast aging

Senile osteoporosis is mainly caused by osteoblasts attenuation, which results in reduced bone mass and disrupted bone remodeling. Numerous studies have focused on the regulatory role of m6A modification in osteoporosis; however, most of the studies have investigated the differentiation of bone marrow mesenchymal stem cells (BMSCs), while the direct regulatory mechanism of m6A on osteoblasts remains unknown. This study revealed that the progression of senile osteoporosis is closely related to the downregulation of m6A modification and methyltransferase-like 3 (METTL3). Overexpression of METTL3 inhibits osteoblast aging. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that METTL3 upregulates the stability of Hspa1a mRNA, thereby inhibiting osteoblast aging. Moreover, the results demonstrated that METTL3 enhances the stability of Hspa1a mRNA via m6A modification to regulate osteoblast aging. Notably, YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) participates in stabilizing Hspa1a mRNA in the METTL3-mediated m6A modification process, rather than the well-known degradation function. Mechanistically, METTL3 increases the stability of Hspa1a mRNA in a YTHDF2-dependent manner to inhibit osteoblast aging. Our results confirmed the significant role of METTL3 in osteoblast aging and suggested that METTL3 could be a potential therapeutic target for senile osteoporosis.

Foot-and-mouth disease virus VP1 degrades YTHDF2 through autophagy to regulate IRF3 activity for viral replication

ABSTRACT Many viruses, including foot-and-mouth disease virus (FMDV), can promote the degradation of host proteins through macroautophagy/autophagy, thereby promoting viral replication. However, the regulatory mechanism between autophagy and innate immune responses is not fully understood during FMDV infection. Here, we found that the host GTPBP4/NOG1 (GTP binding protein 4) is a negative regulator of innate immune responses. GTPBP4 deficiency promotes the antiviral innate immune response, resulting in the ability of GTPBP4 to promote FMDV replication. Meanwhile, GTPBP4-deficient mice are more resistant to FMDV infection. To antagonize the host’s antiviral immunity, FMDV structural protein VP1 promotes the expression of GTPBP4, and the 209th site of VP1 is responsible for this effect. Mechanically, FMDV VP1 promotes autophagy during virus infection and interacts with and degrades YTHDF2 (YTH N6-methyladenosine RNA binding protein F2) in an AKT-MTOR-dependent autophagy pathway, resulting in an increase in GTPBP4 mRNA and protein levels. Increased GTPBP4 inhibits IRF3 binding to the Ifnb/Ifn-β promoter, suppressing FMDV-induced type I interferon production. In conclusion, our study revealed an underlying mechanism of how VP1 negatively regulates innate immunity through the autophagy pathway, which would contribute to understanding the negative regulation of host innate immune responses and the function of GTPBP4 and YTHDF2 during FMDV infection. Abbreviation: 3-MA:3-methyladenine; ACTB: actin beta; ATG: autophagy related; ChIP:chromatin immunoprecipitation; CQ: chloroquine; DAPI:4’,6-diamidino-2-phenylindole; dpi: days post-infection; EV71:enterovirus 71; FMDV: foot-and-mouth disease virus; GTPBP4/NOG1: GTPbinding protein 4; HIF1A: hypoxia inducible factor 1 subunit alpha;hpt:hours post-transfection; IFNB/IFN-β:interferon beta; IRF3: interferon regulatory factor 3; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAVS: mitochondriaantiviral signaling protein; MOI: multiplicity of infection; MTOR:mechanistic target of rapamycin kinase; m6A: N(6)-methyladenosine;qPCR:quantitativePCR; SIRT3:sirtuin 3; SQSTM1/p62: sequestosome 1; STING1: stimulator ofinterferon response cGAMP interactor 1; siRNA: small interfering RNA;TBK1: TANK binding kinase 1; TCID50:50% tissue culture infectious doses; ULK1: unc-51 like autophagyactivating kinase 1; UTR: untranslated region; WT: wild type; YTHDF2:YTH N6-methyladenosine RNA binding protein F2

KCTD15 acts as an anti-tumor factor in colorectal cancer cells downstream of the demethylase FTO and the m6A reader YTHDF2

Potassium Channel Tetramerization Domain Containing 15 (KCTD15) participates in the carcinogenesis of several solid malignancies; however, its role in colorectal cancer (CRC) remains unclear. Here we find that KCTD15 exhibits lower expression in CRC tissues as compared to para-carcinoma tissues. Tetracycline (tet)-induced overexpression and knockdown of KCTD15 confirms KCTD15 as an anti-proliferative and pro-apoptotic factor in CRC both in vitro and in xenografted tumors. N6-methyladenosine (m6A) is known to affect the expression, stabilization, and degradation of RNAs with this modification. We demonstrate that upregulation of fat mass and obesity-associated protein (FTO), a classical m6A eraser, prevents KCTD15 mRNA degradation in CRC cells. Less KCTD15 RNA is recognized by m6A ‘reader’ YTH N6-Methyladenosine RNA Binding Protein F2 (YTHDF2) in FTO-overexpressed cells. Moreover, KCTD15 overexpression decreases protein expression of histone deacetylase 1 (HDAC1) but increases acetylation of critical tumor suppressor p53 at Lys373 and Lys382. Degradation of p53 is delayed in CRC cells post-KCTD15 overexpression. We further show that the regulatory effects of KCTD15 on p53 are HDAC1-dependent. Collectively, we conclude that KCTD15 functions as an anti-growth factor in CRC cells, and its expression is orchestrated by the FTO-YTHDF2 axis. Enhanced p53 protein stabilization may contribute to KCTD15’s actions in CRC cells.

YTHDF2-regulated matrilin-3 mitigates post-reperfusion hemorrhagic transformation in ischemic stroke via the PI3K/AKT pathway.

Hemorrhagic transformation can complicate ischemic strokes after recanalization treatment within a time window that requires early intervention. To determine potential therapeutic effects of matrilin-3, rat cerebral ischemia-reperfusion was produced using transient middle cerebral artery occlusion (tMCAO); intracranial hemorrhage and infarct volumes were assayed through hemoglobin determination and 2,3,5-triphenyltetrazoliumchloride (TTC) staining, respectively. Oxygen-glucose deprivation (OGD) modeling of ischemia was performed on C8-D1A cells. Interactions between matrilin-3 and YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) were determined using RNA immunoprecipitation assay and actinomycin D treatment. Reperfusion after tMCAO modeling increased hemorrhage, hemoglobin content, and infarct volumes; these were alleviated by matrilin treatment. Matrilin-3 was expressed at low levels and YTHDF2 was expressed at high levels in ischemic brains. In OGD-induced cells, matrilin-3 was negatively regulated by YTHDF2. Matrilin-3 overexpression downregulated p-PI3K/PI3K, p-AKT/AKT, ZO-1, VE-cadherin and occludin, and upregulated p-JNK/JNK in ischemic rat brains; these effects were reversed by LY294002 (a PI3K inhibitor). YTHDF2 knockdown inactivated the PI3K/AKT pathway, inhibited inflammation and decreased blood-brain barrier-related protein levels in cells; these effects were reversed by matrilin-3 deficiency. These results indicate that YTHDF2-regulated matrilin-3 protected ischemic rats against post-reperfusion hemorrhagic transformation via the PI3K/AKT pathway and that matrilin may have therapeutic potential in ischemic stroke.

YTHDF1's Regulatory Involvement in Breast Cancer Prognosis, Immunity, and the ceRNA Network.

YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), an m6A reader, has a role in the development and progression of breast cancer as well as the immunological microenvironment. The networks of competing endogenous RNA in cancer have received much attention in research. In tumor gene therapy, the regulatory networks of m6A and competing endogenous RNA are increasingly emerging as a new route. We evaluated the relationship between the YTHDF1 expression, overall survival, and clinicopathology of breast cancer using TCGA, PrognoScan, and other datasets. We used Western blot to demonstrate that YTHDF1 is substantially expressed in breast cancer tissues. Furthermore, we explored YTHDF1's functions in the tumor mutational burden, microsatellite instability, and tumor microenvironment. Our findings indicate that YTHDF1 is a critical component of the m6A regulatory proteins in breast cancer and may have a particular function in the immunological microenvironment. Crucially, we investigated the relationship between YTHDF1 and the associated competitive endogenous RNA regulatory networks, innovatively creating three such networks (Dehydrogenase/Reductase 4-Antisense RNA 1-miR-378g-YTHDF1, HLA Complex Group 9-miR-378g-YTHDF1, Taurine Up-regulated 1-miR-378g-YTHDF1). Furthermore, we showed that miR-378g could inhibit the expression of YTHDF1, and that miR-378g/YTHDF1 could impact MDA-MB-231 proliferation. We speculate that YTHDF1 may serve as a biomarker for poor prognosis and differential diagnosis, impact the growth of breast cancer cells via the ceRNA network axis, and be a target for immunotherapy against breast cancer.

LINC00707 impairs the Natural Killer cell antitumour activity in hepatocellular carcinoma through decreasing YTHDF2 stability.

Hepatocellular carcinoma (HCC) is the fifth most frequently diagnosed cancer and ranks third in cancer-related fatalities. The recognized involvement of long noncoding RNAs (lncRNAs) in several cancer types, including HCC, inspired this study to explore a novel lncRNA's functional importance in the progression of HCC. To achieve this, lncRNA microarray analysis was conducted on three distinct sets of HCC tissues, revealing LINC00707 as the most significantly upregulated lncRNA. Further research into its biological functions has revealed that LINC00707 acts as an oncogene, driving HCC progression by enhancing the proliferation, migration and invasion of HCC cells. Mechanistic insights were provided, demonstrating that LINC00707 interacts with YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2), thus facilitating the ubiquitination-dependent degradation of the YTHDF2 protein. Furthermore, LINC00707 was found to influence the cytotoxicity of NK-92MI cells against HCC cells through its interactions with YTHDF2. These findings significantly contribute to a deeper understanding of the role played by LINC00707 in the progression of HCC.

FOXM1 Participates in Scleral Remodeling in Myopia by Upregulating APOA1 Expression Through METTL3/YTHDF2.

Apolipoprotein A1 (APOA1) is a potential crucial protein and treatment goal for pathological myopia in humans. This study set out to discover the function of APOA1 in scleral remodeling in myopia and its underlying mechanisms. A myopic cell model was induced using hypoxia. Following loss- and gain-of function experiments, the expression of the myofibroblast transdifferentiation-related and collagen production-related factors Forkhead box M1 (FOXM1), APOA1, and methyltransferase-like 3 (METTL3) in the myopic cell model was examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The proliferation and apoptosis were determined by Cell Counting Kit-8 assay and flow cytometry, respectively. Chromatin immunoprecipitation (ChIP) was employed to examine FOXM1 enrichment in the METTL3 promoter, methylated RNA immunoprecipitation (Me-RIP) to examine the N6-methyladenosine (m6A) modification level of APOA1, and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to examine the binding between METTL3 and APOA1. Hypoxia-induced human scleral fibroblasts (HSFs) had high APOA1 and FOXM1 expression and low METTL3 expression. FOXM1 knockdown elevated METTL3 expression and downregulated APOA1 expression. FOXM1 was enriched in METTL3 promoter. APOA1 or FOXM1 knockdown or METTL3 overexpression reversed the hypoxia-induced elevation in vinculin, paxillin, and α-smooth muscle actin (α-SMA) levels and apoptosis and the reduction in collagen, type I, alpha 1 (COL1A1) level and cell proliferation in HSFs. METTL3 or YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) knockdown or APOA1 overexpression reversed the impacts of FOXM1 knockdown on vinculin, paxillin, α-SMA, and COL1A1 expression and cell proliferation and apoptosis. FOXM1 elevated the m6A methylation level of APOA1 by repressing METTL3 transcription and enhanced APOA1 mRNA stability and transcription by reducing the YTHDF2-recognized m6A methylated transcripts.

METTL3-mediated m6A modification of SOX4 regulates osteoblast proliferation and differentiation via YTHDF3 recognition

N6-methyladenosine (m6A), the most prevalent internal modification in mRNA, is related to the pathogenesis of osteoporosis (OP). Although methyltransferase Like-3 (METTL3), an m6A transferase, has been shown to mitigate OP progression, the mechanisms of METTL3-mediated m6A modification in osteoblast function remain unclear. Here, fluid shear stress (FSS) induced osteoblast proliferation and differentiation, resulting in elevated levels of METTL3 expression and m6A modification. Through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and Transcriptomic RNA Sequencing (RNA-seq), SRY (Sex Determining Region Y)-box 4 (SOX4) was screened as a target of METTL3, whose m6A-modified coding sequence (CDS) regions exhibited binding affinity towards METTL3. Further functional experiments demonstrated that knockdown of METTL3 and SOX4 hampered osteogenesis, and METTL3 knockdown compromised SOX4 mRNA stability. Via RNA immunoprecipitation (RIP) assays, we further confirmed the direct interaction between METTL3 and SOX4. YTH N6-Methyladenosine RNA Binding Protein 3 (YTHDF3) was identified as the m6A reader responsible for modulating SOX4 mRNA and protein levels by affecting its degradation. Furthermore, in vivo experiments demonstrated that bone loss in an ovariectomized (OVX) mouse model was reversed through the overexpression of SOX4 mediated by adeno-associated virus serotype 2 (AAV2). In conclusion, our research demonstrates that METTL3-mediated m6A modification of SOX4 plays a crucial role in regulating osteoblast proliferation and differentiation through its recognition by YTHDF3. Our research confirms METTL3-m6A-SOX4-YTHDF3 as an essential axis and potential mechanism in OP.

Betaine alleviates cognitive impairment induced by homocysteine through attenuating NLRP3-mediated microglial pyroptosis in an m6A-YTHDF2-dependent manner

Dementia, with homocysteine (Hcy) as an important risk factor, is a severe public health problem in the aging society. Betaine serves as a methyl donor and plays an important role in reducing Hcy. However, the effects and mechanisms of betaine on Hcy-induced cognitive impairment remain unclear. Firstly, SD rats were injected with Hcy (400 μg/kg) through vena caudalis, and betaine (2.5 % w/v) was supplemented via drinking water for 14 days. Betaine supplementation could attenuate Hcy-induced cognitive impairment in the Y maze and novel object recognition tests by repairing brain injury. Meanwhile, microglial activation was observed to be inhibited by betaine supplementation using immunofluorescence and sholl analysis. Secondly, HMC3 cells were treated with betaine, which was found to decrease the ROS level, ameliorate cell membrane rupture, reduce the release of LDH, IL-18 and IL-1β, and attenuate the damage of microglia to neurons. Mechanistically, betaine alleviates cognitive impairment by inhibiting microglial pyroptosis via reducing the expressions of NLRP3, ASC, pro-caspase-1, cleaved-caspase-1, GSDMD, GSDMD-N, IL-18 and IL-1β. Betaine treatment can increase SAM/SAH ratio, confirming its enhancement on methylation capacity. Furthermore, betaine treatment was found to enhance N6-methyladenosine (m6A) modification of NLRP3 mRNA, and reduced the NLRP3 mRNA stability through increasing the expression of the m6A reader YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). Finally, silencing YTHDF2 could reverse the inhibitory effect of betaine on pyroptosis. Our data demonstrated that betaine attenuated Hcy-induced cognitive impairment by suppressing microglia pyroptosis via inhibiting the NLRP3/caspase-1/GSDMD pathway in an m6A-YTHDF2-dependent manner.

Putrescine promotes MMP9-induced angiogenesis in skeletal muscle through hydrogen peroxide/METTL3 pathway

Blood vessels play a crucial role in the development of skeletal muscle, ensuring the supply of nutrients and oxygen. Putrescine, an essential polyamine for eukaryotic cells, has an unclear impact on skeletal muscle angiogenesis. In this study, we observed lower vessel density and reduced putrescine level in the muscle of low-birth-weight piglet models, and identified a positive correlation between putrescine content and muscle vessel density. Furthermore, putrescine was found to promote angiogenesis in skeletal muscle both in vitro and in vivo by targeting matrix metalloproteinase 9 (MMP9). On a mechanistic level, putrescine augmented the expression of methyltransferase like 3 (METTL3) by attenuating hydrogen peroxide production, thereby increasing the level of N6-methyladenosine (m6A)-modified MMP9 mRNA. This m6A-modified MMP9 mRNA was subsequently recognized and bound by the YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), enhancing the stability of MMP9 mRNA and its protein expression, consequently accelerating angiogenesis in skeletal muscle. In summary, our findings suggest that putrescine enhances MMP9-mediated angiogenesis in skeletal muscle via the hydrogen peroxide/METTL3 pathway.

Circ-0000953 deficiency exacerbates podocyte injury and autophagy disorder by targeting Mir665-3p-Atg4b in diabetic nephropathy

ABSTRACT Circular RNAs (circRNAs) are special non-coding RNA (ncRNA) molecules that play a significant role in many diseases. However, the biogenesis and regulation of circRNAs in diabetic nephropathy (DN) are largely unknown. Here, we investigated the expression profile of circRNAs in kidney of DN mice through circular RNA sequencing (circRNA-seq). The renal biopsy samples of patients with DN had low circ -0,000,953 expression, which was significantly associated with renal function. Furthermore, loss-of-function and gain-of-function experiments were carried out to prove the role of circ -0,000,953 in DN. Podocyte conditional knockin (cKI) or systemic overexpression of circ -0,000,953 alleviated albuminuria and restored macroautophagy/autophagy in kidney of diabetic mice. However, circ -0,000,953 knockdown exacerbated albuminuria and podocyte injury. Mechanistically, we found circ -0,000,953 directly binds to Mir665-3p-Atg4b to perform its function. Silencing of Mir665-3p or overexpression of Atg4b recovered podocyte autophagy both in vitro and in vivo. To examine the cause of circ -0,000,953 downregulation in DN, bioinformatics prediction found that circ -0,000,953 sequence has a high possibility of containing an m6A methylation site. Additionally, METTL3 was proved to regulate the expression and methylation level of circ -0,000,953 through YTHDF2 (YTH N6-methyladenosine RNA binding protein 2). In conclusion, this study revealed that circ -0,000,953 regulates podocyte autophagy by targeting Mir665-3p-Atg4b in DN. Therefore, circ -0,000,953 is a potential biomarker for prevention and cure of DN. Abbreviation: CCL2/MCP-1: C-C motif chemokine ligand 2; ceRNA: competing endogenous RNA; circRNA: circular RNA; cKI: conditional knockin; cKO: conditional knockout; CRE: creatinine; DM: diabetes mellitus; DN: diabetic nephropathy; ESRD: end-stage renal disease; HG: high glucose; IF: immunofluorescence; MAP1LC3/LC3B: microtubule-associated protein 1 light chain 3 beta; MPC5: mouse podocyte clone 5; MTECs: mouse tubular epithelial cells; MTOR: mechanistic target of rapamycin kinase; NC: normal control; ncRNA: non-coding RNA; NPHS1: nephrosis 1, nephrin; NPHS2: nephrosis 2, podocin; PAS: periodic acid-Schiff; RELA/p65: v-rel reticuloendotheliosis viral oncogene homolog A (avian); SDs: slit diaphragm proteins; Seq: sequencing; STZ: streptozotocin; SV40: SV40-MES13-cells, mouse mesangial cell line; T1D: type 1 diabetes mellitus; T2D: type 2 diabetes mellitus; TEM: transmission electron microscopy; TNF/TNF-α: tumor necrosis factor; VECs: vascular endothelial cells; WT1: WT1 transcription factor; YTHDF2: YTH N6-methyladenosine RNA binding protein 2.

METTL3 suppresses invasion of lung cancer via SH3BP5 m6A modification

The metastasis of lung cancer poses a major clinical challenge, and m6A modification has been implicated in regulating the invasive capabilities of tumor cells. However, the mechanisms underlying m6A modification in lung cancer metastasis are not well understood. This study aims to explore the biological functions and molecular mechanisms of methyltransferase-like 3 (METTL3) in lung cancer. In this study, METTL3 were found to be downregulated in lung cancer tissues. Functionally, METTL3 inhibited the migration and invasion abilities of lung cancer cells in vitro. Furthermore, SH3 domain binding protein 5 (SH3BP5) was identified as a downstream target of METTL3. Overexpression of SH3BP5 suppressed the invasive capacity of lung cancer cells, and this regulation was m6A-dependent. Finally, we discovered that YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) mediated stability is responsible for maintaining the m6A modification of SH3BP5 mRNA. Overall, our study provides insights into the critical role of METTL3-mediated m6A modification and m6A-dependent regulatory mechanisms in the progression of human lung cancer. We demonstrated that METTL3 regulates the mRNA stability of SH3BP5 in a YTHDF1-dependent manner, thereby impacting the invasive capacity of lung cancer cells.

Epitranscriptional regulation of TGF-β pseudoreceptor BAMBI by m6A/YTHDF2 drives extrinsic radioresistance.

Activation of TGF-β signaling serves as an extrinsic resistance mechanism that limits the potential for radiotherapy. Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) antagonizes TGF-β signaling and is implicated in cancer progression. However, the molecular mechanisms of BAMBI regulation in immune cells and its impact on antitumor immunity after radiation have not been established. Here, we show that ionizing radiation (IR) specifically reduces BAMBI expression in immunosuppressive myeloid-derived suppressor cells (MDSCs) in both murine models and humans. Mechanistically, YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2) directly binds and degrades Bambi transcripts in an N6-methyladenosine-dependent (m6A-dependent) manner, and this relies on NF-κB signaling. BAMBI suppresses the tumor-infiltrating capacity and suppression function of MDSCs via inhibiting TGF-β signaling. Adeno-associated viral delivery of Bambi (AAV-Bambi) to the tumor microenvironment boosts the antitumor effects of radiotherapy and radioimmunotherapy combinations. Intriguingly, combination of AAV-Bambi and IR not only improves local tumor control, but also suppresses distant metastasis, further supporting its clinical translation potential. Our findings uncover a surprising role of BAMBI in myeloid cells, unveiling a potential therapeutic strategy for overcoming extrinsic radioresistance.

#326 : Multi-Omics Analysis Reveals Translational Landscapes and Regulations in Mouse and Human Oocyte Aging

Background and Aims: Abnormal resumption of meiosis and decreased oocyte quality are hallmarks of maternal aging and ultimately lead to infertility. Transcriptional silencing makes translational control an urgent task during meiosis resumption in maternal aging. However, insights into aging-related translational characteristics and underlying mechanisms are limited. Method: Here, using multi-omics analysis of oocytes, including single-cell proteomics, ultrasensitive translatomics and transcriptomics, we profiled the multi-omics landscapes of mouse and human oocyte aging. The role and mechanisms underlying oocyte aging were explored and verified using immunofluorescence staining, electroporation, RNA immunoprecipitation (RIP) sequencing, quantitative PCR (qPCR), methylated RIP, and dual-luciferase reporter assay etc. Results: We found that translatomics during aging was related to changes in the proteome and revealed decreased translational efficiency with aging phenotypes in mouse oocytes. Notably, the translational efficiency decrease was associated with the N6-methyladenosine (m6A) modification of transcripts. We clarified that the expression of the m6A reader YTH N6-methyladenosine RNA-binding protein 3 (YTHDF3) was significantly decreased in aged oocytes and inhibited oocyte meiotic maturation. YTHDF3 intervention perturbed the translatome of oocytes and suppressed the translational efficiency. We further identified Hells as an aging-associated maternal factor whose translation was suppressed during oocyte aging in a YTHDF3-dependent manner. Moreover, we profiled the translational landscape in human oocyte aging, and observed the same translational changes of DNA methylation regulatory enzymes and different regulation of histone modifications between human and mice oocytes aging. Translation efficiency was not changed in human oocytes aging. Due to translational silence of YTHDF3 in human oocytes, translation activity was not associated with m6A modification, but was related to alternative splicing factors, such as SRSF6. Conclusion: Together, our findings profiled the specific translational landscapes during oocyte aging in mice and human and uncovered non-conservative regulators on translation control in meiosis resumption and maternal aging.

EGFR promotes ALKBH5 nuclear retention to attenuate N6-methyladenosine and protect against ferroptosis in glioblastoma

Growth factor receptors rank among the most important oncogenic pathways, but pharmacologic inhibitors often demonstrate limited benefit as monotherapy. Here, we show that epidermal growth factor receptor (EGFR) signaling repressed N6-methyladenosine (m6A) levels in glioblastoma stem cells (GSCs), whereas genetic or pharmacologic EGFR targeting elevated m6A levels. Activated EGFR induced non-receptor tyrosine kinase SRC to phosphorylate the m6A demethylase, AlkB homolog 5 (ALKBH5), thereby inhibiting chromosomal maintenance 1 (CRM1)-mediated nuclear export of ALKBH5 to permit sustained mRNA m6A demethylation in the nucleus. ALKBH5 critically regulated ferroptosis through m6A modulation and YTH N6-methyladenosine RNA binding protein (YTHDF2)-mediated decay of the glutamate-cysteine ligase modifier subunit (GCLM). Pharmacologic targeting of ALKBH5 augmented the anti-tumor efficacy of EGFR and GCLM inhibitors, supporting an EGFR-ALKBH5-GCLM oncogenic axis. Collectively, EGFR reprograms the epitranscriptomic landscape through nuclear retention of the ALKBH5 demethylase to protect against ferroptosis, offering therapeutic paradigms for the treatment of lethal cancers.

LncRNA FTO-IT1 promotes glycolysis and progression of hepatocellular carcinoma through modulating FTO-mediated N6-methyladenosine modification on GLUT1 and PKM2

BackgroundLong non-coding RNAs (LncRNAs) have been extensively studied to play essential roles in tumor progression. However, more in-depth studies are waiting to be solved on how lncRNAs regulate the progression of hepatocellular carcinoma (HCC).MethodsDifferent expression levels of lncRNAs in HCC cells were compared by analysis of Gene Expression Omnibus and The Cancer Genome Atlas databases. The effects of lncRNA FTO Intronic Transcript 1 (FTO-IT1) on HCC cells were assessed by gain- and loss-of-function experiments. Colony formation assay, Edu assay, glucose uptake and lactic acid production assay were performed to evaluate the regulation of proliferation and glycolysis of HCC cells by FTO-IT1. The binding between protein interleukin enhancer binding factor 2/3 (ILF2/ILF3) and FTO-IT1 was determined by RNA pull-down, mass spectroscopy and RNA immunoprecipitation experiments. RNA stability assay, quantitative reverse transcription PCR and Western blot were employed to determine the regulatory mechanisms of FTO-IT1 on fat mass and obesity-associated (FTO). Methylated RNA immunoprecipitation assay was used to assessed the regulation of key enzymes of glycolysis by FTO. The role of FTO-IT1/FTO in vivo was confirmed via xenograft tumor model.ResultsLncRNA FTO-IT1, an intronic region transcript of FTO gene, was highly expressed in HCC and associated with poor prognosis of patients with HCC. FTO-IT1 was related to proliferation and glycolysis of HCC cells, and contributed to the malignant progression of HCC by promoting glycolysis. Mechanistically, FTO-IT1 induced stabilization of FTO mRNA by recruiting ILF2/ILF3 protein complex to 3’UTR of FTO mRNA. As a demethylase for N6-methyladenosine (m6A), FTO decreased m6A modification on mRNAs of glycolysis associated genes including GLUT1, PKM2, and c-Myc which alleviated the YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated mRNA degradation. Therefore, the upregulated expression of FTO-IT1 leaded to overexpression of GLUT1, PKM2, and c-Myc by which enhanced glycolysis of HCC. Meanwhile, it was found that c-Myc transcriptional regulated expression of FTO-IT1 by binding to its promoter area under hypo-glucose condition, forming a reciprocal loop between c-Myc and FTO-IT1.ConclusionsThis study identified an important role of the FTO-IT1/FTO axis mediated m6A modification of glycolytic genes contributed to glycolysis and tumorigenesis of HCC, and FTO-IT1 might be served as a new therapeutic target for HCC.

YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia†.

Genomic integrity is critical for sexual reproduction, ensuring correct transmission of parental genetic information to the descendant. To preserve genomic integrity, germ cells have evolved multiple DNA repair mechanisms, together termed as DNA damage response. The RNA N6-methyladenosine is the most abundant mRNA modification in eukaryotic cells, which plays important roles in DNA damage response, and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) is a well-acknowledged N6-methyladenosine reader protein regulating the mRNA decay and stress response. Despite this, the correlation between YTHDF2 and DNA damage response in germ cells, if any, remains enigmatic. Here, by employing a Ythdf2-conditional knockout mouse model as well as a Ythdf2-null GC-1 mouse spermatogonial cell line, we explored the role and the underlying mechanism for YTHDF2 in spermatogonial DNA damage response. We identified that, despite no evident testicular morphological abnormalities under the normal circumstance, conditional mutation of Ythdf2 in adult male mice sensitized germ cells, including spermatogonia, to etoposide-induced DNA damage. Consistently, Ythdf2-KO GC-1 cells displayed increased sensitivity and apoptosis in response to DNA damage, accompanied by the decreased SET domain bifurcated 1 (SETDB1, a histone methyltransferase) and H3K9me3 levels. The Setdb1 knockdown in GC-1 cells generated a similar phenotype, but its overexpression in Ythdf2-null GC-1 cells alleviated the sensitivity and apoptosis in response to DNA damage. Taken together, these results demonstrate that the N6-methyladenosine reader YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia, which provides novel insights into the mechanisms underlying spermatogonial genome integrity maintenance and therefore contributes to safe reproduction.

Targeting YTHDF2 inhibits tumorigenesis of diffuse large B-cell lymphoma through ACER2-mediated ceramide catabolism

IntroductionEpigenetic alterations play crucial roles in diffuse large B-cell lymphoma (DLBCL). Disturbances in lipid metabolism contribute to tumor progression. However, studies in epigenetics, especially its critical regulator YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), on lipid metabolism regulation in DLBCL are unidentified. ObjectivesElucidate the prognostic value and biological functions of YTHDF2 in DLBCL and illuminate the underlying epigenetic regulation mechanism of lipid metabolism by YTHDF2 in DLBCL development. MethodsThe expression and clinical value of YTHDF2 in DLBCL were performed in public databases and clinical specimens. The biological functions of YTHDF2 in DLBCL were determined in vivo and in vitro through overexpression and CRISPR/Cas9-mediated knockout of YTHDF2. RNA sequencing, lipidomics, methylated RNA immunoprecipitation sequencing, RNA immunoprecipitation-qPCR, luciferase activity assay, and RNA stability experiments were used to explore the potential mechanism by which YTHDF2 contributed to DLBCL progression. ResultsYTHDF2 was highly expressed in DLBCL, and related to poor prognosis. YTHDF2 overexpression exerted a tumor-promoting effect in DLBCL, and knockdown of YTHDF2 restricted DLBCL cell proliferation, arrested cell cycle in the G2/M phase, facilitated apoptosis, and enhanced drug sensitivity to ibrutinib and venetoclax. In addition, YTHDF2 knockout drastically suppressed tumor growth in xenograft DLBCL models. Furthermore, a regulatory role of YTHDF2 in ceramide metabolism was identified in DLBCL cells. Exogenous ceramide effectively inhibited the malignant phenotype of DLBCL cells in vitro. The binding of YTHDF2 to m6A sites on alkaline ceramidase 2 (ACER2) mRNA promoted its stability and expression. Enhanced ACER2 expression hydrolyzed ceramides, disrupting the balance between ceramide and sphingosine-1-phosphate (S1P), activating the ERK and PI3K/AKT pathways, and leading to DLBCL tumorigenesis. ConclusionThis study demonstrated that YTHDF2 contributed to the progression of DLBCL by regulating ACER2-mediated ceramide metabolism in an m6A-dependent manner, providing novel insights into targeted therapies.

YTHDF1 shapes “cold” tumor and inhibits CD8+ T cells infiltration and function in breast cancer

While YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) was recognized as a crucial contributor in the development and immune-related regulation of various types of tumors, its function in the immune response of breast cancer has largely remained uninvestigated. Through analysis of public databases, we found YTHDF1 as a highly expressed gene in breast cancers and confirmed this finding in breast cancer cells and clinical specimens from our center. Subsequently, we examined the link between YTHDF1 expression and immune cells and molecules by utilizing immune-related public databases and algorithm. We further validated our findings through cellular and animal experiments, as well as RNA sequencing. YTHDF1 was found highly expressed in tumor tissues of breast cancer, which negatively correlated with patient survival. The downregulation of YTHDF1 promoted the expression of pro-inflammatory markers and improved the anti-cancer ability of immune cells in breast cancer. RNA sequencing analysis revealed that YTHDF1 knockdown resulted in enrichment of differential genes in signal transduction pathways. Additionally, in vitro experiments showed that immune cells had higher cytotoxicity against breast cancer cells with decreased YTHDF1 expression. Moreover, in vivo studies indicated that YTHDF1 promoted breast cancer growth while inhibiting CD8+ T cell infiltration and function. Our study demonstrates that YTHDF1 plays a crucial role in establishing a “cold” tumor microenvironment in breast cancer by inhibiting the release of pro-inflammatory cytokines from cancer cells. As a result, the infiltration and functional differentiation of anti-tumor CD8+ T cells are hindered, ultimately resulting in the immune evasion of breast cancer.