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Post-translational modification of RNA m6A demethylase ALKBH5 regulates ROS-induced DNA damage response.

ABSTRACT: Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from DNA damage and apoptosis during reactive oxygen species (ROS)-induced stress. We find that ROS significantly induces global mRNA N6-methyladenosine (m6A) levels by modulating ALKBH5 post-translational modifications (PTMs), leading to the rapid and efficient induction of thousands of genes involved in a variety of biological processes including DNA damage repair. Mechanistically, ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5 m6A demethylase activity by blocking substrate accessibility. Moreover, ERK/JNK/ALKBH5-PTMs/m6A axis is activated by ROS in hematopoietic stem/progenitor cells (HSPCs) in vivo in mice, suggesting a physiological role of this molecular pathway in the maintenance of genome stability in HSPCs. Together, our study uncovers a molecular mechanism involving ALKBH5 PTMs and increased mRNA m6A levels that protect genomic integrity of cells in response to ROS.

SUBMITTER: Yu F

PROVIDER: S-EPMC8191756 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Post-translational Modification of RNA m6A Demethylase ALKBH5 Regulates ROS-induced DNA Damage Response

Project description:Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from DNA damage and apoptosis during reactive oxygen species (ROS)-induced stress. We find that ROS significantly induces global mRNA N6-methyladenosine (m6A) levels by modulating ALKBH5 post-translational modifications (PTMs), leading to the rapid and efficient induction of thousands of genes involved in a variety of biological processes including DNA damage repair. Mechanistically, ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5 m6A demethylase activity by blocking substrate accessibility. Moreover, ERK/JNK/ALKBH5-PTMs/m6A axis is activated by ROS in hematopoietic stem/progenitor cells (HSPCs) in vivo in mice, suggesting a physiological role of this molecular pathway in the maintenance of genome stability in HSPCs. Together, our study uncovers a molecular mechanism involving ALKBH5 PTMs and increased mRNA m6A levels that protect genomic integrity of cells in response to ROS.

2021-04-29 | GSE144620 | GEO

Post-translational Modification of RNA m6A Demethylase ALKBH5 Regulates ROS-induced DNA Damage Response

Project description:Post-translational Modification of RNA m6A Demethylase ALKBH5 Regulates ROS-induced DNA Damage Response

| PRJNA604206 | ENA

RNA demethylase ALKBH5 promotes tumorigenesis of t (8;21) acute myeloid leukemia via ITPA m6A modification.

Project description:BackgroundAlthough t (8;21) is in fact considered a good risk acute myeloid leukemia (AML), only 60% of the patients live beyond 5 years after diagnosis. Studies have shown that RNA demethylase ALKBH5 promotes leukemogenesis. However, the molecular mechanism and clinical significance of ALKBH5 in t (8;21) AML have not been elucidated.MethodsThe expression of ALKBH5 was assessed in t (8;21) AML patients via qRT-PCR and western blot. The proliferative activity of these cells was examined through CCK-8 or colony-forming assays, while flow cytometry approaches were used to examine apoptotic cell rates. The in vivo role of ALKBH5 promoting leukemogenesis was assessed using t (8;21) murine model, CDX, and PDX models. RNA sequencing, m6A RNA methylation assay, RNA immunoprecipitation, and luciferase reporter assay were used to explore the molecular mechanism of ALKBH5 in t (8;21) AML.ResultsALKBH5 is highly expressed in t (8;21) AML patients. Silencing ALKBH5 suppresses the proliferation and promotes the apoptosis of patient-derived AML cells and Kasumi-1 cells. With integrated transcriptome analysis and wet-lab confirmation, we found that ITPA is a functionally important target of ALKBH5. Mechanistically, ALKBH5 demethylates ITPA mRNA and increases its mRNA stability, leading to enhanced ITPA expression. Furthermore, transcription factor TCF15, specifically expressed in leukemia stem/initiating cells (LSCs/LICs), is responsible for the dysregulated expression of ALKBH5 in t (8;21) AML.ConclusionOur work uncovers a critical function for the TCF15/ALKBH5/ITPA axis and provides insights into the vital roles of m6A methylation in t (8;21) AML.

| S-EPMC10007764 | biostudies-literature

Demethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification.

Project description:BackgroundGastric cancer (GC) is one of the most pernicious tumors that seriously harm human healthcare. GC metastasis is one of the prime cause of failed cancer treatment, but correlation between N6-methyladenosine (m6A) and GC metastasis was less reported.MethodsMethylated RNA immunoprecipitation sequencing (MeRIP-seq) of GC tissues was conducted. Quantitative real-time PCR (qRT-PCR), western blotting and immunohistochemistry (IHC) were taken to determine the expression of ALKBH5 in GC tissues and cell lines. RNA-seq together with MeRIP-qRT-PCR was used to screen the target gene of ALKBH5. RNA pulldown, mass spectrometry and RNA immunoprecipitation (RIP) were used to search the "reader" protein of target gene. The mechanism was also validated via a tail vein injection method for lung metastasis model.ResultsDecreased expression of ALKBH5 was detected in GC samples, and it was correlated with clinical tumor distal metastasis and lymph node metastasis. ALKBH5 interference promoted metastasis of GC cells and this effect was closely related to the demethylase activity of ALKBH5. PKMYT1, as a downstream target of ALKBH5, promoted invasion and migration in GC. Caused by ALKBH5 knockdown or its demethylase activity mutation, upregulated expression of PKMYT1 indicated that ALKBH5 modulates expression of PKMYT1 in an m6A-dependent manner. IGF2BP3 helped stabilize the mRNA stability of PKMYT1 via its m6A modification site.ConclusionsThis study established an ALKBH5-PKMYT1-IGF2BP3 regulation system in metastasis, representing a new therapeutic target for GC metastasis.

| S-EPMC8812266 | biostudies-literature

The DEAD-Box RNA Helicase DDX3 Interacts with m6A RNA Demethylase ALKBH5.

Project description:DDX3 is a member of the family of DEAD-box RNA helicases. DDX3 is a multifaceted helicase and plays essential roles in key biological processes such as cell cycle, stress response, apoptosis, and RNA metabolism. In this study, we found that DDX3 interacted with ALKBH5, an m6A RNA demethylase. The ATP domain of DDX3 and DSBH domain of ALKBH5 were indispensable to their interaction with each other. Furthermore, DDX3 could modulate the demethylation of mRNAs. We also showed that DDX3 regulated the methylation status of microRNAs and there was an interaction between DDX3 and AGO2. The dynamics of m6A RNA modification is still a field demanding further investigation, and here, we add a link by showing that RNA demethylation can be regulated by proteins such as DDX3.

| S-EPMC5733242 | biostudies-literature

Genistein Ameliorates Renal Fibrosis Through Regulation Snail via m6A RNA Demethylase ALKBH5.

Project description:Renal tubule-interstitial fibrosis is related to chronic kidney disease progression and a typical feature of the aging kidney. Epigenetic modifications of fibrosis-prone genes regulate the development of renal fibrosis. As a kind of "epigenetic diet", soy isoflavone genistein was reported to have renal protective action and epigenetic-modulating effects. However, its renal protection role and underlying mechanisms are yet to be fully clarified. Herein, we showed that genistein exhibits a demonstrable anti-fibrotic effect on kidney in vivo UUO (unilateral ureteral occlusion) model and renal epithelial cells in vitro model. The mechanism is strongly associated with epithelial-to-mesenchymal transition and m6A RNA demethylase ALKBH5. Mouse fibrotic kidneys induced by UUO exhibited adverse expression of renal fibrosis-related proteins and significant increases in the total m6A level. As an eraser, ALKBH5 showed severer suppression in the renal fibrosis process. However, genistein pretreatment restored ALKBH5 loss remarkably and reduced renal fibrosis, abnormal protein, and inflammatory markers. The examination of possible mechanisms revealed that genistein promoted ALKBH5 and maybe induced the level of mRNA m6A methylation in some epithelial-to-mesenchymal transition-related transcription factors. We found snail was the critical regulator and critical for the protective role of genistein. To verify the relationship between ALKBH5 and snail, we generated knockdown and overexpression of ALKBH5 cells in vitro. ALKBH5 knockdown enhanced the mesenchymal phenotype marker α-smooth muscle actin and snail expression. In agreement, overexpression ALKBH5 increased epithelial adhesion molecule E-cadherin and reduced snail expression. In conclusion, genistein increased renal ALKBH5 expression in UUO-induced renal fibrosis and reduced RNA m6A levels and ameliorates renal damages.

| S-EPMC7751752 | biostudies-literature

Rational Design of Novel Anticancer Small-Molecule RNA m6A Demethylase ALKBH5 Inhibitors.

Project description:The RNA 6-N-methyladenosine (m6A) demethylase ALKBH5 has been shown to be oncogenic in several cancer types, including leukemia and glioblastoma. We present here the target-tailored development and first evaluation of the antiproliferative effects of new ALKBH5 inhibitors. Two compounds, 2-[(1-hydroxy-2-oxo-2-phenylethyl)sulfanyl]acetic acid (3) and 4-{[(furan-2-yl)methyl]amino}-1,2-diazinane-3,6-dione (6), with IC50 values of 0.84 μM and 1.79 μM, respectively, were identified in high-throughput virtual screening of the library of 144 000 preselected compounds and subsequent verification of hits in an m6A antibody-based enzyme-linked immunosorbent assay (ELISA) enzyme inhibition assay. The effect of these compounds on the proliferation of selected target cancer cell lines was then measured. In the case of three leukemia cell lines (HL-60, CCRF-CEM, and K562) the cell proliferation was suppressed at low micromolar concentrations of inhibitors, with IC50 ranging from 1.38 to 16.5 μM. However, the effect was low or negligible in the case of another leukemia cell line, Jurkat, and the glioblastoma cell line A-172. These results demonstrate the potential of ALKBH5 inhibition as a cancer-cell-type-selective antiproliferative strategy.

| S-EPMC8158789 | biostudies-literature

The m6A RNA Demethylase ALKBH5 Promotes Radioresistance and Invasion Capability of Glioma Stem Cells.

Project description:Recurrence of GBM is thought to be due to GBMSCs, which are particularly chemo-radioresistant and characterized by a high capacity to invade normal brain. Evidence is emerging that modulation of m6A RNA methylation plays an important role in tumor progression. However, the impact of this mRNA modification in GBM is poorly studied. We used patient-derived GBMSCs to demonstrate that high expression of the RNA demethylase, ALKBH5, increases radioresistance by regulating homologous recombination (HR). In cells downregulated for ALKBH5, we observed a decrease in GBMSC survival after irradiation likely due to a defect in DNA-damage repair. Indeed, we observed a decrease in the expression of several genes involved in the HR, including CHK1 and RAD51, as well as a persistence of γ-H2AX staining after IR. We also demonstrated in this study that ALKBH5 contributes to the aggressiveness of GBM by favoring the invasion of GBMSCs. Indeed, GBMSCs deficient for ALKBH5 exhibited a significant reduced invasion capability relative to control cells. Our data suggest that ALKBH5 is an attractive therapeutic target to overcome radioresistance and invasiveness of GBMSCs.

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m6A demethylase ALKBH5 controls CD4+ T cell pathogenicity and promotes autoimmunity.

Project description:N6-methyladenosine (m6A) modification is dynamically regulated by "writer" and "eraser" enzymes. m6A "writers" have been shown to ensure the homeostasis of CD4+ T cells, but the "erasers" functioning in T cells is poorly understood. Here, we reported that m6A eraser AlkB homolog 5 (ALKBH5), but not FTO, maintains the ability of naïve CD4+ T cells to induce adoptive transfer colitis. In addition, T cell-specific ablation of ALKBH5 confers protection against experimental autoimmune encephalomyelitis. During the induced neuroinflammation, ALKBH5 deficiency increased m6A modification on interferon-γ and C-X-C motif chemokine ligand 2 messenger RNA (mRNA), thus decreasing their mRNA stability and protein expression in CD4+ T cells. These modifications resulted in attenuated CD4+ T cell responses and diminished recruitment of neutrophils into the central nervous system. Our findings reveal an unexpected specific role of ALKBH5 as an m6A eraser in controlling the pathogenicity of CD4+ T cells during autoimmunity.

| S-EPMC8208713 | biostudies-literature

Decreased expression of m6A demethylase ALKBH5 in decidua contributes to preeclampsia via m6A-CORIN-HuR pathway.

Project description: Not available

| S-EPMC10308088 | biostudies-literature