ABSTRACT: N6-methyladenosine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as a new mammalian demethylase that oxidatively removes the m6A modification in mRNA in vitro and inside cells. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1552 differentially expressed genes which cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. We show that Alkbh5-deficiency impacts the expression levels of some of these mRNAs, supporting the observed phenotype. The discovery of this new RNA demethylase strongly suggests that the reversible m6A modification plays fundamental and broad functions in mammalian cells. RNA-seq in two cell types
Project description:N6-methyladenosine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as a new mammalian demethylase that oxidatively removes the m6A modification in mRNA in vitro and inside cells. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1552 differentially expressed genes which cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. We show that Alkbh5-deficiency impacts the expression levels of some of these mRNAs, supporting the observed phenotype. The discovery of this new RNA demethylase strongly suggests that the reversible m6A modification plays fundamental and broad functions in mammalian cells. RNA-seq in two cell types
Project description:N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.
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.
Project description:N6-Methyladenosine (m6A) is the most prevalent internal modification in mammalian mRNAs. Although m6A is important in many biological processes, its roles in the placenta are unclear. Methods: Levels of global mRNA m6A methylation and ALKBH5 expression in recurrent miscarriage (RM) patients were determined using quantitative reverse transcription-PCR (qRT-PCR), m6A RNA methylation quantification, and immunohistochemical methods. Using ALKBH5 overexpression and knockdown methods, we determined the role of ALKBH5 in trophoblast invasion at the maternal interface through trophoblasts and an extravillous explant culture experiments. Furthermore, the regulation of CYR61 by ALKBH5 was explored by RNA-sequencing coupled with methylated RNA immunoprecipitation. Results: We found that the level of global mRNA m6A methylation was significantly decreased in placental villous tissue from RM patients, while ALKBH5 expression was specifically unregulated. Furthermore, we demonstrated that ALKBH5 knockdown in human trophoblast promoted trophoblast invasion. Conversely, overexpression of ALKBH5 inhibited cell invasion. ALKBH5 knockdown promoted trophoblast invasion in villous explant culture experiments, while overexpression of ALKBH5 repressed these effects. Furthermore, we clarified that ALKBH5 inhibited trophoblast invasion by regulating CYR61 mRNA stability, and this RNA regulation is m6A dependent. Mechanistic analyses showed that decreased ALKBH5 in trophoblast increased the half-life of CYR61 mRNA and promoted steady-state CYR61 mRNA expression levels. Conclusions: We elucidated the functional roles of ALKBH5 and mRNA m6A methylation in trophoblast and identified a novel RNA regulatory mechanism, providing a basis for further exploration of broad RNA epigenetic regulatory patterns in RM diseases.
Project description:Background:Osteosarcoma (OS) is one of the most common malignant bone tumors. Plasmacytoma variant translocation 1 (PVT1) is a well-known oncogenic long noncoding RNA (lncRNA). However, to date, the regulatory mechanism of PVT1 upregulation in OS remains unknown. Methods:qRT-PCR was carried out to test the expression level of PVT1 and ALKBH5. RNA immunoprecipitation (RIP) and RNA pull-down assays were performed to detect the interaction of PVT1 with ALKBH5 and YTHDF2. Methylated RNA immune-precipitation (MeRIP) was used to examine the N 6-methyladenosine (m6A) modification of PVT1 transcript. Results:In this study, we found that PVT1 expression was upregulated in OS tissues and cells and significantly related with clinical stage, tumor size, and prognosis of patients with OS. Further investigation revealed that N 6-methyladenosine (m6A) demethylase ALKBH5 could associate with PVT1 and suppress its degradation. ALKBH5 decreased the m6A modification of PVT1, thus inhibiting the binding of reader protein YTHDF2 in PVT1. Functionally, ALKBH5-mediated PVT1 upregulation promoted the OS cell proliferation in vitro and tumor growth in vivo. Conclusions:Our study suggests that ALKBH5-mediated m6A modification of PVT1 contributes to OS tumorigenesis.
Project description:BACKGROUND:N6-methyladenosine (m6A) is the most abundant reversible methylation modification of eukaryotic mRNA, and it plays vital roles in tumourigenesis. This study aimed to explore the role of the m6A demethylase ALKBH5 in pancreatic cancer (PC). METHODS:The expression of ALKBH5 and its clinicopathological impact were evaluated in PC cohorts. The effects of ALKBH5 on the biological characteristics of PC cells were investigated on the basis of gain-of-function and loss-of-function analyses. Subcutaneous and orthotopic models further uncovered the role of ALKBH5 in tumour growth. mRNA and m6A sequencing and assays of m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the targeted effect of ALKBH5 on PER1. P53-binding sites in the ALKBH5 promoter were investigated by ChIP and luciferase assays to reveal the interplay between ALKBH5 and PER1-activated ATM-CHK2-P53/CDC25C signalling. RESULTS:ALKBH5 loss characterized the occurrence and poor clinicopathological manifestations in patients with PC. Overexpression of ALKBH5 reduced tumoural proliferative, migrative, invasive activities in vitro and ameliorated tumour growth in vivo, whereas ALKBH5 knockdown facilitated PC progression. Mechanistically, ALKBH5 posttranscriptionally activated PER1 by m6A demethylation in an m6A-YTHDF2-dependent manner. PER1 upregulation led to the reactivation of ATM-CHK2-P53/CDC25C signalling, which inhibited cell growth. P53-induced activation of ALKBH5 transcription acted as a feedback loop regulating the m6A modifications in PC. CONCLUSION:ALKBH5 serves as a PC suppressor by regulating the posttranscriptional activation of PER1 through m6A abolishment, which may highlight a demethylation-based approach for PC diagnosis and therapy.
Project description:The dynamic and reversible N6-methyladenosine (m6A) RNA modification installed and erased by N6-methyltransferases and demethylases regulates gene expression and cell fate. We show that the m6A demethylase ALKBH5 is highly expressed in glioblastoma stem-like cells (GSCs). Silencing ALKBH5 suppresses the proliferation of patient-derived GSCs. Integrated transcriptome and m6A-seq analyses revealed altered expression of certain ALKBH5 target genes, including the transcription factor FOXM1. ALKBH5 demethylates FOXM1 nascent transcripts, leading to enhanced FOXM1 expression. Furthermore, a long non-coding RNA antisense to FOXM1 (FOXM1-AS) promotes the interaction of ALKBH5 with FOXM1 nascent transcripts. Depleting ALKBH5 and FOXM1-AS disrupted GSC tumorigenesis through the FOXM1 axis. Our work uncovers a critical function for ALKBH5 and provides insight into critical roles of m6A methylation in glioblastoma.
Project description:N6-methyladenosine (m6A) mRNA modifications play critical roles in various biological processes. However, no study addresses the role of m6A in macroautophagy/autophagy. Here, we show that m6A modifications are increased in H/R-treated cardiomyocytes and ischemia/reperfusion (I/R)-treated mice heart. We found that METTL3 (methyltransferase like 3) is the primary factor involved in aberrant m6A modification. Silencing METTL3 enhances autophagic flux and inhibits apoptosis in H/R-treated cardiomyocytes. However, overexpression of METTL3 or inhibition of the RNA demethylase ALKBH5 has an opposite effect, suggesting that METTL3 is a negative regulator of autophagy. Mechanistically, METTL3 methylates TFEB, a master regulator of lysosomal biogenesis and autophagy genes, at two m6A residues in the 3'-UTR, which promotes the association of the RNA-binding protein HNRNPD with TFEB pre-mRNA and subsequently decreases the expression levels of TFEB. Further experiments show that autophagic flux enhanced by METTL3 deficiency is TFEB dependent. In turn, TFEB regulates the expression levels of METTL3 and ALKBH5 in opposite directions: it induces ALKBH5 and inhibits METTL3. TFEB binds to the ALKBH5 promoter and activates its transcription. In contrast, inhibition of METTL3 by TFEB does not involve transcriptional repression but rather downregulation of mRNA stability, thereby establishing a negative feedback loop. Together, our work uncovers a critical link between METTL3-ALKBH5 and autophagy, providing insight into the functional importance of the reversible mRNA m6A methylation and its modulators in ischemic heart disease. Abbreviations: ACTB, actin beta; ALKBH5, alkB homolog 5, RNA demethylase; ANXA5, annexin A5; ATG, autophagy-related; BafA, bafilomycin A1; CASP3, caspase 3; ELAVL1, ELAV like RNA binding protein 1; FTO, FTO, alpha-ketoglutarate dependent dioxygenase; GFP, green fluorescent protein; GST, glutathione S-transferase; HNRNPD, heterogeneous nuclear ribonucleoprotein D; H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; LAD, left anterior descending; m6A, N6-methyladenosine; MEFs, mouse embryo fibroblasts; Mer, mutated estrogen receptor domains; METTL3, methyltransferase like 3; METTL14, methyltransferase like 14; mRFP, monomeric red fluorescent protein; MTORC1, mechanistic target of rapamycin kinase complex 1; NMVCs, neonatal mouse ventricular cardiomyocytes; PCNA, proliferating cell nuclear antigen; PE, phosphatidylethanolamine; PI, propidium iodide; PTMs, post-translational modifications; PVDF, polyvinylidenedifluoride; RIP, RNA-immunoprecipitation; siRNA, small interfering RNA; SQSTM1, sequestosome 1; TFEB, transcription factor EB; TUBA: tublin alpha; WTAP, WT1 associated protein; YTHDF, YTH N6-methyladenosine RNA binding protein.
Project description:BACKGROUND:The importance of mRNA methylation erased by ALKBH5 in mRNA biogenesis, decay, and translation control is an emerging research focus. Ectopically activated YAP is associated with the development of many human cancers. However, the mechanism whereby ALKBH5 regulates YAP expression and activity to inhibit NSCLC tumor growth and metastasis is not clear. METHODS:Protein and transcript interactions were analyzed in normal lung cell and NSCLC cells. Gene expression was evaluated by qPCR and reporter assays. Protein levels were determined by immunochemical approaches. Nucleic acid interactions and status were analyzed by immunoprecipitation. Cell behavior was analyzed by standard biochemical tests. The m6A modification was analyzed by MeRIP. RESULTS:Our results show that YAP expression is negatively correlated with ALKBH5 expression and plays an opposite role in the regulation of cellular proliferation, invasion, migration, and EMT of NSCLC cells. ALKBH5 reduced m6A modification of YAP. YTHDF3 combined YAP pre-mRNA depending on m6A modification. YTHDF1 and YTHDF2 competitively interacted with YTHDF3 in an m6A-independent manner to regulate YAP expression. YTHDF2 facilitated YAP mRNA decay via the AGO2 system, whereas YTHDF1 promoted YAP mRNA translation by interacting with eIF3a; both these activities are regulated by m6A modification. Furthermore, ALKBH5 decreased YAP activity by regulating miR-107/LATS2 axis in an HuR-dependent manner. Further, ALKBH5 inhibited tumor growth and metastasis in vivo by reducing the expression and activity of YAP. CONCLUSIONS:The presented findings suggest m6A demethylase ALKBH5 inhibits tumor growth and metastasis by reducing YTHDFs-mediated YAP expression and inhibiting miR-107/LATS2-mediated YAP activity in NSCLC. Moreover, effective inhibition of m6A modification of ALKBH5 might constitute a potential treatment strategy for lung cancer.
Project description:Background:The modification and regulation of N6-methyladenosine (m6A) at mRNA level can affect the development and progression in various tumors. ALKBH5, as an m6A demethylase, plays different roles in tumors by regulating the m6A modification of mRNA. However, its role in renal cell carcinoma (RCC) remains unclear. Methods:First, levels of ALKBH5 in RCC tissues and cell lines were verified by qRT-PCR and western blot. We analyzed the relationship between ALKBH5 and the clinicopathological characteristics of RCC patients and the influence of ALKBH5 on the prognosis of patients. Then we generated ALBKH5-overexpression, ALBKH5-knockdown stable RCC cell lines and their control cell lines. Through cell proliferation assay, colony formation assay, cell invasion and tumor migration assay, cell cycle assay and xenograft studies, we studied the ALKBH5 roles in RCC cell lines. AURKB was predicted to be its potential target based on TCGA database analysis and verified by western blot. The role of AURKB in RCC was verified by TCGA database and Kaplan-Meier analysis with TMA immunohistochemical analysis. Finally, the specific molecular mechanism of ALKBH5 targeting AURKB was explored by dual-luciferase reporter assay, RNA immunoprecipitation (RIP), m6A dot-blot assay, m6A RNA Immunoprecipitation (MeRIP) assay, and mRNA stability assay. Results:We found that ALKBH5 was highly expressed in both RCC tumor tissues and cell lines. Clinicopathological analysis showed that high ALKBH5 expression was associated with larger tumor volume (P=0.017) and higher TNM staging (P=0.006), and worse prognosis (log rank: P=0.0199). The cellular functional assays showed that stably overexpression ALKBH5 could promote the cell proliferation, colony formation, cell migration and cell invasion of renal cell carcinoma cells in vitro and promote tumor growth in vivo. In contrast, ALKBH5 knocked down inhibited cell proliferation, colony formation, migration and invasion of renal cell carcinoma cells in vitro. Based on TCGA database analysis, AURKB was predicted highly expressed in RCC and a potential target of ALKBH5. Both database prediction and TMA immunohistochemical analysis supported that AURKB could affect the prognosis of RCC patients (P values of 5.5e-08 and 0.0004, respectively) and was regulated by ALKBH5 expression level. Subsequent mechanism experiments showed that ALKBH5 regulated the expression of AURKB by regulating the stability of AURKB mRNA in the m6A-dependent manner, and finally promoted cell proliferation. Furthermore, we found that hypoxia-induced HIF could up-regulate both expressions of AURKB and ALKBH5. Conclusions:Our findings suggest that ALKBH5 may play a carcinogenic role in renal cell carcinoma by stabilizing AURKB mRNA in a m6A-dependent manner. These data suggest that ALKBH5 may play a key role in RCC and targeting the ALKBH5 signaling pathway may be a promising strategy for the treatment of RCC.