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:Neutrophils are key immune components in rheumatoid arthritis (RA), but the role of ALKBH5 in neutrophil function and RA progression remains unclear. METTL3, METTL14, WTAP, ALKBH5, FTO, YTHDF2, autophagy related gene were investigated by RT-qPCR. Autophagy, LC3B were respectively detected by flow cytometry analysis, Western blotting. Correlation analyses between ALKBH5 levels and RA disease activity, autophagy were performed, and receiver operating characteristic (ROC) curves were constructed to evaluate its predictive value. Univariate analysis and multivariate regression analysis were used to analyze the risk factors and construct predictive model. Sh-ALKBH5 lentiviral vectors was constructed and used to infect HL-60 cell line. The role of ALKBH5 in autophagy regulation was explored using RNA immunoprecipitation with next generation sequencing (RIP-Seq), m6A RNA Methylation Analysis, m6A immunocoprecipitation-quantitative polymerase chain reaction (MeRIP-qPCR) and Actinomycin D treatment. Results showed significantly reduced ALKBH5 levels in RA neutrophils compared to healthy controls (HC) and ankylosing spondylitis (AS) patients, correlating with RA disease activity. A novel predictive model incorporating ALKBH5, hemoglobin (HGB), and lymphocyte percentage (L%) exhibited enhanced efficacy in distinguishing RA patients from HC (AUC = 0.937) and AS patients (AUC = 0.943), and could reflect RA severity and activity. Additionally, autophagy levels in neutrophils were highest in RA synovial fluid, followed by RA peripheral blood, and lowest in HC peripheral blood, with ALKBH5 expression showing the opposite trend. Moreover, ALKBH5 negatively correlated with neutrophil autophagy and the silencing of ALKBH5 in HL-60 enhanced the autophagy. Silencing ALKBH5 increased m6A level of ATG7 mRNA and ATG7 mRNA stability. Mechanistically, ALKBH5 inhibited neutrophil autophagy through mediating m6A modification of the ATG7 mRNA. In conclusion, these findings demonstrate that ALKBH5 regulates neutrophil autophagy in RA, and the ALKBH5-HGB-L% model holds potential as a diagnostic and disease activity indicator for RA.
Project description:Since m6A demethylases (FTO and ALKBH5) have been reported to be involved in pre-mRNA splicing regulation, we hypothesized that dynamic m6A distribution during mRNA maturation might involve removal of m6A in internal exons by FTO or ALKBH5 accompanied by splicing factors. To explore this we performed pull-down assays coupled with protein mass spectrometry.
Project description:Alkylation repair homolog protein 5 (ALKBH5) binds to RNA and mediates RNA m6A demethylation. However, whether ALKBH5 binds to DNA is largely unknown. In this study, our CUT&Tag assay identified 12333 peaks in ALKBH5-overexpressing Hepa1-6 cells, indicating that ALKBH5 can bind to DNA.
Project description:Alkylation repair homolog protein 5 (ALKBH5) binds to RNA and mediates RNA m6A demethylation. However, whether ALKBH5 binds to DNA is largely unknown. In this study, our CUT&RUN data show that ALKBH5 can bind to DNA in Hepa1-6 cells.
Project description:N6-methyladenonsine (m6A) modification locates ubiquitously in mammalian mRNA, and profoundly impacts various physiological processes and pathogenesis. However, the precise involvement of m6A in early endoderm development has yet to be fully elucidated. Here, we reported that depletion of the m6A demethylase ALKBH5 in human embryonic stem cells (hESCs) severely impaired definitive endoderm (DE) differentiation. Within this process, ALKBH5-/- hESCs failed to undergo the primitive streak (PS) intermediate transition, which is considered as a prelude to endoderm specification. Mechanistically, we demonstrated that ALKBH5 deficiency induced m6A hypermethylation around the 3’ untranslated region (3’UTR) of GATA6 transcripts and destabilized GATA6 mRNA in a YTHDF2-dependent manner. Moreover, dysregulation of GATA6 expression ablated its occupancy with critical regulators of Wnt/β-catenin signaling pathway, thereby disrupting the signaling logic underlying DE formation. Overall, our findings unveil a mechanism whereby the ALKBH5-GATA6-WNT/β-catenin axis modulates human in vitro DE induction, and present novel insights on m6A modification in early embryonic development.
Project description:ALKBH5 play important role in regulation of trophoblast invasion, we examined the downstream genes of ALKBH5 via transcriptome sequencing
Project description:ALKBH5 is the RNA N(6)-methyladenosine (m6A) demethylase. To understhand the function and mechnism of ALKBH5 in human acute myeloid leukemia, we compared the translational efficiency in wild-type and ALKBH5-knock-down MOLM-13 cells.
Project description:We here report ALKBH5, a m6A RNA demethylase, as a crucial oncogene in multiple myeloma (MM). Using various MM models, we demonstrated a critical requirement of ALKBH5 for MM cell proliferation in vitro and in vivo. To identify the potential mRNA targets of ALKBH5, we conducted m6A-seq with mRNA samples enriched from MM cells with or without ALKBH5 knockdown.