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:To investigate the function of ALKBH5 in the regulation of maternal RNA decay during mice oocyte meiosis, we established Alkbh5 knockout mice and analyzed the m6A level changes between control and Alkbh5 knockout oocyte at GV stage
Project description:Objectives: To investigate the role of ALKBH5 in fibroblasts during post-myocardial infarction (MI) repair. Background: N6-methyladenosine (m6A) mRNA modification has been shown to play an important role in cardiovascular diseases. The RNA demethylase, AlkB homolog 5 (ALKBH5), is an m6A "eraser" that is responsible for decreased m6A methylation. However, its role in cardiac fibroblasts during the post-MI healing process remains elusive. Methods: MI was mimicked by permanent left anterior descending artery ligation in global ALKBH5-knockout, ALKBH5-knockin, and fibroblast-specific ALKBH5-knockout mice to study the function of ALKBH5 during post-MI collagen repair. Methylated RNA immunoprecipitation sequencing was performed to explore potential ALKBH5 targets. Results: Dramatic alterations in ALKBH5 expression were observed during the early stage post-MI and in hypoxic fibroblasts. Global ALKBH5 knockin reduced infarct size and improved cardiac function after MI. The global and fibroblast-specific ALKBH5-knockout mice both exhibited low survival rates along with poor collagen repair, impaired cardiac function, and cardiac rupture. Both in vivo and in vitro ALKBH5 loss led to impaired fibroblast activation and decreased collagen deposition. Additionally, hypoxia, but not TGF-β1 or Ang II, upregulated ALKBH5 expression in myofibroblasts in a HIF-1α-dependent transcriptional manner. Mechanistically, ALKBH5 promoted the stability of ErbB4 mRNA and the degradation of ST14 mRNA via m6A demethylation. Fibroblast-specific ErbB4 overexpression ameliorated the impaired fibroblast-to-myofibroblast transformation and poor post-MI repair due to ALKBH5 knockout. Conclusions: Fibroblast ALKBH5 positively regulates post-MI healing via post-transcriptional modification and stabilization of ErbB4 mRNA in an m6A-dependent manner. Targeting ALKBH5/ERBB4 may be a potential therapeutic option for post-MI cardiac rupture.
Project description:To investigate the function of ALKBH5 in the regulation of maternal RNA decay during mice oocyte meiosis, we established Alkbh5 knockout mice and analyzed the transcriptome changes between control and Alkbh5 knockout oocyte at different meiotic maturation stages
Project description:To investigate the function of ALKBH5 in the regulation of keratinocyte cellular function, we established HaCAT cell lines in which ALKBH5 has been knocked down by siRNA. We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells from duplicate experiments.
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. Here, 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 in vitro and in vivo. Integrated transcriptome and m6A-seq analyses revealed altered expression of select ALKBH5 target genes, including FOXM1, a critical transcription factor for the ALKBH5-dependent cell cycle gene expression. ALKBH5 binds to and demethylates FOXM1 nascent transcripts, leading to enhanced FOXM1 expression. Further, a long noncoding RNA antisense to the FOXM1 (FOXM1-AS) interacts with ALKBH5 and FOXM1 nascent transcripts and promotes their interaction. Depleting ALKBH5 and FOXM1-AS disrupted GSC tumorigenesis through the FOXM1 axis. Our work uncovers a novel function for ALKBH5 in maintaining GSC tumorigenicity and provides insight into critical roles of the m6A RNA methylation in human brain tumor.
