Oxidative demethylase ALKBH5 repairs DNA alkylation damage and protects against alkylation-induced toxicity

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. Copyright © 2013 Elsevier Inc. All rights reserved.

Cancer is one of the leading causes of death, and there is great interest in understanding the underlying molecular mechanisms involved in the pathogenesis and progression of individual tumors. We used systems-level approaches to analyze the genome-wide transcriptome of the protein-coding genes of 17 major cancer types with respect to clinical outcome. A general pattern emerged: Shorter patient survival was associated with up-regulation of genes involved in cell growth and with down-regulation of genes involved in cellular differentiation. Using genome-scale metabolic models, we show that cancer patients have widespread metabolic heterogeneity, highlighting the need for precise and personalized medicine for cancer treatment. All data are presented in an interactive open-access database (www.proteinatlas.org/pathology) to allow genome-wide exploration of the impact of individual proteins on clinical outcomes.

N(6)-methyladenosine (m(6)A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m(6)A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxia-inducible factor (HIF)-1α- and HIF-2α-dependent expression of AlkB homolog 5 (ALKBH5), an m(6)A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m(6)A residue in the 3'-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF- and ALKBH5-dependent manner. Insertion of the NANOG 3'-UTR into a luciferase reporter gene led to regulation of luciferase activity by O2, HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment.