Human AlkB Homologue 5 Is a Nuclear 2-Oxoglutarate Dependent Oxygenase and a Direct Target of Hypoxia-Inducible Factor 1α (HIF-1α)

Abstract

Human 2-oxoglutarate oxygenases catalyse a range of biological oxidations including the demethylation of histone and nucleic acid substrates and the hydroxylation of proteins and small molecules. Some of these processes are centrally involved in regulation of cellular responses to hypoxia. The ALKBH proteins are a sub-family of 2OG oxygenases that are defined by homology to the Escherichia coli DNA-methylation repair enzyme AlkB. Here we report evidence that ALKBH5 is probably unique amongst the ALKBH genes in being a direct transcriptional target of hypoxia inducible factor-1 (HIF-1) and is induced by hypoxia in a range of cell types. We show that purified recombinant ALKBH5 is a bona fide 2OG oxygenase that catalyses the decarboxylation of 2OG but appears to have different prime substrate requirements from those so far defined for other ALKBH family members. Our findings define a new class of HIF-transcriptional target gene and suggest that ALKBH5 may have a role in the regulation of cellular responses to hypoxia.

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

“…While the Ten‐Eleven Translocation (TET) protein family of dioxygenases primarily mediates oxidation of m 5 C in nuclear DNA and has also been implicated in histone modification, most of the members of the AlkB‐like Fe(II)/alpha‐ketoglutarate‐dependent dioxygenases (ALKBH) have been shown to act on RNA (reviewed in Shen et al , 2014; Fedeles et al , 2015; Li et al , 2015; Ougland et al , 2015). These include FTO (ALKBH9) that is implicated together with ALKBH5 in the oxidative removal of several modifications including 6‐methyladenosine (m 6 A) from RNA and ALKBH8 that is involved in the generation of 5‐methoxycarbonylmethyluridine (mcm 5 U) in cytoplasmic tRNAs (Fu et al , 2010a,b; Songe‐Møller et al , 2010; Jia et al , 2011; Thalhammer et al , 2011; Berulava et al , 2013; Zheng et al , 2013). So far, only ALKBH7, which was suggested to act on protein substrates during necrosis (Fu et al , 2013; Solberg et al , 2013; Wang et al , 2014), and ALKBH1/ABH1 have been reported to localise to mitochondria; however, the cellular localisation of ABH1 has been a matter of debate (Pan et al , 2008; Westbye et al , 2008; Ougland et al , 2012).…”

Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m 6 A-demethylation of NANOG mRNA

“…Even before its identification as an RNA demethylase, hypoxic induction of ALKBH5 expression was reported (36), but, at that time, the functional significance of this observation was not known. In the study reported here, we tested the hypothesis that exposure of breast cancer cells to hypoxia is sufficient to stimulate HIFdependent ALKBH5 expression, leading to decreased m 6 A methylation of NANOG mRNA, increased NANOG mRNA stability, increased NANOG protein levels, and BCSC enrichment, thereby establishing hypoxia as a major physiological stimulus in the tumor microenvironment that directly connects regulation of m 6 A RNA methylation to the specification and maintenance of BCSCs.…”

Structures of Human ALKBH5 Demethylase Reveal a Unique Binding Mode for Specific Single-stranded N6-Methyladenosine RNA Demethylation

“…E. coli AlkB, ALKBH1, and ALKBH3 prefer single-stranded nucleic acids, and ALKBH2 acts on double-stranded DNAs (21)(22)(23)(24)(25). ALKBH5 exhibits a strict preference for m 6 A ssRNA (8,30), whereas FTO can demethylate m 6 A ssRNA, as well as m 3 T ssDNA (9,29). To understand why ALKBH5 acts on singlestranded, but not double-stranded nucleic acids, we compared the structure of ALKBH5 with that of ALKBH2-dsDNA and found that a rigid loop in ALKBH5 will cause a steric clash with dsDNA.…”

“…ALKBH5 is reported to catalyze N-demethylation of m 6 A ssRNA or ssDNA, but not other tested modified nucleotides (8,30).…”