Human AlkB homolog ABH8 Is a tRNA methyltransferase required for wobble uridine modification and DNA damage survival

Abstract

tRNA nucleosides are extensively modified to ensure their proper function in translation. However, many of the enzymes responsible for tRNA modifications in mammals await identification. Here, we show that human AlkB homolog 8 (ABH8) catalyzes tRNA methylation to generate 5-methylcarboxymethyl uridine (mcm(5)U) at the wobble position of certain tRNAs, a critical anticodon loop modification linked to DNA damage survival. We find that ABH8 interacts specifically with tRNAs containing mcm(5)U and that purified ABH8 complexes methylate RNA in vitro. Significantly, ABH8 depletion in human cells reduces endogenous levels of mcm(5)U in RNA and increases cellular sensitivity to DNA-damaging agents. Moreover, DNA-damaging agents induce ABH8 expression in an ATM-dependent manner. These results expand the role of mammalian AlkB proteins beyond that of direct DNA repair and support a regulatory mechanism in the DNA damage response pathway involving modulation of tRNA modification.

FIG. 1.

Human ABH8 contains a tRNA methyltransferase motif and localizes to the cytoplasm. (A) Schematic of the human AlkB homolog (ABH) family of nonheme iron/2-oxoglutarate-dependent dioxygenase proteins. (B) Annotated gene tree of ABH8. The maximum likelihood phylogenetic tree of ABH8 and homologous proteins was generated via TreeBeST (tree building guided by species tree) using the Ensembl genome database. The complete tree with bootstrap values and distances is available on request. S. pombe, Schizosaccharomyces pombe; K. lactis, Kluyveryomyces lactis. (C) Subcellular localization of green fluorescent protein-tagged ABH8. Human 293T embryonic kidney cells were transfected with constructs expressing the indicated proteins and visualized by fluorescence microscopy. The nucleus was visualized by DAPI (4′,6-diamidino-2-phenylindole) staining. As a comparison, GFP alone was analyzed alongside.

FIG. 2.

Purification and analysis of human ABH8 complexes. (A) Schematic of ABH8 variants and domains. RRM, RNA recognition motif; 2OG, 2-oxoglutarate; Fe(II), iron; SAM, S-adenosyl-l-methionine; AlkB, dioxygenase domain; MT, methyltransferase. (B) Protein profile of purified ABH8 complexes by silver stain analysis. Arrows indicate proteins specifically identified in purified ABH8 samples. (C) Confirmation and characterization of interactions between ABH8 with TRiC subunits and Trm112. (D) Purification of ABH8 complexes from HeLa human cells.

FIG. 3.

ABH8 does not demethylate canonical AlkB DNA substrates. The full-length ABH8 and domain variants were tested for oxidative demethylase activity on DNA substrates using a restriction enzyme-mediated oligonucleotide demethylase assay (see Materials and Methods). Purified ABH8 variants were incubated with a 49-mer DNA oligonucleotide containing 1-methyladenine (1-meA), 3-methylcytosine (3-meC), or ethenoadenine (ethenoA) (Table 1), followed by digestion with the methylation-sensitive restriction enzyme DpnII and gel electrophoresis. Unrepaired substrate and repaired product are indicated. The ABH2 protein, a known DNA/RNA demethylase, was included as a control.

FIG. 4.

ABH8 catalyzes RNA methylation. Shown is in vitro methyltransferase activity of purified ABH8 variants on the indicated substrates. A mock purification sample (control) or purified ABH8 variants were incubated with purified human RNA enriched for tRNAs (from control or ABH8-depleted cells), DNA oligonucleotides, or bovine serum albumin protein followed by methyl donor removal and measurement of the eluted samples via scintillation counting. Methyltransferase activity is expressed as increase relative to the control. The average ¹⁴C incorporation for each sample is listed in Table 3.

FIG. 5.

ABH8 interacts with a subset of tRNAs containing known modified wobble uridine residues. (A) Denaturing gel electrophoresis and Sybr gold stain of ABH8-associated RNAs. Arrows denote the migration pattern of the indicated RNAs, and the asterisk represents an ABH8-associated RNA species. (B) RNA blot hybridization analysis of ABH8-associated RNAs with the indicated oligonucleotide probes. The presence of a modified uridine (mcm⁵U) in the equivalent yeast tRNA is indicated on the right.

FIG. 6.

ABH8 is required for maintaining mcm⁵U levels in vivo. (A) Confirmation of ABH8 depletion in human cells by immunoblot analysis. The depletion of ABH8 is expressed as a percentage relative to the control shRNA cell line normalized to the GAPDH loading control. (B) Identification of cm⁵U and mcm⁵U by HPLC-coupled tandem quadrupole mass spectrometry. Shown are extracted ion chromatograms of molecular species consistent with cm⁵U and mcm⁵U nucleobases based upon theoretical m/z. (C) Detection and comparison of cm⁵U and mcm⁵U levels in small RNAs purified from control and ABH8-depleted human cells. The levels of cm⁵U and mcm⁵U nucleosides were detected by mass spectrometry and quantified by integration of the normalized peak intensity for each nucleoside signal.

FIG. 7.

Depletion of ABH8 diminishes cellular survival after exposure to DNA-damaging agents. (A) Colony formation assay of human cell lines after treatment with the indicated doses of MMS. (B and C) Viability of control and ABH8-depleted human cell lines after treatment with the indicated doses of MMS or bleomycin as measured by trypan blue dye exclusion or by the WST-1 cell proliferation and viability assay. (D) Verification of full-length ABH8 expression in human cell lines. Whole-cell extracts prepared from the indicated human cell lines were immunoblotted with antibody probes against the respective proteins. The asterisk denotes a protein that cross-reacts with the anti-ABH8 antibody. (E and F) Viability of human cell lines after treatment with MMS or bleomycin. (G) Real-time RT-PCR analysis of ABH8 transcripts from the indicated human cells. (H) Confirmation of URM1 depletion in human cells. (I) Viability of control and URM1-depleted human cell lines after treatment with MMS.