Human AlkB Homolog ABH8 Is a tRNA Methyltransferase Required for Wobble Uridine Modification and DNA Damage Survival

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02138, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 03/2010; 30(10):2449-59. DOI: 10.1128/MCB.01604-09
Source: PubMed


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 (mcm5U) 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 mcm5U and that purified ABH8 complexes methylate RNA in vitro. Significantly, ABH8 depletion in human cells reduces endogenous levels of mcm5U 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.

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    • "In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm5U), or derivatives thereof, in the wobble position, which are believed to restrict wobbling and improve translational efficiency. Recently, the ALKBH8 member of the AlkB protein family was reported to methylate 5-carboxymethyluridine (cm5U) and 5-carboxymethyl-2-thiouridine (cm5s2U) and, thus, participate in the maturation of the tRNASec, tRNAGlu and tRNAArg [47, 48]. It is interesting to note that AlkB proteins were initially characterised for their ability to remove alkylation damage in DNA [49]. "
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    ABSTRACT: The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.
    Cellular and Molecular Life Sciences CMLS 02/2014; 71(13). DOI:10.1007/s00018-014-1562-y · 5.81 Impact Factor
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    • "The results presented in this study provide comprehensive profiles of the proteins required for cellular recovery after exposure to a range of alkylating, oxidizing and also non-genotoxic compounds. Many of the identified toxicity-modulating proteins and pathways, such as the DNA repair cluster needed after alkylating agent treatment, have already been heavily studied [28]. However, novel pathways needed for recovery also came to light, such as the requirement of aromatic amino acid synthesis (reviewed in [29]) following exposure to the oxidizing agents menadione (MEN) and hydroquinone (HYQ). "
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    ABSTRACT: Toxicity screening of compounds provides a means to identify compounds harmful for human health and the environment. Here, we further develop the technique of genomic phenotyping to improve throughput while maintaining specificity. We exposed cells to eight different compounds that rely on different modes of action: four genotoxic alkylating (methyl methanesulfonate (MMS), N-Methyl-N-nitrosourea (MNU), N,N'-bis(2-chloroethyl)-N-nitroso-urea (BCNU), N-ethylnitrosourea (ENU)), two oxidizing (2-methylnaphthalene-1,4-dione (menadione, MEN), benzene-1,4-diol (hydroquinone, HYQ)), and two non-genotoxic (methyl carbamate (MC) and dimethyl sulfoxide (DMSO)) compounds. A library of S. cerevisiae 4,852 deletion strains, each identifiable by a unique genetic 'barcode', were grown in competition; at different time points the ratio between the strains was assessed by quantitative high throughput 'barcode' sequencing. The method was validated by comparison to previous genomic phenotyping studies and 90% of the strains identified as MMS-sensitive here were also identified as MMS-sensitive in a much lower throughput solid agar screen. The data provide profiles of proteins and pathways needed for recovery after both genotoxic and non-genotoxic compounds. In addition, a novel role for aromatic amino acids in the recovery after treatment with oxidizing agents was suggested. The role of aromatic acids was further validated; the quinone subgroup of oxidizing agents were extremely toxic in cells where tryptophan biosynthesis was compromised.
    PLoS ONE 09/2013; 8(9):e73736. DOI:10.1371/journal.pone.0073736 · 3.23 Impact Factor
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    • "Interestingly, Sua5/YrdC mutants are also defective in telomere length homeostasis in the budding yeast (14,15). As the posttranscriptional modification of tRNAs can influence seemingly unrelated cellular processes such as the DNA damage response and apoptosis (16–18) by modulating tRNA structure and function (19–21), it is possible that the pleiotropic effects of KEOPS and Sua5 mutations may be due entirely to defects in t6A metabolism, although this hypothesis has yet to be proven formally. "
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    ABSTRACT: The universally conserved Kae1/Qri7/YgjD and Sua5/YrdC protein families have been implicated in growth, telomere homeostasis, transcription and the N6-threonylcarbamoylation (t(6)A) of tRNA, an essential modification required for translational fidelity by the ribosome. In bacteria, YgjD orthologues operate in concert with the bacterial-specific proteins YeaZ and YjeE, whereas in archaeal and eukaryotic systems, Kae1 operates as part of a larger macromolecular assembly called KEOPS with Bud32, Cgi121, Gon7 and Pcc1 subunits. Qri7 orthologues function in the mitochondria and may represent the most primitive member of the Kae1/Qri7/YgjD protein family. In accordance with previous findings, we confirm that Qri7 complements Kae1 function and uncover that Qri7 complements the function of all KEOPS subunits in growth, t(6)A biosynthesis and, to a partial degree, telomere maintenance. These observations suggest that Kae1 provides a core essential function that other subunits within KEOPS have evolved to support. Consistent with this inference, Qri7 alone is sufficient for t(6)A biosynthesis with Sua5 in vitro. In addition, the 2.9 Å crystal structure of Qri7 reveals a simple homodimer arrangement that is supplanted by the heterodimerization of YgjD with YeaZ in bacteria and heterodimerization of Kae1 with Pcc1 in KEOPS. The partial complementation of telomere maintenance by Qri7 hints that KEOPS has evolved novel functions in higher organisms.
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