The formation of diverse tRNA modifications requires many cellular pathways involving numerous enzymes. In mammals, several putative tRNA methyltransferases have been identified by sequence homology with bacterial or yeast proteins (11
), but the majority of these enzymes remain uncharacterized, and their requirement in tRNA methylation awaits verification. Interestingly, the DNMT2 enzyme of the DNA cytosine methyltransferase family is one of the few known mammalian proteins with a confirmed role in tRNA methylation (20
). DNMT2 methylates tRNAAsp
using a DNA methyltransferase-like catalytic mechanism, but the function of this modification is unknown. Here, we show that human ABH8 protein contains an active RNA methyltransferase domain that is essential for maintaining the levels of a critical tRNA modification in human cells.
In addition to tRNA methylation, the DNA damage sensitivity phenotype of ABH8-depleted cells suggests a role for ABH8 in DNA damage survival through the regulation of tRNA modification, potentially in concert with any oxidative demethylase activity conferred by the AlkB dioxygenase domain of ABH8. Support for this notion comes first from our confirmation that ABH8 localizes primarily to the cytoplasm and second from our finding that ABH8 protects against bleomycin, a damaging agent that does not methylate nucleic acids. Future studies will be devoted to understanding the relative contributions of the ABH8 dioxygenase and methyltransferase domains to survival after DNA damage.
In yeast, mRNA transcripts encoding stress response proteins are significantly enriched for codons decoded by tRNAs containing the Trm9p-dependent mcm5
U wobble modification (6
). The mcm5
U wobble base generated by Trm9 has been shown to modulate tRNA-mRNA pairing and enhance binding with the cognate codon (2
). Thus, tRNA modification enzymes can directly regulate the translation of specific proteins by modulating a subset of codon-anticodon interactions. Our results indicate that a conserved mechanism in which ABH8-catalyzed tRNA modification regulates the translation of specific proteins that are essential for surviving genotoxic stress could be operating in human cells. Notably, previous reports have demonstrated the preferential translation of mRNAs encoding DNA damage response and repair proteins in human cells after cellular stress (37
). Due to the presence of mcm5
U in the wobble position of tRNASec(UGA)
, ABH8 could also modulate the specific translation of the entire repertoire of selenocysteine proteins. It will be of great interest to identify the specific proteins whose translation is regulated by tRNA modification catalyzed by ABH8.
The fusion of a putative dioxygenase motif to an active methyltransferase domain in ABH8 suggests the intriguing possibility of reversible RNA modification. While the substrates of the ABH8 dioxygenase motif remain to be discovered, ABH8 could demethylate wobble uridines or other modified nucleosides in tRNA under particular conditions. Alternatively, given the RNA repair capacity demonstrated by certain AlkB proteins (1
), ABH8 could reverse aberrant methylation of tRNA, either caused by spurious methylase activity of the methyltransferase domain or induced by endogenous or exogenous alkylating agents. Alkylating agents have been shown to alter or inactivate mRNA, tRNA, or rRNA function, leading to ribosome stalling, miscoding, or translational blocks, with the production of truncated or mutant polypeptides (32
). While TRiC could function as a chaperone for the correct folding of a multidomain protein such as ABH8, it could also represent a mechanism by which incompletely synthesized translation products caused by a stalled ribosome are stabilized by TRiC while ABH8 rescues the damaged RNAs.
The potential substrates of ABH8 could also encompass protein targets since the AlkB enzymes belong to a superfamily of iron-dependent dioxygenases that include the JmjC domain histone demethylases, which have an enzymatic mechanism identical to that of AlkB to demethylate lysine residues in histone proteins (35
). In addition, oxidative cleavage of amino acids by iron-dependent dioxygenases such as cysteine dioxygenase plays important roles in maintaining proper levels of particular amino acids (25
). In the case of ABH8, oxidative cleavage of specific amino acids or proteins could occur if their levels are upregulated under particular conditions of stress or damage.
Of significance, ABH8 is highly expressed in many urothelial cancers, with a positive correlation between ABH8 expression and high-grade, invasive carcinomas (46
). Consistent with these observations, silencing of ABH8 significantly suppresses the angiogenesis and growth of bladder cancers in vivo
). Thus, tRNA modifications catalyzed by methyltransferases such as ABH8 could be an important factor in the growth and survival of both normal and transformed cells.