Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA

Abstract

Uridine at the wobble position of tRNA is usually modified, and modification is required for accurate and efficient protein translation. In eukaryotes, wobble uridines are modified into 5-methoxycarbonylmethyluridine (mcm5U), 5-carbamoylmethyluridine (ncm5U) or derivatives thereof. Here, we demonstrate, both by in vitro and in vivo studies, that the Arabidopsis thaliana methyltransferase AT1G31600, denoted by us AtTRM9, is responsible for the final step in mcm5U formation, thus representing a functional homologue of the Saccharomyces cerevisiae Trm9 protein. We also show that the enzymatic activity of AtTRM9 depends on either one of two closely related proteins, AtTRM112a and AtTRM112b. Moreover, we demonstrate that AT1G36310, denoted AtALKBH8, is required for hydroxylation of mcm5U to (S)-mchm5U in tRNAGlyUCC, and has a function similar to the mammalian dioxygenase ALKBH8. Interestingly, atalkbh8 mutant plants displayed strongly increased levels of mcm5U, and also of mcm5Um, its 2′-O-ribose methylated derivative. This suggests that accumulated mcm5U is prone to further ribose methylation by a non-specialized mechanism, and may challenge the notion that the existence of mcm5U- and mcm5Um-containing forms of the selenocysteine-specific tRNASec in mammals reflects an important regulatory process. The present study reveals a role in for several hitherto uncharacterized Arabidopsis proteins in the formation of modified wobble uridines.