Here, we demonstrate that METTL2A and 2B interact with DALRD3, a previously uncharacterized protein harboring a putative anticodon-binding domain found in arginyl tRNA synthetases. Using gene editing, we show that loss of DARLD3 expression in human cells abolishes m3C formation in arginine tRNAs that can be rescued with re-expression of full-length DALRD3. Strikingly, we find that DALRD3 is mutated in human patients exhibiting a severe form of epileptic encephalopathy and that these patients no longer exhibit m3C modification in their arginine tRNAs. Altogether, this study uncovers an unanticipated role for the DALRD3 protein in the recognition of specific arginine tRNAs for METTL2-catalyzed m3C formation and implicates the m3C modification in proper neurological function.
