Identification of the enzymes responsible for m2,2G and acp3U formation on cytosolic tRNA from insects and plants

Abstract

Posttranscriptional modification of tRNA is critical for efficient protein translation and proper cell growth, and defects in tRNA modifications are often associated with human disease. Although most of the enzymes required for eukaryotic tRNA modifications are known, many of these enzymes have not been identified and characterized in several model multicellular eukaryotes. Here, we present two related approaches to identify the genes required for tRNA modifications in multicellular organisms using primer extension assays with fluorescent oligonucleotides. To demonstrate the utility of these approaches we first use expression of exogenous genes in yeast to experimentally identify twoTRM1orthologs capable of formingN2,N2-dimethylguanosine (m2,2G) on residue 26 of cytosolic tRNA in the model plantArabidopsis thaliana. We also show that a predicted catalytic aspartate residue is required for function in each of the proteins. We next use RNA interference in culturedDrosophila melanogastercells to identify the gene required for m2,2G26formation on cytosolic tRNA. Additionally, using these approaches we experimentally identifyD.melanogastergeneCG10050as the corresponding ortholog of humanDTWD2, which encodes the protein required for formation of 3-amino-3-propylcarboxyuridine (acp3U) on residue 20a of cytosolic tRNA. We further show thatA.thalianageneAT2G41750can form acp3U20bon anA.thalianatRNA expressed in yeast cells, and that the aspartate and tryptophan residues in the DXTW motif of this protein are required for modification activity. These results demonstrate that these approaches can be used to study tRNA modification enzymes.