Posttranslational tRNA modifications are essential for protein translation and proper cell growth. Defects in human tRNA modifications are associated with diseases such as cancer, type two diabetes, neurological disorders, and mitochondrial-linked disorders. The 3-amino-3-propylcarboxyuridine (acp3U) modification on tRNA is found in bacteria, plants, and animals, but not in yeast. DTWD proteins were recently identified as the enzymes responsible for acp3U modifications on eukaryotic tRNA. DTWD2 was shown to be responsible for the acp3U modification at residue 20a in human cells. We determined that DTWD2 forms acp3U20a in D. melanogaster cells. In A. thaliana, there are three homologs of human DTWD2 which are DTWD2A (AT2G41750), DTWD2B (AT5G54880), and TapT-like (AT1G03687). Our lab was able to identify that the aspartate and tryptophan residues of the DTWD2A protein in A. thaliana are required for modification activity and we were able to determine that it forms acp3U at position 20b on the tRNA. This was determined by expressing A. thaliana tRNA and A. thaliana DTWD2 genes in trm1Δ met22Δ yeast cells and testing by primer extension with fluorescent oligonucleotides. It is the current aim of this work to utilize the methods used to identify DTWD2A as the enzyme responsible for acp3U at position 20b to identify the substrates of the DTWD2B and Tap-T like proteins. From this research we hope to better understand gene expression in multiple model multicellular eukaryotes.

Identification of the enzyme required for the acp3U modification of plastid tRNA

ACS Spring Meeting 2021

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