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Identification of the enzymes responsible for m2,2G and acp3U formation on cytosolic tRNA from insects and plants
Author(s) -
Holly M. Funk,
Ruoxia Zhao,
Maggie Thomas,
Sarah M. Spigelmyer,
Nichlas J. Sebree,
Regan O. Bales,
Jamison B. Burchett,
Justen B. Mamaril,
Patrick A. Limbach,
Michael P. Guy
Publication year - 2020
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0242737
Subject(s) - transfer rna , biology , multicellular organism , biochemistry , gene , drosophila melanogaster , rna , translation (biology) , arabidopsis thaliana , messenger rna , mutant
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 two TRM1 orthologs capable of forming N2 , N2 -dimethylguanosine (m 2,2 G) on residue 26 of cytosolic tRNA in the model plant Arabidopsis 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 cultured Drosophila melanogaster cells to identify the gene required for m 2,2 G 26 formation on cytosolic tRNA. Additionally, using these approaches we experimentally identify D . melanogaster gene CG10050 as the corresponding ortholog of human DTWD2 , which encodes the protein required for formation of 3-amino-3-propylcarboxyuridine (acp 3 U) on residue 20a of cytosolic tRNA. We further show that A . thaliana gene AT2G41750 can form acp 3 U 20b on an A . thaliana tRNA 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.

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