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Identification of the enzymes responsible for m(2,2)G and acp(3)U formation on cytosolic tRNA from insects and plants
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...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704012/ https://www.ncbi.nlm.nih.gov/pubmed/33253256 http://dx.doi.org/10.1371/journal.pone.0242737 |
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author | Funk, Holly M. Zhao, Ruoxia Thomas, Maggie Spigelmyer, Sarah M. Sebree, Nichlas J. Bales, Regan O. Burchett, Jamison B. Mamaril, Justen B. Limbach, Patrick A. Guy, Michael P. |
author_facet | Funk, Holly M. Zhao, Ruoxia Thomas, Maggie Spigelmyer, Sarah M. Sebree, Nichlas J. Bales, Regan O. Burchett, Jamison B. Mamaril, Justen B. Limbach, Patrick A. Guy, Michael P. |
author_sort | Funk, Holly M. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7704012 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-77040122020-12-03 Identification of the enzymes responsible for m(2,2)G and acp(3)U formation on cytosolic tRNA from insects and plants Funk, Holly M. Zhao, Ruoxia Thomas, Maggie Spigelmyer, Sarah M. Sebree, Nichlas J. Bales, Regan O. Burchett, Jamison B. Mamaril, Justen B. Limbach, Patrick A. Guy, Michael P. PLoS One Research Article 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. Public Library of Science 2020-11-30 /pmc/articles/PMC7704012/ /pubmed/33253256 http://dx.doi.org/10.1371/journal.pone.0242737 Text en © 2020 Funk et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Funk, Holly M. Zhao, Ruoxia Thomas, Maggie Spigelmyer, Sarah M. Sebree, Nichlas J. Bales, Regan O. Burchett, Jamison B. Mamaril, Justen B. Limbach, Patrick A. Guy, Michael P. Identification of the enzymes responsible for m(2,2)G and acp(3)U formation on cytosolic tRNA from insects and plants |
title | Identification of the enzymes responsible for m(2,2)G and acp(3)U formation on cytosolic tRNA from insects and plants |
title_full | Identification of the enzymes responsible for m(2,2)G and acp(3)U formation on cytosolic tRNA from insects and plants |
title_fullStr | Identification of the enzymes responsible for m(2,2)G and acp(3)U formation on cytosolic tRNA from insects and plants |
title_full_unstemmed | Identification of the enzymes responsible for m(2,2)G and acp(3)U formation on cytosolic tRNA from insects and plants |
title_short | Identification of the enzymes responsible for m(2,2)G and acp(3)U formation on cytosolic tRNA from insects and plants |
title_sort | identification of the enzymes responsible for m(2,2)g and acp(3)u formation on cytosolic trna from insects and plants |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704012/ https://www.ncbi.nlm.nih.gov/pubmed/33253256 http://dx.doi.org/10.1371/journal.pone.0242737 |
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