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Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In additio...

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Autores principales: Borland, Kayla, Diesend, Jan, Ito-Kureha, Taku, Heissmeyer, Vigo, Hammann, Christian, Buck, Amy H., Michalakis, Stylianos, Kellner, Stefanie
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356711/
https://www.ncbi.nlm.nih.gov/pubmed/30621251
http://dx.doi.org/10.3390/genes10010026
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author Borland, Kayla
Diesend, Jan
Ito-Kureha, Taku
Heissmeyer, Vigo
Hammann, Christian
Buck, Amy H.
Michalakis, Stylianos
Kellner, Stefanie
author_facet Borland, Kayla
Diesend, Jan
Ito-Kureha, Taku
Heissmeyer, Vigo
Hammann, Christian
Buck, Amy H.
Michalakis, Stylianos
Kellner, Stefanie
author_sort Borland, Kayla
collection PubMed
description Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.
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spelling pubmed-63567112019-02-04 Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis Borland, Kayla Diesend, Jan Ito-Kureha, Taku Heissmeyer, Vigo Hammann, Christian Buck, Amy H. Michalakis, Stylianos Kellner, Stefanie Genes (Basel) Article Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue. MDPI 2019-01-05 /pmc/articles/PMC6356711/ /pubmed/30621251 http://dx.doi.org/10.3390/genes10010026 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Borland, Kayla
Diesend, Jan
Ito-Kureha, Taku
Heissmeyer, Vigo
Hammann, Christian
Buck, Amy H.
Michalakis, Stylianos
Kellner, Stefanie
Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis
title Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis
title_full Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis
title_fullStr Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis
title_full_unstemmed Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis
title_short Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis
title_sort production and application of stable isotope-labeled internal standards for rna modification analysis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356711/
https://www.ncbi.nlm.nih.gov/pubmed/30621251
http://dx.doi.org/10.3390/genes10010026
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