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Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics
The elucidation of the biological significance of RNA post-transcriptional modifications is hampered by the dearth of effective high-throughput sequencing approaches for detecting, locating, and tracking their levels as a function of predetermined experimental factors. With the goal of confronting t...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478354/ https://www.ncbi.nlm.nih.gov/pubmed/25995446 http://dx.doi.org/10.1261/rna.049429.114 |
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author | Rose, Rebecca E. Quinn, Ryan Sayre, Jackie L. Fabris, Daniele |
author_facet | Rose, Rebecca E. Quinn, Ryan Sayre, Jackie L. Fabris, Daniele |
author_sort | Rose, Rebecca E. |
collection | PubMed |
description | The elucidation of the biological significance of RNA post-transcriptional modifications is hampered by the dearth of effective high-throughput sequencing approaches for detecting, locating, and tracking their levels as a function of predetermined experimental factors. With the goal of confronting this knowledge gap, we devised a strategy for completing global surveys of all ribonucleotide modifications in a cell, which is based on the analysis of whole cell extracts by direct infusion electrospray ionization mass spectrometry (ESI-MS). Our approach eschews chromatographic separation to promote instead the direct application of MS techniques capable of providing detection, differentiation, and quantification of post-transcriptional modifications (PTMs) in complex ribonucleotide mixtures. Accurate mass analysis was used to carry out database-aided identification of PTMs, whereas multistep tandem mass spectrometry (MS(n)) and consecutive reaction monitoring (CRM) provided the necessary structural corroboration. We demonstrated that heat-map plots afforded by ion mobility spectrometry mass spectrometry (IMS-MS) can provide comprehensive modification profiles that are unique for different cell types and metabolic states. We showed that isolated tRNA samples can be used as controlled sources of PTMs in standard-additions quantification. Intrinsic internal standards enable direct comparisons of heat-maps obtained under different experimental conditions, thus offering the opportunity to evaluate the global effects of such conditions on the expression levels of all PTMs simultaneously. This type of comparative analysis will be expected to support the investigation of the system biology of RNA modifications, which will be aimed at exploring mutual correlations of their expression levels and providing new valuable insights into their biological significance. |
format | Online Article Text |
id | pubmed-4478354 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-44783542016-07-01 Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics Rose, Rebecca E. Quinn, Ryan Sayre, Jackie L. Fabris, Daniele RNA Methods The elucidation of the biological significance of RNA post-transcriptional modifications is hampered by the dearth of effective high-throughput sequencing approaches for detecting, locating, and tracking their levels as a function of predetermined experimental factors. With the goal of confronting this knowledge gap, we devised a strategy for completing global surveys of all ribonucleotide modifications in a cell, which is based on the analysis of whole cell extracts by direct infusion electrospray ionization mass spectrometry (ESI-MS). Our approach eschews chromatographic separation to promote instead the direct application of MS techniques capable of providing detection, differentiation, and quantification of post-transcriptional modifications (PTMs) in complex ribonucleotide mixtures. Accurate mass analysis was used to carry out database-aided identification of PTMs, whereas multistep tandem mass spectrometry (MS(n)) and consecutive reaction monitoring (CRM) provided the necessary structural corroboration. We demonstrated that heat-map plots afforded by ion mobility spectrometry mass spectrometry (IMS-MS) can provide comprehensive modification profiles that are unique for different cell types and metabolic states. We showed that isolated tRNA samples can be used as controlled sources of PTMs in standard-additions quantification. Intrinsic internal standards enable direct comparisons of heat-maps obtained under different experimental conditions, thus offering the opportunity to evaluate the global effects of such conditions on the expression levels of all PTMs simultaneously. This type of comparative analysis will be expected to support the investigation of the system biology of RNA modifications, which will be aimed at exploring mutual correlations of their expression levels and providing new valuable insights into their biological significance. Cold Spring Harbor Laboratory Press 2015-07 /pmc/articles/PMC4478354/ /pubmed/25995446 http://dx.doi.org/10.1261/rna.049429.114 Text en © 2015 Rose et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. |
spellingShingle | Methods Rose, Rebecca E. Quinn, Ryan Sayre, Jackie L. Fabris, Daniele Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics |
title | Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics |
title_full | Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics |
title_fullStr | Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics |
title_full_unstemmed | Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics |
title_short | Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics |
title_sort | profiling ribonucleotide modifications at full-transcriptome level: a step toward ms-based epitranscriptomics |
topic | Methods |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478354/ https://www.ncbi.nlm.nih.gov/pubmed/25995446 http://dx.doi.org/10.1261/rna.049429.114 |
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