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Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast

Assessing variations in mRNA stability typically involves inhibiting transcription either globally or in a gene-specific manner. Alternatively, mRNA pulse-labeling strategies offer a means to calculate mRNA stability without inhibiting transcription. However, key stress-responsive cell signaling pat...

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Autores principales: Eshleman, Nichole, Luo, Xiangxia, Capaldi, Andrew, Buchan, J. Ross
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913126/
https://www.ncbi.nlm.nih.gov/pubmed/31601735
http://dx.doi.org/10.1261/rna.072892.119
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author Eshleman, Nichole
Luo, Xiangxia
Capaldi, Andrew
Buchan, J. Ross
author_facet Eshleman, Nichole
Luo, Xiangxia
Capaldi, Andrew
Buchan, J. Ross
author_sort Eshleman, Nichole
collection PubMed
description Assessing variations in mRNA stability typically involves inhibiting transcription either globally or in a gene-specific manner. Alternatively, mRNA pulse-labeling strategies offer a means to calculate mRNA stability without inhibiting transcription. However, key stress-responsive cell signaling pathways, which affect mRNA stability, may themselves be perturbed by the approaches used to measure mRNA stability, leading to artifactual results. Here, we have focused on common strategies to measure mRNA half-lives in yeast and determined that commonly used transcription inhibitors thiolutin and 1,10 phenanthroline inhibit TORC1 signaling, PKC signaling, and partially activate HOG signaling. Additionally, 4-thiouracil (4tU), a uracil analog used in mRNA pulse-labeling approaches, modestly induces P-bodies, mRNA–protein granules implicated in storage and decay of nontranslating mRNA. Thiolutin also induces P-bodies, whereas phenanthroline has no effect. Doxycycline, which controls “Tet On/Tet Off” regulatable promoters, shows no impact on the above signaling pathways or P-bodies. In summary, our data argues that broad-acting transcriptional inhibitors are problematic for determining mRNA half-life, particularly if studying the impacts of the TORC1, HOG, or PKC pathway on mRNA stability. Regulatable promoter systems are a preferred approach for individual mRNA half-life studies, with 4tU labeling representing a good approach to global mRNA half-life analysis, despite modestly inducing P-bodies.
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spelling pubmed-69131262021-01-01 Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast Eshleman, Nichole Luo, Xiangxia Capaldi, Andrew Buchan, J. Ross RNA Letter to the Editor Assessing variations in mRNA stability typically involves inhibiting transcription either globally or in a gene-specific manner. Alternatively, mRNA pulse-labeling strategies offer a means to calculate mRNA stability without inhibiting transcription. However, key stress-responsive cell signaling pathways, which affect mRNA stability, may themselves be perturbed by the approaches used to measure mRNA stability, leading to artifactual results. Here, we have focused on common strategies to measure mRNA half-lives in yeast and determined that commonly used transcription inhibitors thiolutin and 1,10 phenanthroline inhibit TORC1 signaling, PKC signaling, and partially activate HOG signaling. Additionally, 4-thiouracil (4tU), a uracil analog used in mRNA pulse-labeling approaches, modestly induces P-bodies, mRNA–protein granules implicated in storage and decay of nontranslating mRNA. Thiolutin also induces P-bodies, whereas phenanthroline has no effect. Doxycycline, which controls “Tet On/Tet Off” regulatable promoters, shows no impact on the above signaling pathways or P-bodies. In summary, our data argues that broad-acting transcriptional inhibitors are problematic for determining mRNA half-life, particularly if studying the impacts of the TORC1, HOG, or PKC pathway on mRNA stability. Regulatable promoter systems are a preferred approach for individual mRNA half-life studies, with 4tU labeling representing a good approach to global mRNA half-life analysis, despite modestly inducing P-bodies. Cold Spring Harbor Laboratory Press 2020-01 /pmc/articles/PMC6913126/ /pubmed/31601735 http://dx.doi.org/10.1261/rna.072892.119 Text en © 2020 Eshleman 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 Letter to the Editor
Eshleman, Nichole
Luo, Xiangxia
Capaldi, Andrew
Buchan, J. Ross
Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast
title Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast
title_full Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast
title_fullStr Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast
title_full_unstemmed Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast
title_short Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast
title_sort alterations of signaling pathways in response to chemical perturbations used to measure mrna decay rates in yeast
topic Letter to the Editor
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913126/
https://www.ncbi.nlm.nih.gov/pubmed/31601735
http://dx.doi.org/10.1261/rna.072892.119
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