TY - JOUR
T1 - Alterations of signaling pathways in response to chemical perturbations used to measure mRNA decay rates in yeast
AU - Eshleman, Nichole
AU - Luo, Xiangxia
AU - Capaldi, Andrew
AU - Buchan, J. Ross
N1 - Funding Information:
This work was funded in part by a grant to J.R.B. from the National Institute of General Medical Sciences (NIGMS, R01-GM1145664), and a T32 training grant (5T32GM008659-18) awarded to the MCB department at the University of Arizona.
Publisher Copyright:
© 2020 Eshleman et al.
PY - 2020
Y1 - 2020
N2 - Assessing variations inmRNAstability 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.
AB - Assessing variations inmRNAstability 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.
KW - Hog1
KW - P-bodies
KW - PKC
KW - TORC1
KW - mRNA decay
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U2 - 10.1261/rna.072892.119
DO - 10.1261/rna.072892.119
M3 - Article
C2 - 31601735
AN - SCOPUS:85076793587
SN - 1355-8382
VL - 26
SP - 10
EP - 18
JO - RNA
JF - RNA
IS - 1
ER -