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Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos
Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression o...
Autores principales: | , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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Public Library of Science
2010
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2868900/ https://www.ncbi.nlm.nih.gov/pubmed/20485548 http://dx.doi.org/10.1371/journal.pone.0010618 |
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author | Zhang, Yuyan Yang, Shaoli Liu, Ming Song, Chunxia Wu, Ning Ling, Peixue Chu, Edward Lin, Xiukun |
author_facet | Zhang, Yuyan Yang, Shaoli Liu, Ming Song, Chunxia Wu, Ning Ling, Peixue Chu, Edward Lin, Xiukun |
author_sort | Zhang, Yuyan |
collection | PubMed |
description | Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 µM 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 µM ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5′-UTR of TS mRNA, which corresponded to nt 13–32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors. |
format | Text |
id | pubmed-2868900 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-28689002010-05-19 Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos Zhang, Yuyan Yang, Shaoli Liu, Ming Song, Chunxia Wu, Ning Ling, Peixue Chu, Edward Lin, Xiukun PLoS One Research Article Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 µM 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 µM ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5′-UTR of TS mRNA, which corresponded to nt 13–32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors. Public Library of Science 2010-05-12 /pmc/articles/PMC2868900/ /pubmed/20485548 http://dx.doi.org/10.1371/journal.pone.0010618 Text en Zhang 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, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Zhang, Yuyan Yang, Shaoli Liu, Ming Song, Chunxia Wu, Ning Ling, Peixue Chu, Edward Lin, Xiukun Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos |
title | Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos |
title_full | Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos |
title_fullStr | Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos |
title_full_unstemmed | Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos |
title_short | Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos |
title_sort | interaction between thymidylate synthase and its cognate mrna in zebrafish embryos |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2868900/ https://www.ncbi.nlm.nih.gov/pubmed/20485548 http://dx.doi.org/10.1371/journal.pone.0010618 |
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