Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

doi:10.1371/journal.pone.0010618

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	Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos
	Zhang, Yuyan 1,3; Yang, Shaoli 1; Liu, Ming 1; Song, Chunxia 1; Wu, Ning 1,3; Ling, Peixue 4; Chu, Edward 2; Lin, Xiukun 1; Zhang, YY, Chinese Acad Sci, Inst Oceanol, Qingdao, Peoples R China
	2010-05-12
发表期刊	PLOS ONE
ISSN	1932-6203
卷号	5 期号:5 页码:e10618
文章类型	Article
摘要	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 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M 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.; 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 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M 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.
关键词	Binding Protein Autoregulation Degradation Expression Induction Mechanism Residues Cancer Cells
学科领域	Biology ; Multidisciplinary Sciences
DOI	10.1371/journal.pone.0010618
URL	查看原文
收录类别	SCI
语种	英语
WOS记录号	WOS:000277563400021
引用统计	被引频次：12[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1745
专题	实验海洋生物学重点实验室
通讯作者	Zhang, YY, Chinese Acad Sci, Inst Oceanol, Qingdao, Peoples R China
作者单位	1.Chinese Acad Sci, Inst Oceanol, Qingdao, Peoples R China 2.Yale Univ, Sch Med, Yale Canc Ctr, New Haven, CT USA 3.Chinese Acad Sci, Grad Sch, Beijing, Peoples R China 4.Inst Biopharmaceut Shandong Prov, Jinan, Peoples R China
推荐引用方式 GB/T 7714	Zhang, Yuyan,Yang, Shaoli,Liu, Ming,et al. Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos[J]. PLOS ONE,2010,5(5):e10618.
APA	Zhang, Yuyan.,Yang, Shaoli.,Liu, Ming.,Song, Chunxia.,Wu, Ning.,...&Zhang, YY, Chinese Acad Sci, Inst Oceanol, Qingdao, Peoples R China.(2010).Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos.PLOS ONE,5(5),e10618.
MLA	Zhang, Yuyan,et al."Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos".PLOS ONE 5.5(2010):e10618.

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