PUBLICATION

Interaction between thymidylate synthase and its cognate mRNA in zebrafish embryos

Authors

Zhang, Y., Yang, S., Liu, M., Song, C., Wu, N., Ling, P., Chu, E., and Lin, X.

ID

ZDB-PUB-100525-10

Date

2010

Source

PLoS One 5(5): e10618 (Journal)

Registered Authors

Keywords

Zebrafish, Embryos, Messenger RNA, RNA structure, RNA stem-loop structure, RNA-binding proteins, Protein interactions, Protein translation

MeSH Terms

Mutation/genetics
Thiophenes/pharmacology
Protein Binding/drug effects
RNA, Messenger/chemistry
RNA, Messenger/genetics
RNA, Messenger/metabolism
Time Factors
Quinazolines/pharmacology
Molecular Sequence Data
Animals
Gene Expression Regulation, Enzymologic/drug effects
Base Sequence
Protein Biosynthesis/drug effects
Protein Stability/drug effects
Fluorouracil/pharmacology
Zebrafish/embryology*
Zebrafish/genetics*
Embryo, Nonmammalian/drug effects
Embryo, Nonmammalian/enzymology*
Nucleic Acid Conformation
Gene Expression Profiling
RNA Stability/drug effects
Thymidylate Synthase/genetics*
Thymidylate Synthase/metabolism*

PubMed

20485548 Full text @ PLoS One

Abstract

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 microM 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 microM 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.

Genes / Markers

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