Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA
Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA
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DOI：
10.1093/nar/28.6.1381
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发表时间：
2000-03-15
影响因子：
14.9
通讯作者：
Chu, E
中科院分区：
文献类型：
--
作者：
Lin, XK;Parsels, LA;Chu, E
胸苷酸合酶 CTS) 通过与自身 mRNA 上的两个不同序列相互作用而发挥 RNA 结合蛋白的作用，其中一个位点位于人 TS mRNA 的 5' 上游区域，而第二个位点位于对应于 nt 434-634 的蛋白质编码区内。本文鉴定了对应于 nt 480-550 的 70 nt RNA 序列，其与 TS 蛋白的亲和力与全长相似。 TS mRNA 和 TS434-634 RNA，体外翻译研究证实该序列对于 TS 的翻译自动调节作用至关重要，为了记录体内生物学意义，将该区域内包含的 TS 序列克隆到荧光素酶报告质粒的 5' 端，并使用 H630 人结肠癌细胞进行瞬时转染实验，在用 p644/TS434-634 或p644/TS480-550 中，与仅用 p644 质粒转染的细胞相比，荧光素酶活性降低了 2.5 倍，根据 RNase 保护和 RT-PCR 分析确定，每种条件下的荧光素酶 mRNA 水平均相同。 TS 核糖核蛋白复合物的免疫沉淀揭示了转染的 H630 细胞中 TS 蛋白和 TS480-550 RNA 之间的直接相互作用。仅在用 p644/TS434-634 和 p644/TS480-550 转染的细胞中，用 5-氟尿苷处理导致荧光素酶活性增加近 2 倍。本研究在 TS mRNA 的蛋白质编码区鉴定出具有体外和体内翻译调节活性的 70 nt TS 反应元件。
Thymidylate synthase CTS) functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, One site is located in the 5'-upstream region of human TS mRNA while the second site is located within the protein coding region corresponding to nt 434-634, In this paper, a 70 nt RNA sequence, corresponding to nt 480-550, was identified that binds TS protein with an affinity similar to that of full-length TS mRNA and TS434-634 RNA, In vitro translation studies confirmed that this sequence is critical for the translational autoregulatory effects of TS, To document in vivo biological significance, TS sequences contained within this region were cloned onto the 5'-end of a luciferase reporter plasmid and transient transfection experiments were performed using H630 human colon cancer cells, In cells transfected with p644/TS434-634 or p644/TS480-550, luciferase activity was decreased 2.5-fold when compared to cells transfected with p644 plasmid alone, Luciferase mRNA levels were identical for each of these conditions as determined by RNase protection and RT-PCR analysis. Immunoprecipitation of TS ribonucleoprotein complexes revealed a direct interaction between TS protein and TS480-550 RNA in transfected H630 cells. Treatment with 5-fluorouridine resulted in a nearly 2-fold increase in luciferase activity only in cells transfected with p644/TS434-634 and p644/TS480-550. This study identifies a 70 nt TS response element in the protein coding region of TS mRNA with in vitro and in vivo translational regulatory activity.