Tai Ningwen, Schmitz John C, Chen Tian-min, Chu Edward
Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA.
Biochem J. 2004 Mar 15;378(Pt 3):999-1006. doi: 10.1042/BJ20031396.
Previous studies have shown that human DHFR (dihydrofolate reductase), in addition to its critical role in DNA biosynthesis, functions as an RNA-binding protein. The interaction between DHFR and its own mRNA results in translational repression. In this study, we characterized the cis-acting elements on human DHFR mRNA that are required for the DHFR mRNA-DHFR protein interaction. Using a series of gel-shift and nitrocellulose filter-binding assays, a 164 nt RNA sequence, corresponding to nt 401-564, was identified within the coding region that binds to DHFR protein with an affinity similar to that of full-length DHFR mRNA. To document in vivo biological activity, various DHFR sequences contained within the coding region were cloned on to the 5' end of a luciferase reporter plasmid, and transient transfection experiments were performed using human colon cancer RKO cells. In cells transfected with p644/DHFR:401-564, luciferase activity was decreased by 50% when compared with cells transfected with the p644 plasmid alone. Luciferase mRNA levels were identical under each of these conditions, as determined by Northern-blot analysis. In cells transfected with p644/DHFR:401-564, luciferase activity was restored to almost 100% of control when cells were treated with the antifolate analogue methotrexate or with a short-interfering RNA targeting DHFR mRNA. These findings provide evidence that the DHFR 401-564 sequence is a DHFR-response element. In vitro and in vivo studies further localized this cis-element to an 82 nt sequence corresponding to nt 401-482. This work provides new insights into critical elements that mediate RNA-protein interactions.
先前的研究表明，人类二氢叶酸还原酶（DHFR）除了在DNA生物合成中发挥关键作用外，还作为一种RNA结合蛋白发挥作用。DHFR与其自身mRNA之间的相互作用导致翻译抑制。在本研究中，我们对人类DHFR mRNA上DHFR mRNA-DHFR蛋白相互作用所需的顺式作用元件进行了表征。通过一系列凝胶迁移和硝酸纤维素滤膜结合试验，在编码区内鉴定出一个164 nt的RNA序列，对应于第401-564位核苷酸，该序列与DHFR蛋白结合的亲和力与全长DHFR mRNA相似。为了证明体内生物学活性，将编码区内包含的各种DHFR序列克隆到荧光素酶报告质粒的5'端，并使用人结肠癌RKO细胞进行瞬时转染实验。与单独转染p644质粒的细胞相比，转染p644/DHFR:401-564的细胞中荧光素酶活性降低了50%。通过Northern印迹分析确定，在每种条件下荧光素酶mRNA水平相同。在用抗叶酸类似物甲氨蝶呤或靶向DHFR mRNA的短干扰RNA处理细胞后，转染p644/DHFR:401-564的细胞中荧光素酶活性恢复到对照的近100%。这些发现提供了证据，证明DHFR 401-564序列是一个DHFR反应元件。体外和体内研究进一步将该顺式元件定位到一个82 nt的序列，对应于第401-482位核苷酸。这项工作为介导RNA-蛋白质相互作用的关键元件提供了新的见解。
