Tai Ningwen, Schmitz John C, Chen Tian-Min, O'Neill Michelle B, Chu Edward
Department of Medicine and Pharmacology, Developmental Therapeutic Program, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA.
Biochem Biophys Res Commun. 2008 May 9;369(3):795-800. doi: 10.1016/j.bbrc.2007.09.044. Epub 2007 Sep 21.
Human dihydrofolate reductase (DHFR) is a critical target in cancer chemotherapy. Previous studies showed that an 82-nt RNA fragment within the DHFR mRNA protein-coding region functions as a DHFR cis-acting response element. In this study, we further investigated the key elements contained within this sequence that are required for the DHFR mRNA-DHFR protein interaction. Using enzymatic foot-printing assays and RNA-binding experiments, we isolated a 27-nt sequence (DHFR27, corresponding to nts 407-433), which bound with high affinity and specificity to human DHFR to form a ribonucleoprotein complex. In vivo transient transfection experiments using a luciferase reporter system revealed that DHFR27 RNA could repress the luciferase expression in a DHFR-dependent manner when placed upstream of luciferase mRNA. This work provides new insights into the essential molecular elements that mediate RNA-protein interactions.
人二氢叶酸还原酶（DHFR）是癌症化疗中的关键靶点。先前的研究表明，DHFR mRNA蛋白质编码区域内一个82个核苷酸的RNA片段作为DHFR顺式作用反应元件发挥作用。在本研究中，我们进一步研究了该序列中DHFR mRNA与DHFR蛋白相互作用所需的关键元件。通过酶足迹分析和RNA结合实验，我们分离出一个27个核苷酸的序列（DHFR27，对应于第407 - 433位核苷酸），它与人DHFR以高亲和力和特异性结合形成核糖核蛋白复合物。使用荧光素酶报告系统进行的体内瞬时转染实验表明，当DHFR27 RNA置于荧光素酶mRNA上游时，它可以以DHFR依赖的方式抑制荧光素酶的表达。这项工作为介导RNA - 蛋白质相互作用的基本分子元件提供了新的见解。
