Xiao X, Tang Y S, Mackins J Y, Sun X L, Jayaram H N, Hansen D K, Antony A C
Division of Hematology-Oncology, Department of Medicine, and the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and the Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 46202-5254, USA.
J Biol Chem. 2001 Nov 2;276(44):41510-7. doi: 10.1074/jbc.M106824200. Epub 2001 Aug 29.
The interaction of an 18-base cis-element in the 5'-untranslated region of human folate receptor (FR)-alpha mRNA with a cytosolic trans-factor protein is critical for the translation of FR (Sun, X.-L., and Antony, A. C. (1996) J. Biol. Chem. 271, 25539-25547). This trans-factor was isolated to apparent homogeneity as a 43- and 38-kDa doublet from human placenta using poly(U)-Sepharose, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-elution as major purification steps. Amino acid microsequencing of two cyanogen bromide-generated peptide fragments of the 43-kDa trans-factor revealed complete identity with 43-kDa heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1). Purified specific rabbit anti-hnRNP E1 peptide antibodies (generated against a synthetic oligopeptide that was not represented in microsequenced peptides of the trans-factor) also recognized the purified trans-factor on Western blots. Conversely, the 18-base FR RNA cis-element also bound hnRNP E1 protein on Northwestern blots. Moreover, a 19-base RNA cis-element in the 3'-untranslated region of 15-lipoxygenase mRNA that is known to bind hnRNP E1 also interacted with placental 43-kDa trans-factor. In addition, several murine tissues containing a hnRNP E1-related protein (also known as alphaCP-1) readily interacted with the 18-base FR RNA cis-element. Finally, anti-hnRNP E1 antibodies specifically inhibited translation of FR in vitro in a dose-dependent manner, and the antibody effect could be reversed in a dose-dependent manner by either purified trans-factor or hnRNP E1. Collectively, the data favor identity of the FR mRNA-binding trans-factor and hnRNP E1, confirm its critical role in the translation of FR, and highlight yet another role of multifunctional hnRNP E1 in eukaryotic mRNA regulation.
人叶酸受体（FR）-α mRNA的5'-非翻译区中一个18碱基顺式元件与一种胞质反式作用因子蛋白的相互作用对FR的翻译至关重要（Sun，X.-L.，和Antony，A. C.（1996）《生物化学杂志》271，25539 - 25547）。该反式作用因子通过聚（U）-琼脂糖从人胎盘中分离至表观均一，呈现为43 kDa和38 kDa的双峰，随后以制备性十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和电洗脱作为主要纯化步骤。对43 kDa反式作用因子的两个溴化氰生成的肽片段进行氨基酸微序列分析，结果显示与43 kDa异质性核核糖核蛋白E1（hnRNP E1）完全相同。纯化的特异性兔抗hnRNP E1肽抗体（针对反式作用因子微序列分析肽中未出现的合成寡肽产生）在蛋白质印迹法中也能识别纯化的反式作用因子。相反，18碱基的FR RNA顺式元件在蛋白质 - RNA杂交印迹法中也能结合hnRNP E1蛋白。此外，15 - 脂氧合酶mRNA的3'-非翻译区中已知能结合hnRNP E1的一个19碱基RNA顺式元件也与胎盘43 kDa反式作用因子相互作用。另外，几种含有hnRNP E1相关蛋白（也称为αCP - 1）的鼠组织很容易与18碱基的FR RNA顺式元件相互作用。最后，抗hnRNP E1抗体在体外以剂量依赖的方式特异性抑制FR的翻译，并且纯化的反式作用因子或hnRNP E1能以剂量依赖的方式逆转抗体的作用。总体而言，这些数据支持FR mRNA结合反式作用因子与hnRNP E1的一致性，证实了其在FR翻译中的关键作用，并突出了多功能hnRNP E1在真核生物mRNA调控中的又一作用。
