Fialcowitz-White Elizabeth J, Brewer Brandy Y, Ballin Jeff D, Willis Chris D, Toth Eric A, Wilson Gerald M
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
J Biol Chem. 2007 Jul 20;282(29):20948-59. doi: 10.1074/jbc.M701751200. Epub 2007 May 21.
The RNA-binding factor HuR is a ubiquitously expressed member of the Hu protein family that binds and stabilizes mRNAs containing AU-rich elements (AREs). Hu proteins share a common domain organization of two tandemly arrayed RNA recognition motifs (RRMs) near the N terminus, followed by a basic hinge domain and a third RRM near the C terminus. In this study, we engineered recombinant wild-type and mutant HuR proteins lacking affinity tags to characterize their ARE-binding properties. Using combinations of electrophoretic mobility shift and fluorescence anisotropy-based binding assays, we show that HuR can bind ARE substrates as small as 13 nucleotides with low nanomolar affinity, but forms cooperative oligomeric protein complexes on ARE substrates of at least 18 nucleotides in length. Analyses of deletion mutant proteins indicated that RRM3 does not contribute to high affinity recognition of ARE substrates, but is required for cooperative assembly of HuR oligomers on RNA. Finally, the hinge domain between RRM2 and RRM3 contributes significant binding energy to HuR.ARE complex formation in an ARE length-dependent manner. The hinge does not enhance RNA-binding activity by increased ion pair formation despite extensive positive charge within this region, and it does not thermodynamically stabilize protein folding. Together, the results define distinct roles for the HuR hinge and RRM3 domains in formation of cooperative HuR.ARE complexes in solution.
RNA结合因子HuR是Hu蛋白家族中一种广泛表达的成员，它能结合并稳定含有富AU元件（ARE）的mRNA。Hu蛋白在N端附近具有两个串联排列的RNA识别基序（RRM）的共同结构域组织，随后是一个碱性铰链结构域和C端附近的第三个RRM。在本研究中，我们构建了缺乏亲和标签的重组野生型和突变型HuR蛋白，以表征它们与ARE的结合特性。通过电泳迁移率变动分析和基于荧光偏振的结合分析相结合，我们发现HuR能以低纳摩尔亲和力结合小至13个核苷酸的ARE底物，但在长度至少为18个核苷酸的ARE底物上形成协同寡聚蛋白复合物。缺失突变蛋白的分析表明，RRM3对ARE底物的高亲和力识别没有贡献，但对于HuR寡聚体在RNA上的协同组装是必需的。最后，RRM2和RRM3之间的铰链结构域以ARE长度依赖性方式为HuR-ARE复合物的形成贡献了显著的结合能。尽管该区域有大量正电荷，但铰链并没有通过增加离子对的形成来增强RNA结合活性，并且它在热力学上也不稳定蛋白质折叠。总之，这些结果确定了HuR铰链和RRM3结构域在溶液中形成协同HuR-ARE复合物中的不同作用。
