Park S, Myszka D G, Yu M, Littler S J, Laird-Offringa I A
Norris Cancer Center, University of Southern California, Keck School of Medicine, Los Angeles 90089-9176, USA.
Mol Cell Biol. 2000 Jul;20(13):4765-72. doi: 10.1128/MCB.20.13.4765-4772.2000.
Human neuron-specific RNA-binding protein HuD belongs to the family of Hu proteins and consists of two N-terminal RNA recognition motifs (RRM1 and -2), a hinge region, and a C-terminal RRM (RRM3). Hu proteins can bind to AU-rich elements in the 3' untranslated regions of unstable mRNAs, causing the stabilization of certain transcripts. We have studied the interaction between HuD and prototype mRNA instability elements of the sequence UU(AUUU)(n)AUU using equilibrium methods and real-time kinetics (surface plasmon resonance using a BIACORE). We show that a single molecule of HuD requires at least three AUUU repeats to bind tightly to the RNA. Deletion of RRM1 reduced the K(d) by 2 orders of magnitude and caused a decrease in the association rate and a strong increase in the dissociation rate of the RNA-protein complex, as expected when a critical RNA-binding domain is removed. In contrast, deletion of either RRM2 or -3, which only moderately reduced the affinity, caused marked increases in the association and dissociation rates. The slower binding and stabilization of the complex observed in the presence of all three RRMs suggest that a change in the tertiary structure occurs during binding. The individual RRMs bind poorly to the RNA (RRM1 binds with micromolar affinity, while the affinities of RRM2 and -3 are in the millimolar range). However, the combination of RRM1 and either RRM2 or RRM3 in the context of the protein allows binding with a nanomolar affinity. Thus, the three RRMs appear to cooperate not only to increase the affinity of the interaction but also to stabilize the formed complex. Kinetic effects, similar to those described here, could play a role in RNA binding by many multi-RRM proteins and may influence the competition between proteins for RNA-binding sites and the ability of RNA-bound proteins to be transported intracellularly.
人类神经元特异性RNA结合蛋白HuD属于Hu蛋白家族，由两个N端RNA识别基序（RRM1和RRM2）、一个铰链区和一个C端RRM（RRM3）组成。Hu蛋白可与不稳定mRNA的3'非翻译区富含AU的元件结合，从而使某些转录本得以稳定。我们使用平衡方法和实时动力学（利用BIACORE进行表面等离子体共振）研究了HuD与序列UU(AUUU)(n)AUU的原型mRNA不稳定元件之间的相互作用。我们发现，单个HuD分子至少需要三个AUUU重复序列才能与RNA紧密结合。正如去除关键RNA结合结构域时所预期的那样，RRM1的缺失使解离常数（K(d)）降低了2个数量级，并导致RNA-蛋白质复合物的结合速率降低以及解离速率大幅增加。相比之下，RRM2或RRM3的缺失仅适度降低了亲和力，但却导致结合和解离速率显著增加。在所有三个RRM存在的情况下观察到的复合物结合和稳定较慢，这表明结合过程中发生了三级结构的变化。单个RRM与RNA的结合能力较差（RRM1以微摩尔亲和力结合，而RRM2和RRM3的亲和力在毫摩尔范围内）。然而，在蛋白质背景下，RRM1与RRM2或RRM3的组合能够以纳摩尔亲和力结合。因此，这三个RRM似乎不仅协同作用以增加相互作用的亲和力，还能稳定形成的复合物。类似于本文所述的动力学效应可能在许多多RRM蛋白的RNA结合中发挥作用，并可能影响蛋白质之间对RNA结合位点的竞争以及RNA结合蛋白在细胞内运输的能力。
