Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
Nucleic Acids Res. 2011 Sep 1;39(17):7730-9. doi: 10.1093/nar/gkr446. Epub 2011 Jun 17.
eIF4a3, a DEAD-box protein family member, is a component of the exon junction complex which assembles on spliced mRNAs. The protein also acts as a transcript-selective translational repressor of selenoprotein synthesis during selenium deficiency. Selenocysteine (Sec) incorporation into selenoproteins requires a Sec Insertion Sequence (SECIS) element in the 3' untranslated region. During selenium deficiency, eIF4a3 binds SECIS elements from non-essential selenoproteins, preventing Sec insertion. We identified a molecular signature for the eIF4a3-SECIS interaction using RNA gel shifts, surface plasmon resonance and enzymatic foot printing. Our results support a two-site interaction model, where eIF4a3 binds the internal and apical loops of the SECIS. Additionally, the stability of the complex requires uridine in the SECIS core. In terms of protein requirements, the two globular domains of eIF4a3, which are connected by a linker, are both critical for SECIS binding. Compared to full-length eIF4a3, the two domains in trans bind with a lower association rate but notably, the uridine is no longer important for complex stability. These results provide insight into how eIF4a3 discriminates among SECIS elements and represses translation.
eIF4a3 是 DEAD-box 蛋白家族的成员，是在拼接的 mRNA 上组装的外显子结合复合物的一个组成部分。该蛋白还作为硒缺乏时硒蛋白合成的转录选择性翻译抑制剂。硒代半胱氨酸（Sec）掺入硒蛋白需要在 3'非翻译区的 Sec 插入序列（SECIS）元件。在硒缺乏时，eIF4a3 结合非必需硒蛋白的 SECIS 元件，防止 Sec 插入。我们使用 RNA 凝胶迁移、表面等离子体共振和酶足迹分析鉴定了 eIF4a3-SECIS 相互作用的分子特征。我们的结果支持双位点相互作用模型，其中 eIF4a3 结合 SECIS 的内部和顶端环。此外，该复合物的稳定性需要 SECIS 核心中的尿嘧啶。就蛋白质要求而言，由接头连接的 eIF4a3 的两个球状结构域对于 SECIS 结合都是至关重要的。与全长 eIF4a3 相比，两个结构域以反式结合，其结合速率较低，但值得注意的是，复合物的稳定性不再依赖于尿嘧啶。这些结果提供了关于 eIF4a3 如何区分 SECIS 元件和抑制翻译的见解。
