Saha Baishakhi, Haizel Solomon A, Goss Dixie J
Department of Chemistry, Hunter College, City University of New York, New York, New York, USA.
PhD. Program in Biochemistry, The Graduate Center of the City University of New York, New York, New York, USA; Center for Genomics and Systems Biology, New York University, New York, New York, USA.
J Biol Chem. 2024 Nov;300(11):107866. doi: 10.1016/j.jbc.2024.107866. Epub 2024 Oct 9.
Protein translation is globally downregulated under stress conditions. Many proteins that are synthesized under stress conditions use a cap-independent translation initiation pathway. A subset of cellular mRNAs that encode for these proteins contain stable secondary structures within their 5'UTR, and initiate cap-independent translation using elements called cap-independent translation enhancers or internal ribosome entry sites within their 5'UTRs. The interaction among initiation factors such as eukaryotic initiation factor 4E (eIF4E), eIF4A, and eIF4GI, especially in regulating the eIF4F complex during noncanonical translation initiation of different 5'UTR mRNAs, is poorly understood. Here, equilibrium-binding assays, CD studies and in vitro translation assays were used to elucidate the recruitment of these initiation factors to the highly structured 5'UTRs of fibroblast-growth factor 9 (FGF-9) and hypoxia inducible factor 1 subunit alpha (HIF-1α) encoding mRNAs. We showed that eIF4A and eIF4E enhanced eIF4GI's binding affinity to the uncapped 5'UTR of HIF-1α mRNA, inducing conformational changes in the protein/RNA complex. In contrast, these factors have no effect on the binding of eIF4GI to the 5'UTR of FGF-9 mRNA. Recently, Izidoro et al. reported that the interaction of 42nt unstructured RNA to human eIF4F complex is dominated by eIF4E and ATP-bound state of eIF4A. Here, we show that structured 5'UTR mRNA binding mitigates this requirement. Based on these observations, we describe two possible cap-independent translation mechanisms for FGF-9 and HIF-1α encoding mRNAs used by cells to mitigate cellular stress conditions.
在应激条件下，蛋白质翻译在整体上受到下调。许多在应激条件下合成的蛋白质使用不依赖帽结构的翻译起始途径。编码这些蛋白质的细胞mRNA的一个子集在其5'非翻译区（5'UTR）内含有稳定的二级结构，并利用其5'UTR内称为不依赖帽结构的翻译增强子或内部核糖体进入位点的元件起始不依赖帽结构的翻译。起始因子如真核起始因子4E（eIF4E）、eIF4A和eIF4GI之间的相互作用，尤其是在不同5'UTR mRNA的非经典翻译起始过程中对eIF4F复合物的调节作用，目前了解甚少。在这里，采用平衡结合测定、圆二色光谱研究和体外翻译测定来阐明这些起始因子与成纤维细胞生长因子9（FGF-9）和缺氧诱导因子1α亚基（HIF-1α）编码mRNA的高度结构化5'UTR的结合情况。我们发现，eIF4A和eIF4E增强了eIF4GI对HIF-1α mRNA无帽5'UTR的结合亲和力，诱导了蛋白质/RNA复合物的构象变化。相比之下，这些因子对eIF4GI与FGF-9 mRNA的5'UTR的结合没有影响。最近，伊齐多罗等人报道，42nt无结构RNA与人类eIF4F复合物的相互作用主要由eIF4E和eIFA的ATP结合状态主导。在这里，我们表明结构化的5'UTR mRNA结合减轻了这一需求。基于这些观察结果，我们描述了细胞用于减轻细胞应激条件的FGF-9和HIF-1α编码mRNA的两种可能的不依赖帽结构的翻译机制。
