Thermodynamically Favorable Interactions between eIF4E Binding Domain of eIF4GI with Structured 5'-Untranslated Regions Drive Cap-Independent Translation of Selected mRNAs.
Thermodynamically Favorable Interactions between eIF4E Binding Domain of eIF4GI with Structured 5'-Untranslated Regions Drive Cap-Independent Translation of Selected mRNAs.
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eIF4GI 的 eIF4E 结合域与结构化 5-非翻译区域之间的热力学有利相互作用驱动所选 mRNA 的帽独立翻译。
DOI：
10.1021/acs.biochem.3c00125
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发表时间：
2023
期刊：
影响因子：
2.9
通讯作者：
Goss,DixieJ
中科院分区：
文献类型：
--
作者：
Saha,Baishakhi;Bhardwaj,Usha;Goss,DixieJ
在细胞应激条件下，尤其是在多种癌症中，典型的帽依赖的翻译被抑制，并且已知的细胞内α的子集(例如编码成纤维细胞生长因子-9、缺氧诱导因子-1和p53等的那些)以帽非依赖性的方式翻译。人类eIF4GI特异性地与这些mRNAs的高度结构化的5‘-非翻译区(5’UTRs)结合，以促进帽子无关的翻译。这些蛋白质-RNA相互作用的热力学还没有被探索，这些信息将有助于理解基本的相互作用和潜在的治疗药物设计。利用基于荧光猝灭的分析和定点突变，我们确定了三个eIF4GI结构与成纤维细胞生长因子-9、缺氧诱导因子-1α和p53mRNA的5‘UTRs结合的热力学性质。这三个结构被设计来探索eIF4GI的eIF4E结合域的重要性，它已经被证明在结合和选择性方面是重要的。含有eIF4E结合域的eIF4GI557-1599具有更高的结合焓(−21到−14kJ·mol-1)，表明氢键增加；而对于没有eIF4E结合结构域的eIF4GI682-1599，结合是熵有利的(TΔS/ΔG为46-85%)，表明疏水力和/或特异性较低。第三个结构中，一簇带正电的氨基酸被改变为中性氨基酸，显示出中间性质。圆二色谱证实了eIF4E结合区在eIF4GI与mRNAs之间通过构象变化形成稳定键的重要作用。总之，这些数据有助于更好地理解eIF4GI-mRNA识别中涉及的分子力，并阐明对于设计介导这些相互作用的小分子非常重要的性质。
During cellular stress conditions, particularly those seen in multiple cancers, canonical cap-dependent translation is suppressed and a subset of cellular mRNAs (e.g., those encoding FGF-9, HIF-1α, and p53, among others) is known to translate in a cap-independent manner. Human eIF4GI specifically binds to the highly structured 5′-untranslated regions (5′UTRs) of these mRNAs to promote cap-independent translation. The thermodynamics of these protein–RNA interactions have not been explored, and such information will aid in understanding the basic interactions and in potential design of therapeutic drugs. Using fluorescence quenching-based assays and site-directed mutagenesis, we determined the thermodynamic properties of three eIF4GI constructs binding to the 5′UTRs of FGF-9, HIF-1α, and p53 mRNA. These three constructs were designed to explore the importance of the eIF4E binding domain of eIF4GI, which has been shown to be important in binding and selectivity. eIF4GI557–1599, containing the eIF4E binding domain, had higher binding enthalpy (−21 to −14 kJ mol–1higher), suggesting increased hydrogen bonding, whereas for eIF4GI682–1599lacking the eIF4E binding domain, binding was entropically favored (TΔS/ΔG of 46–85%), suggesting hydrophobic forces and/or less specific binding. A third construct where a cluster of positively charged amino acids was changed to neutral amino acids showed intermediate properties. Circular dichroism spectra confirmed the significant role of eIF4E binding domain in stable bond formation between eIF4GI and mRNAs via conformational changes. Together, these data contribute to a better understanding of the molecular forces involved in eIF4GI-mRNA recognition and elucidate properties important for the design of small molecules to mediate these interactions.