Atomic-Resolution Analysis of eIF3-Mediated Translation Control

项目总结 蛋白质的生物合成直接将细胞中的基因型与表型结合在一起，它的调节是细胞的中心。 生理学。我们对蛋白质合成分子机制的理解经历了一场革命 过去十年，建立在结构生物学和系统生物学快速发展的基础上。然而，重要的是 与翻译中的动态事件有关的问题仍然没有得到回答，因为这些事件的暂时性 使得它们很难被分离出来。在这个应用中，我们建议破译分子机制是如何 人类真核细胞翻译起始因子3(EIF3)调节翻译起始。我们最近发现 人类的eIF3在翻译中起着多重作用。人类eIF3通常帮助组装翻译 起始密码子上的预引发复合体，也直接控制特定mRNAs的翻译。我们 发现eIF3可以激活或抑制mRNA的翻译，这取决于eIF3与 M RNA的5‘非翻译区(5’非编码区)。此外，我们发现eIF3含有一个亚单位--EIF3D-- 以RNA结构依赖的方式将5‘-m7G帽结合到某些mRNA上。这些发现表明 EIF3可以整合多个信号来控制单个mRNA的翻译输出，很像 转录中的中介复合体。 本应用程序的目标建立在这些开创性结果的基础上，以解决以下基本问题 EIF3功能。我们建议确定eIF3介导的专门化控制的结构基础 翻译，使用低温电子显微镜(Cryo-EM)和体外生物化学。我们还将调查如何 受eIF3调控的mRNAs是在活细胞中构建的，并剖析了这些基因的功能重要性 EIF3调控的结构和这些mRNAs的翻译能力。最后，我们将使用系统 用生物学方法探讨eIF3如何参与N-6介导的翻译调控 MRNAs中的甲基腺苷(M6A)修饰。通过将冷冻-EM的进步与我们在人类方面的专业知识相结合 细胞工程学，我们处于一个独特的位置来揭示eIF3对翻译的分子贡献 在人类身上开始。综上所述，该应用程序的三个目标建立在对 EIF3的结构和功能在前一个资助期内获得，并解决翻译中的关键机制 这种控制可能在人类生物学中广泛存在。从长远来看，我们对分子机制的洞察 在人类细胞中用于指导eIF3介导的特定mRNAs的激活和抑制可能为 用于开发新的小分子和基于细胞的疗法。

PROJECT SUMMARY Protein biosynthesis directly couples genotype to phenotype in the cell, and its regulation is central to cellular physiology. Our understanding of the molecular mechanism of protein synthesis has undergone a revolution in the last decade, built on rapid advances in structural biology and systems biology. However, important questions relating to dynamic events in translation remain unanswered, as the transient nature of these events makes them difficult to isolate. In this application, we propose to decipher the molecular mechanisms of how eukaryotic translation initiation factor 3 (eIF3) in humans regulates translation initiation. We recently discovered that human eIF3 serves multiple roles in translation. Human eIF3 generally helps assemble translation preinitiation complexes at the start codon, but also directly controls the translation of specific mRNAs. We found that eIF3 can either activate or repress the translation of an mRNA, depending on how eIF3 binds to the mRNA's 5' untranslated region (5' UTR). Furthermore, we discovered that eIF3 harbors a subunit–EIF3D–that binds the 5'-m7G cap on certain mRNAs in an RNA structure-dependent manner. These discoveries indicate that eIF3 may integrate multiple signals to control the translational output of individual mRNAs, much like the Mediator complex in transcription. The aims in this application build on these groundbreaking results to address fundamental questions of how eIF3 functions. We propose to determine the structural basis for eIF3-mediated control of specialized translation, using cryo-electron microscopy (cryo-EM) and in vitro biochemistry. We will also probe how mRNAs regulated by eIF3 are structured in living cells, and dissect the functional importance of these structures to eIF3 regulation and the translational capacity of these mRNAs. Finally, we will use systems biological approaches to explore how eIF3 contributes to the regulation of translation mediated by N-6- methyladenosine (m6A) modifications in mRNAs. By combining advances in cryo-EM to our expertise in human cell engineering, we are in a unique position to unravel the molecular contributions of eIF3 to translation initiation in humans. Taken together, the three aims of this application build on the fundamental insights into eIF3 structure and function obtained in the prior funding period, and address key mechanisms in translational control that could be widespread in human biology. In the long run, our insights into the molecular mechanisms used in human cells to direct eIF3-mediated activation and repression of specific mRNAs could pave the way for the development of new small-molecule and cell-based therapeutics.