Gentry Riley C, Ide Nicholas A, Comunale Victoria M, Hartwick Erik W, Kinz-Thompson Colin D, Gonzalez Ruben L
Department of Biological Sciences, Columbia University, New York, NY, USA.
Department of Chemistry, Columbia University, New York, NY, USA.
Nature. 2025 Jan;637(8046):736-743. doi: 10.1038/s41586-024-08304-0. Epub 2024 Dec 11.
During translation initiation, mRNA molecules must be identified and activated for loading into a ribosome. In this rate-limiting step, the heterotrimeric protein eukaryotic initiation factor eIF4F must recognize and productively interact with the 7-methylguanosine cap at the 5' end of the mRNA and subsequently activate the message. Despite its fundamental, regulatory role in gene expression, the molecular events underlying cap recognition and mRNA activation remain unclear. Here we generate a single-molecule fluorescence imaging system to examine the dynamics with which eIF4F discriminates productive and non-productive locations on full-length, native mRNA molecules. At the single-molecule level, we observe stochastic sampling of eIF4F along the length of the mRNA and identify allosteric communication between the eIF4F subunits that ultimately drive cap-recognition and subsequent activation of the message. Our experiments uncover functions for each subunit of eIF4F and we conclude by presenting a model for mRNA activation that precisely defines the composition of the activated message. This model provides a general framework for understanding how mRNA molecules may be discriminated from one another and how other RNA-binding proteins may control the efficiency of translation initiation.
在翻译起始过程中，mRNA分子必须被识别并激活，以便加载到核糖体中。在这个限速步骤中，异源三聚体蛋白真核起始因子eIF4F必须识别mRNA 5'端的7-甲基鸟苷帽并与之有效相互作用，随后激活该信使分子。尽管eIF4F在基因表达中具有基础性的调节作用，但帽识别和mRNA激活背后的分子事件仍不清楚。在这里，我们构建了一个单分子荧光成像系统，以研究eIF4F区分全长天然mRNA分子上的有效和无效位置的动力学过程。在单分子水平上，我们观察到eIF4F沿着mRNA长度的随机采样，并确定了eIF4F亚基之间的变构通讯，这种通讯最终驱动帽识别和随后的信使分子激活。我们的实验揭示了eIF4F每个亚基的功能，最后我们提出了一个mRNA激活模型，该模型精确地定义了被激活信使分子的组成。这个模型为理解如何区分mRNA分子以及其他RNA结合蛋白如何控制翻译起始效率提供了一个通用框架。
