Hong Hea Jin, Guevara Matthew G, Li Siyu, Liu Yankang, Huang Alexandra N, Lin Eric, Neal Arrmund, Xu Duo, Hai Rong, Zandi Roya, O'Leary Seán E
Department of Biochemistry, University of California Riverside, Riverside, CA 92521.
Department of Physics and Astronomy, California State Polytechnic University, Pomona, CA 91768.
bioRxiv. 2025 Jun 27:2025.06.26.660926. doi: 10.1101/2025.06.26.660926.
Efficient eukaryotic messenger RNA translation requires dynamic collaboration between the three subunits of initiation factor 4F (eIF4F, eIF4E•G•A), which recognises and activates mRNA at its 5' cap structure for ribosome recruitment. Despite its high biological and pharmacological importance, the dynamics of full human eIF4F-mRNA engagement remain largely uncharacterised, hindering mechanistic understanding of translation initiation and its regulation. Here we observed human eIF4F activity with single-molecule fluorescence assays that directly visualise mRNA cap recognition by its eIF4E subunit. Unexpectedly, we find that inherently transient eIF4E-cap binding is repressed by full-length human eIF4G, predominantly through its C-terminus, representing an unanticipated role for eIF4G as a central rate-limiting factor in the eIF4F complex. This repression is relieved by nucleotide-bound eIF4A in the eIF4F heterotrimer, placing eIF4A as a crucial determinant of efficient cap recognition for translation. Molecular dynamics simulations reveal that electrostatic modulation of eIF4E-mRNA interaction allows eIF4G to control the cap-recognition frequency. Our findings also indicate that intrinsic eIF4F-mRNA dynamics are insufficient to support cap-tethered ribosomal scanning to locate translation start sites. They illuminate fundamental design-principle differences for the overall mechanism and division of labour among eIF4F subunits during mRNA recognition in humans and yeast.
高效的真核生物信使核糖核酸（mRNA）翻译需要起始因子4F（eIF4F，即eIF4E•G•A）的三个亚基之间进行动态协作，该因子在mRNA的5'帽结构处识别并激活mRNA，以招募核糖体。尽管其具有很高的生物学和药理学重要性，但完整的人类eIF4F与mRNA结合的动力学在很大程度上仍未得到充分表征，这阻碍了对翻译起始及其调控机制的理解。在这里，我们通过单分子荧光测定法观察了人类eIF4F的活性，该方法可直接可视化其eIF4E亚基对mRNA帽的识别。出乎意料的是，我们发现全长人类eIF4G主要通过其C末端抑制了固有的瞬时eIF4E-帽结合，这代表了eIF4G作为eIF4F复合物中核心限速因子的一个意外作用。eIF4F异源三聚体中与核苷酸结合的eIF4A可缓解这种抑制作用，使eIF4A成为有效帽识别以进行翻译的关键决定因素。分子动力学模拟表明，eIF4E与mRNA相互作用的静电调节使eIF4G能够控制帽识别频率。我们的研究结果还表明，内在的eIF4F-mRNA动力学不足以支持帽连接的核糖体扫描以定位翻译起始位点。它们阐明了人类和酵母在mRNA识别过程中eIF4F亚基的整体机制和分工的基本设计原则差异。
