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bioRxiv · 10.1101/2025.06.26.660926

Dynamics and Regulation of mRNA Cap Recognition by Human eIF4F

Hong, H.·Guevara, M. G.·Li, S.·Liu, Y.·Huang, A. N.·Lin, E.·Neal, A.·Xu, D.·Hai, R.·Zandi, R.·O'Leary, S. E.
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Abstract

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.

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Hong, H., Guevara, M. G., Li, S., Liu, Y., Huang, A. N., Lin, E., Neal, A., Xu, D., Hai, R., Zandi, R., O'Leary, S. E.. 2025-06-27. Dynamics and Regulation of mRNA Cap Recognition by Human eIF4F. https://doi.org/10.1101/2025.06.26.660926

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