Heterogeneous Dynamics of Protein-RNA Interactions across Transcriptome-Derived Messenger RNA Populations

Dynamic RNA-protein interactions underpin numerous molecular control mechanisms in biology. However, relatively little is known about the kinetic landscape of protein interactions with full-length RNAs. The extent to which interaction kinetics vary for the same RNA element across the transcriptome and the molecular determinants of variability therefore remain poorly defined. Moreover, it is unclear how one protein-RNA interaction might be transduced by RNA to kinetically impact a second. We report a parallelized, real-time single-molecule fluorescence assay for protein interaction kinetics on eukaryotic mRNA populations obtained from cells. We observed ∼100-fold heterogeneity for interactions of the translation initiation factor eIF4E with the universal mRNA 5' cap structure, dominated by steric effects on barrier-height variability for association. We also found that an RNA helicase, eIF4A, independently accelerated eIF4E-cap association. These data support a kinetic mechanism for how mRNA can determine the sensitivity of its translation to reduction in cellular eIF4E concentrations. They also support the view that global RNA structure significantly modulates protein-RNA interaction dynamics and can facilitate real-time communication between protein interactions at distinct sites.