mRNA- and factor-driven dynamic variability controls eIF4F-cap recognition for translation initiation

Abstract

mRNA 5′ cap recognition by eIF4F is a key element of eukaryotic translational control. Kinetic differences in eIF4F–mRNA interactions have long been proposed to mediate translation-efficiency differences between mRNAs, and recent transcriptome-wide studies have revealed significant heterogeneity in eIF4F engagement with differentially-translated mRNAs. However, detailed kinetic information exists only for eIF4F interactions with short model RNAs. We developed and applied single-molecule fluorescence approaches to directly observe real-time Saccharomyces cerevisiae eIF4F subunit interactions with full-length polyadenylated mRNAs. We found that eIF4E–mRNA association rates linearly anticorrelate with mRNA length. eIF4G–mRNA interaction accelerates eIF4E–mRNA association in proportion to mRNA length, as does an eIF4F-independent activity of eIF4A, though cap-proximal secondary structure still plays an important role in defining the final association rates. eIF4F–mRNA interactions remained dominated by effects of eIF4G, but were modulated to different extents for different mRNAs by the presence of eIF4A and ATP. We also found that eIF4A-catalyzed ATP hydrolysis ejects eIF4E, and likely eIF4E•eIF4G from the mRNA after initial eIF4F•mRNA complex formation, suggesting a mechanism to prepare the mRNA 5′ end for ribosome recruitment. Our results support a role for mRNA-specific, factor-driven eIF4F association rates in kinetically controlling translation.

Single-molecule visualization of mRNA circularization during translation

“…Conversely, another smFRET study found that partial human eIF4G (557-1137) alone slightly affects eIF4E binding kinetics, but the human eIF4F complex substantially changes the eIF4E-cap association 46 . Interestingly, free eIF4A enhances eIF4E cap accessibility via direct eIF4A-RNA contact, which is similar to the role of eIF4G in eIF4E 25 . Meanwhile, the binding of eIF4F to RNA results in the dissociation of eIF4E from eIF4F shortly after the formation of the eIF4F-RNA complex, but this process is dependent on ATP hydrolysis (0.07-0.08 s -1 ; Fig.…”

“…Recognition of eIF4G increases eIF4E's affinity to a cap or cap analog 44,45 . To study the 5' cap-binding kinetics of eIF4E, smFRET was monitored with fluorescently labeled eIF4E in the absence and presence of eIF4G 25,46,47 (Fig. 2a).…”

“…This observation suggests that PABP may participate in the conformational changes of RNA by directly interacting with mRNA, although its interaction with mRNA itself is relatively weaker than its interaction with the poly(A) tail 48,49 . A follow-up study on the kinetics of the eIF4E-mRNA interaction has been performed more comprehensively using fluorescently labeled eIF4E, eIF4A, eIF4G, and mRNA immobilized on a surface 25 . Direct eIF4G-mRNA interactions b Schematic and intensity-time trace representing the comprehensive eIF4F-cap (mRNA) binding kinetics during translation initiation.…”

“…Interestingly, the rate of eIF4E-mRNA association depends not on the free energy of the secondary structure but on the degree of complexity of the secondary structure. The rate of association between eIF4E and mRNA tends to increase as the total length of mRNA shortens, and the eIF4G-mRNA interaction accelerates the association rate of eIF4E in proportion to the length of mRNA; consequently, less deviation occurs among mRNAs 25 . Conformational changes in a regulator can play a crucial role in circularization.…”

“…It has enabled us to determine the relationship between translation and circularization in cells. Meanwhile, in vitro sm studies on initiation factors have revealed their binding kinetics or conformational change in protein synthesis, thus contributing significantly to our understanding of the molecular processes involved in circularization [25][26][27][28][29] . In this review, we briefly introduce how smFI techniques have been applied to investigate mRNA circularization and discuss the current knowledge of the relationship between translation and mRNA circularization.…”