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

Abstract

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.

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

“…We then delivered Cy5-labeled eIF4E (O'Leary et al, 2013) (Supplemental Figure 1B,C) to the mRNAs. eIF4E delivery led to transient cycles of mRNA-eIF4E single-molecule FRET (Figure 1C), as observed previously (Çetin et al, 2020;O'Leary et al, 2013). FRET was observed uniformly for the present set of 0.4 -2 kb mRNAs, suggesting eIF4E is within ~7 nm of the poly(A) tail when bound to the cap.…”

“…The custom RS instrument was set up as described previously (Chen et al, 2014;Çetin et al, 2020). The RNA to be immobilized was hybridized through its poly(A) tail to (dT)45 conjugated to biotin at its 5ʹ end and Cy3 or Cy3.5 at its 3ʹ end, to act as a FRET donor.…”

“…Raw movie data were extracted and analyzed with an in-house MATLAB processing pipeline as described previously (Chen et al 2014;O'Leary et al, 2013;Çetin et al, 2020). Image files were first converted to fluorescence vs. time traces.…”

Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly

“…The selection of the relatively short recursive introns could be mediated by recognizable remodeled 3D structural scaffolds. Additionally, the variable rate of intron removal could be due to RNA structure-mediated RNA-protein interaction dynamics (5, 36).…”

Ribosome as a Translocase and Helicase

“…Indeed, the single-molecule-resolution fluorescent in situ hybridization (smFISH) and proximity-ligation studies indicate lack of interactions between the distant segments of nuclear mRNAs bound with exon junction protein complexes (EJCs) [ 104 , 107 ]. By contrast, the 5′- and 3′-ends of cytoplasmic mRNAs, which are not actively translated, are co-localized through the formation of intramolecular secondary structure [ 104 , 105 ] that tend to bring mRNA ends in close proximity [ 78 , 108 ].…”