The mechanism of mRNA activation

Abstract

During translation initiation, messenger RNA molecules must be identified and activated for loading into a ribosome. In this rate-limiting step, the heterotrimeric protein eukaryotic initiation factor eIF4F must recognize and productively interact with the 7-methylguanosine cap at the 5' end of the messenger RNA and subsequently activate the message. Despite its fundamental, regulatory role in gene expression, the molecular events underlying cap recognition and messenger RNA activation remain mysterious. Here, we generate a unique, single-molecule fluorescence imaging system to interrogate the dynamics with which eIF4F discriminates productive and non-productive locations on full-length, native messenger RNA molecules. At the single-molecule level, we observe stochastic sampling of eIF4F along the length of the messenger RNA and identify allosteric communication between the eIF4F subunits which ultimately drive cap-recognition and subsequent activation of the message. Our experiments uncover novel functions for each subunit of eIF4F and we conclude by presenting a model for messenger RNA activation which precisely defines the composition of the activated message. This model provides a general framework for understanding how messenger RNA molecules may be discriminated from one another, and how other RNA-binding proteins may control the efficiency of translation initiation.

Extended Data Figure 1.. Purification, labeling, and validation of biochemical components.

(a) SDS-PAGE gel showing all of the purified eIFs required to perform mRNA recruitment gel-shift assays. The inset shows a red fluorescence scan of the outlined portion of the SDS-PAGE gel demonstrating that the factors are fluorescently labeled. (b) Standard mRNA recruitment gel-shift assay showing recruitment of mRNA onto a stable 48S-PIC in the presence of eIF4F. As expected, diminished recruitment is observed in the absence of eIF4B or eIF4E. (c) Titration-regime, anisotropy-based RNA binding experiments performed with either 500nM or 50nM FAM-labeled, 51-nucleotide rpl41a fragment. These experiments show that anisotropy continues to increase beyond a 1:1 molar ratio of mRNA:eIF4G, confirming multiple eIF4G molecules bind the short RNA, as previously observed. Each titration-regime experiment was performed once.

Extended Data Figure 2.. Validation of smFRET between Cy5-eIF4G:E and Cy3-mRNA.

(a) Hypothetical intensities/FRET trajectories showing what is expected to happen if Cy5-eIF4G:E binds at a cap-proximal location on the capped mRNA construct . Upon binding, the green fluorescence intensity decreases while the red intensity increases for the duration of the binding event, leading to an increase in E_FRET while Cy5-eIF4G:E is bound near the donor fluorophore. (b) Shows similar hypothetical trajectories, but in these trajectories Cy5-eIF4G:E binds at a cap-distal location far away from the donor fluorophore. Because the acceptor and donor fluorophores are not near each other, no changes in intensity or E_FRET are detected before photobleaching. (c) Shows an example experimental trajectory for Cy5-eIF4G:E binding to the short, 24-nucleotide 5’ UTR of rpl41a. Because the RNA is short, we expect only a single binding site. At 5nM, we observe transient binding, but at 50nM (d), both the association and dissociation kinetics increase, indicating that eIF4G is able to facilitate its own dissociation. Facilitated dissociation is cartooned in (e) whereby the release of one of RNA binding domains of eIF4G, also called microdissociation, opens up a binding location for a new molecule of eIF4G, which then facilitates the release of the first eIF4G molecule.

Extended Data Figure 3.. Cy5-eIF4G:E facilitates its own dissociation on full-length rpl41a mRNA.

(a) Survival-time analysis showing that the steady-state dissociation (left) of Cy5-eIF4G:E on the capped mRNA construct is biphasic, whereas the pre-steady-state dissociation rate (right) is monophasic, indicating that steady-state dissociation events are a mixture of facilitated and non-facilitated dissociation events. (b) Comparison of the rate constants derived from steady-state and pre-steady-state experiments again demonstrate that eIF4G:E facilitates its own dissociation in the steady-state experiments. Thus, pre-steady-state experiments are more reflective of the inherent dissociation rate of eIF4G:E. However, comparison of the pre-steady-state dissociation rates recorded with and without shuttering of the excitation light source demonstrate that the pre-steady-state dissociation rate is limited by photobleaching. The steady-state dissociation rates plotted are population weighted averages of the two phases shown in (a).

Extended Data Figure 4.. eIF4G:E can “hop” along an mRNA.

Example trajectories taken from pre-steady-state ejection experiments on the capped construct where Cy5-eIF4G:E dissociates from the local position on the mRNA, causing E_FRET to drop to 0, but later recovers, despite the all free eIF4G:E being removed from the flowcell, when the same, or another eIF4G:E bound elsewhere on the mRNA, microdissociates and then “hops” to the local position near the donor fluorphore on the mRNA. These example trajectories are shown for both continuous illumination (a) and excitation light shuttering (b) experiments.

Extended Data Figure 5.. eIF4G:E-Cy5 facilitates its own dissociation.

A bar graph showing the population-weighted average, steady-state dissociation rate of eIF4G:E compared to the pre-steady-state dissociation rate. Similar to Cy5-eIF4G/G:E, free, unbound, eIF4G:E-Cy5 is able to exchange with bound eIF4G:E-Cy5 leading to an increased apparent dissociation rate in steady-state experiments. This serves as additional evidence that eIF4G and eIF4E function as a complex.

Extended Data Figure 6.. Association and dissociation kinetics of Cy5-eIF4G:E in the presence of eIF4A and eIF4B.

(a) The pre-steady-state association rates calculated from a Cy5-eIF4G:E concentration series, also plotted in Figure 1, and the pre-steady-state association rates calculated from a single Cy5-eIF4G:E concentration point of 3 nM in the presence of saturating eIF4A+ATP or eIF4A+eIF4B+ATP. eIF4A and eIF4B do not appear to alter the association kinetics. (b) The steady-state dissociation kinetics calculated from the steady-state portion of the injection experiments for eIF4G:E, eIF4G:E+eIF4A+ATP, and eIF4G:E+eIF4A+eIF4B+ATP. eIF4A and eIF4B enhance the steady-state dissociation rate on every construct except the capped construct in the presence of eIF4E. (c) The equivalent pre-steady-state experiment for those described in (b). eIF4A destabilizes eIF4G/G:E from each mRNA construct in an ATP-dependent manner except in the presence of both eIF4E and the cap. A red line at the highest tick mark on the Y-axis of (b) and (c) denotes the change in axis scale between the two graphs—highlighting that the steady-state dissociation rate far exceeds the pre-steady-state dissociation rate due to facilitated dissociation.

Extended Data Figure 7.. ATP, but not eIF4B, is required for stripping eIF4G from mRNA.

Surface contour plots showing pre-steady-state ejection experiments. These ejection experiments were performed by exchanging all of the free, unbound Cy5-eIF4G:E in the flowcell with buffer lacking Cy5-eIF4G:E, but containing some combination eIF4A, eIF4B and ATP similar to Figure 4. On the left, reactions were performed without eIF4B, while on the right they were performed without ATP. All experiments were performed on the (a) capped, (b) uncapped, (c), capped without eIF4E and (d) internal mRNA constructs. These results show efficient stripping of Cy5-eIF4G:E is not dependent on eIF4B, but is dependent on ATP.

Extended Data Figure 8.. eIF4G:E is stripped from mRNA as a complex.

The equivalent ejection experiments shown in Figure 4 but with the acceptor fluorophore on eIF4E instead of eIF4G. Similar to with Cy5-eIF4G:E, eIF4G:E-Cy5 is stripped from every position except from the cap. This demonstrates that eIF4G:E is stripped as a complex during the search for the cap.

Figure 1.. eIF4G/G:E stably associates with mRNA independent of its binding position or the cap.

(a) Cartoon schematic of the full-length, Cy3-labeled rpl41a mRNA constructs used in this work. (b) Cartoon representation of the stopped flow injection and ejection pre-steady state experiments used to measure k_on and k_off. (c) An example trace taken from a Cy5-eIF4G:E injection experiment is displayed. The trace can be divided into two portions: the first binding event in the trajectory encompasses the pre-steady-state portion, whereas the later portions of the trajectory display steady-state binding and dissociation events until bleaching of the donor fluorophore. On the right, a surface contour plot showing the aggregate behavior of all the trajectories in an injection experiment is displayed. (d) The equivalent example trace and surface contour plot for ejection experiments are shown. Since all of the excess Cy5-eIF4G:E has been removed from the flowcell, only the pre-steady-state behavior can be inferred from ejection experiments. (e) The association rate of Cy5-eIF4G/G:E is linear with respect to concentration and depends more on the presence of eIF4E than on the cap. (f) Quantitation of the pre-steady state association and dissociation rates for Cy5-eIF4G/G:E, demonstrating that eIF4G uniformly binds to mRNA irrespective of the cap or its binding location.

Figure 2.. eIF4E functions in concert with eiF4G irrespective of the cap.

(a) Example trajectories of Cy5-eIF4E binding to the capped mRNA construct in the absence (left) or presence (right) of unlabeled eIF4G. In the absence of eIF4G, Cy5-eIF4E only transiently samples the cap, whereas it stably binds in the presence of eIF4G. (b) Surface contour plots post-synchronized to the steady-state transitions of eIF4E out of the bound state: Time = 0 marks the time at which eIF4E dissociates from the vicinity of the donor fluorophore, such that the negative time axis indicates how long Cy5-eIF4E remains bound before dissociation and the positive time axis shows the delay time between binding events. (c) Quantitation of the association and dissociation rates of Cy5-eIF4E and eIF4G:E-Cy5 from the three mRNA constructs. The association rates for the capped, uncapped, and internal constructs are determined from experiments performed at 5nm eIF4G:E-Cy5, whereas the capped −4G association rate is derived from the slope of the apparent association rates of Cy5-eIF4E performed across three concentrations. The nearly identical rates derived from equivalent experiments performed with Cy5-eIF4G, plotted in Figure 1, are shown as purple dots to aid the comparison between Cy5-eIF4E and Cy5-eIF4G.

Figure 3.. eIF4A strips eIF4G:E from RNA.

Surface contour plots showing ejection experiments where excess, unbound Cy5-eIF4G:E in the flowcell is replaced with buffer lacking Cy5-eIF4G:E or replaced with buffer lacking Cy5-eIF4G:E but supplemented with eIF4A, eIF4B, and ATP (right). Each experiment was recorded at both 10 frames per second (fps) and 1/3 fps to obtain kinetic information in both the seconds (black) and minutes (gray) timescales. These experiments were performed on the (a) capped, (b) uncapped, (c) capped RNA without eIF4E, and (d) internal mRNA constructs. Each experiment begins with Cy5-eIF4G:E bound to the RNA. E_FRET starts at a high value and remains stable until photobleaching in the absence of eIF4A, eIF4B and ATP. However, in the presence of eIF4A, eIF4B and ATP, E_FRET rapidly decays within ~3 seconds (guiding line). This indicates that eIF4A strips eIF4G:E from RNA. eIF4E recognition of the cap (a) is able to resist this stripping. (e) Pre-steady-state off rates were calculated from the individual trajectories of the experiments performed in (a)-(d). These rates show eIF4G is destabilized by 30-40 fold in the presence of eIF4A, eIF4B, and ATP, but eIF4G is resistant to this destabilization in the presence of both eIF4E and the cap.

Figure 4.. The mechanism of mRNA activation.

Our experiments lead us to the following new model of mRNA activation: In the first step, eIF4G:E nonspecifically samples and binds to mRNA. At the second step, the pathway bifurcates into productive (green) or nonproductive (red) pathways. In the non-productive fork (2b), eIF4G:E binds far away from the cap, preventing eIF4E from associating with the cap, allowing eIF4A to recycle the eIF4F complex by either facilitating its intramolecular hopping to another place on the same mRNA molecule (3b) or completely removing it from the RNA (3c). In the productive fork, eIF4G:E binds at the 5’ UTR near the cap, and the eIF4E-cap interaction allosterically signals to eIF4A, through eIF4G, not to strip eIF4G. This stalled eIF4F complex at the cap constitutes the previously elusive activated mRNP, in which we speculate eIF4A may be held in an inactive conformation which can be reactivated by an eIF4B containing 43S-PIC prior to loading the mRNA onto the ribosome (3a). Notably, because eIF4A is present at high concentrations in the cell, this model is agnostic to which step eIF4A binds eIF4G:E, and due to its modest affinity, we speculate it is dynamically sampling the complex throughout the cycle.