A dynamic compositional equilibrium governs mRNA recognition by eIF3

Abstract

Eukaryotic translation initiation factor (eIF) 3 is a multi-subunit protein complex that binds both ribosomes and messenger RNAs (mRNAs) to drive a diverse set of mechanistic steps during translation of an mRNA into the protein it encodes. And yet, a unifying framework explaining how eIF3 performs these numerous activities is lacking. Using single-molecule light scattering microscopy, we demonstrate that Saccharomyces cerevisiae eIF3 is in dynamic exchange between the full complex, subcomplexes, and subunits. By extending our microscopy approach to an in vitro reconstituted eIF3 and complementing it with biochemical assays, we define the subspecies comprising this dynamic compositional equilibrium and show that mRNA binding by eIF3 is not driven by the full complex but instead by the eIF3a subunit within eIF3a-containing subcomplexes. Our findings provide a mechanistic model for the role of eIF3 in mRNA recruitment and establish a mechanistic framework for explaining and investigating the other activities of eIF3.

Keywords: Translation; biochemistry; biophysics; eIF3; mRNA; mRNA recruitment; mass photometry; ribosome; single-molecule; translation initiation; translational regulation.

Figure 1.. eIF3 exists as a dynamic equilibrium between subunits, subcomplexes, and the full complex.

(A) Mass distribution histograms of eIF3_N at 30 nM obtained using mass photometry. The dotted black line indicates the expected ~360 kDa molecular mass for the full eIF3 complex. (B) The equivalent plot as shown in (A) for eIF3_R. (C) Mass distribution histograms of eIF3_N at various concentrations. Dashed grey lines indicate the threshold cutoffs defining ‘subunits’ (0–120 kDa), ‘subcomplexes’ (120–320 kDa), and the ‘full complex’ (320–450 kDa). (D) The equivalent plot as shown in (C) for eIF3_R. (E) Simplified cartoon models denoting the eIF3 subunits, representative subcomplexes, and full complex.

Figure 2.. eIF3_R is biochemically active.

(A) Electrophoretic mobility shift assay (EMSA) comparing eIF3_N and eIF3_R binding to the PIC. An example gel is shown (top) alongside the quantitation across three replicates (bottom), including the apparent binding affinities (K_d^app). (B) EMSA comparing the ability of eIF3_N and eIF3_R to stimulate recruitment of capped rpl41a mRNA. An example gel is shown (left) alongside the quantitation across three replicates (right), including the observed rate constants (k_obs).

Figure 3.. eIF3a is the only subunit that binds mRNA with high affinity.

(A) Fluorescence-anisotropy experiments comparing eIF3_N, eIF3_R, and eIF3a* binding to an mRNA. (B) Fluorescence-anisotropy experiments comparing eIF3a*, eIF3b, eIF3c, and a co-purified eIF3i-eIF3g binding to an mRNA. *The binding curve for eIF3a shown in panels (A) and (B) are the same data shown for comparison. (C) Bar graph of the apparent binding affinities measured for three sequence-distinct mRNAs to eIF3_N, eIF3_R, and eIF3a.

Figure 4.. Distinguishing between transient adsorption and mRNA-specific binding events in mass photometry.

(A) An example frame from mass photometry depicting various events in the field of view. A cartoon illustrating events of transient adsorption to the surface (left) and mRNA-specific binding events (right). (B) Histograms of the mass distribution from mass photometry experiments of 200 nM eIF3_R using derivatized coverslips in the absence (left) and presence (right) of tethered mRNA. (C) The difference mass distribution of 200 nM eIF3_R generated by subtracting the distribution obtained after RNase treatment from that generated before RNase treatment. (D) The mass distribution of 200 nM eIF3_R exhibiting > 0, 1, or 2 events per pixel shown as either raw counts (top) or a normalized population (bottom).

Figure 5.. Comparison of the solution mass distribution to the mRNA-binding mass distribution reveals eIF3a-containing subcomplexes differentially bind mRNA.

(A) Solution mass distribution histogram (top) and the mRNA-binding mass distribution (bottom) of 30 nM eIF3_R. (B) A population difference plot of the mRNA-binding mass distribution relative to the solution mass distribution for 30 nM eIF3_R. (C-F) Population difference plots for eIF3a-containing subcomplexes at 30 nM: 3ab (C), 3ac (D), 3abc (E), and 3abig (F).

Figure 6.. A molecular framework for modulation of eIF3 function by its underlying compositional equilibrium.

In contrast to existing as a stable, compositionally uniform protein complex, eIF3 exists in a dynamic equilibrium between the full complex, various subcomplexes, and free subunits. Additional eIF3-interacting factors likely alter this complex and dynamic compositional equilibrium. The presence eIF3a is required to enable mRNA binding by eIF3 subcomplexes, and eIF3a-containing subcomplexes may bind mRNA with a higher affinity than the full complex. The presence of mRNA thus biases the eIF3 compositional equilibrium to the formation of mRNA-bound eIF3a-containing subcomplexes, which may enable the delivery of mRNAs to free 40S subunits or PICs. Additional modulation of this compositional equilibrium may enable the formation of specific eIF3a-containing subcomplexes capable of dictating the translational fates of specific mRNAs.