Cap-dependent translation initiation monitored in living cells

Abstract

Despite extensive biochemical, genetic, and structural studies, a complete understanding of mRNA translation initiation is still lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-molecule resolution are necessary to fully elucidate regulation of this paramount process. We fused tags suitable for live imaging to eIF4E, eIF4G1 and 4E-BP1 without affecting their function. We combined Fluorescence Correlation Spectroscopy (FCS) and Single-Particle Tracking (SPT) to interrogate the binding dynamics of initiation factors to the 5'cap. Both FCS and SPT were able to detect eIF4E:eIF4G1 binding to the mRNA in the cytoplasm of proliferating cells and neuronal processes. Upon inhibition of phosphorylation by mTOR, 4E-BP1:eIF4E complexes rapidly dissociated from the 5'cap followed by eIF4G1 dissociation. Imaging of the binding dynamics of individual translation factors in living cells revealed the temporal regulation of translation at unprecedented resolution.

Inhibition of coronavirus HCoV-OC43 by targeting the eIF4F complex

“…Notably, autocorrelations for single JF646 SNAPf-eIF4G and JF585 Halo-eIF4E signals demonstrated that their diffusion only marginally slowed in the cytoplasm of cells treated with SBI-756 or SBI-0498 relative to control cells ( Figure 4C ), suggesting that in cellula SBI-756 and SBI-0498 inhibit eIF4E:eIF4G binding without triggering their dissociation from mRNA. On the other hand, autocorrelation obtained in SBI-5844 treated cells, demonstrated that cytoplasmic JF585 Halo-eIF4E molecules diffuse as fast as their nuclear counterparts ( Figure 4C , right panel), mirroring diffusion of free-eIF4E molecules that do not bind the 5′cap ( Gandin et al, 2021 ), suggesting that in cellula SBI-5844 can dissociate mRNAs from the eIF4F complex.…”

“…Because the fluorescent signal is recorded simultaneously, the cross-correlation is a direct indication of the concomitant movement and so interaction of the two molecules as they transit through the focal volume ( Kim et al, 2007 ). FCS and FCCS allow us to detect in real-time translation initiation events in living cells since it can resolve unbound initiation factors (fast diffusion) or mRNA bound (slow diffusion) ( Gandin et al, 2021 ). Halo- and SNAP f -tag suitable for live cell imaging, were fused eIF4E and eIF4G respectively without perturbing their function ( Gandin et al, 2021 ).…”

“…FCS and FCCS allow us to detect in real-time translation initiation events in living cells since it can resolve unbound initiation factors (fast diffusion) or mRNA bound (slow diffusion) ( Gandin et al, 2021 ). Halo- and SNAP f -tag suitable for live cell imaging, were fused eIF4E and eIF4G respectively without perturbing their function ( Gandin et al, 2021 ). FCCS analysis detected simultaneous diffusion of endogenously tagged JF585 Halo-eIF4E and JF646 SNAPf-eIF4G through the imaged focal volume.…”

“…FCS trajectories were obtained using a Leica SP8 Falcon microscope equipped with a Leica APO 86x/N.A. = 1.20 water-immersion objective with the motCORR automated correction collar, as previously described ( Gandin et al, 2021 ). Mouse embryonic stem cells (mESCs) in which Halo and SNAP f tag were inserted into the EIF4E1 and EIF4G1 alleles, respectively, were differentiated into fibroblasts ( Gandin et al, 2021 ).…”

“…= 1.20 water-immersion objective with the motCORR automated correction collar, as previously described ( Gandin et al, 2021 ). Mouse embryonic stem cells (mESCs) in which Halo and SNAP f tag were inserted into the EIF4E1 and EIF4G1 alleles, respectively, were differentiated into fibroblasts ( Gandin et al, 2021 ). Twenty-four hours later, cells were labelled with 100 nM JF585-Halo tag ligand for 10 min and 500 nM JF64-6SNAP f tag ligand for 45 min.…”