Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Summary

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

Abstract

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo oncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

Introduction

Control of gene expression plays a pivotal role in the correct execution of cellular programs such as growth proliferation and differentiation. A regulatory control mechanism can be exerted either at the level of gene transcription or at the level of mRNA translation. In the last decade, it has become increasingly evident that translational control by modulation of the initiation process rather than the later steps of elongation and termination can finely regulate synthesis of specific subsets of proteins that play a wide range of biological functions.

Increased translation of mRNAs involved in survival, anti-autophagic and anti-apoptotic r....

Protocol

HEK293 cell line was used for the protocol and was cultured in Dulbecco's Modified Eagle Medium supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin. Cells were cultured at 37 °C with 5% CO₂ in a humidified environment.

1. Quantitative assessment of eIF4F complex disruption via eIF4E:eIF4G^604-646 complementation assay

1. Cell culture and transient transfection of eIF4E:eIF4G^604-646 complementation assay
   1. Use freshly thawed cells with less than 20 passages for all experiments. On day 1, determine the cell number using a standard cell cou....

Results

In order to validate the sensitivity of the eIF4E:eIF4G^604-646 complementation system, 4EBP1 mediated inhibition of eIF4F complex assembly was assessed by using mTOR inhibitors. By inhibiting mTORC1 kinase dependent phosphorylation of the 4EBP protein family, mTOR inhibition enhances 4EBP1 association to eIF4E and, therefore, eIF4F disassembly¹⁵. Two classes of mechanistically different inhibitor of mTOR kinases were tested: rapalogs (e.g., Rapamycin) th.......

Discussion

The method described in this article utilizes a luciferase-based complementation assay to quantitatively monitor eIF4F complex assembly through direct measurement of eIF4G-eIF4E interaction in live cells. We have provided details for use of eIF4E-eIF4G complementation system and we have also showed that the system is extremely accurate in measuring drug-induced 4EBP1-mediated dissociation of eIF4E-eIF4G interaction¹⁶. In order to facilitate the throughput of this assay, the experimental setup.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
293FT cells	Thermo Fisher Scientific	R70007
Cell culture microplate 96 well, F-Bottom	greiner bio-one	655083
Cell titer Glo 2.0	PROMEGA	G9241
Envision Multilabel Reader	PerkinElmer	not applcable
Finnpipette F2 Multichannel Pipettes 12-channels 30-300 ml	Thermo Fisher Scientific	4662070
Finnpipette F2 Multichannel Pipettes 12-channels 5-50 ml	Thermo Fisher Scientific	4662050
FUGENE6	PROMEGA	E2692
Lipofectamine 3000	Thermo Fisher Scientific	L3000015
NanoBiT PPI Starter Systems	PROMEGA	N2014
Optimem I Reduced Serum Mediun, no phenol red	Thermo Fisher Scientific	11058021
Orbital shaker	Eppendorf	not appicable
γ-Aminophenyl-m7GTP (C10-spacer)-Agarose	Jena Bioscience	AC-155S