IMR Press / FBE / Volume 14 / Issue 3 / DOI: 10.31083/j.fbe1403017
Open Access Original Research
Ferritin Iron Responsive Elements (IREs) mRNA Interacts with eIF4G and Activates In Vitro Translation
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1 Department of Chemistry and Biochemistry, Hunter College of the City University of New York, New York, NY 10065, USA
2 Department of Life Science, College of Science & General Studies, Alfaisal University, 11533 Riyadh, Saudi Arabia
*Correspondence:; (Mateen A. Khan)
Academic Editors: Guoyao Wu and William Konigsberg
Front. Biosci. (Elite Ed) 2022, 14(3), 17;
Submitted: 18 October 2021 | Revised: 17 April 2022 | Accepted: 11 May 2022 | Published: 4 July 2022
Copyright: © 2022 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.

Background: Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to mRNA and promote translation. Translation of ferritin IRE mRNAs is regulated by iron through iron responsive elements (IREs) and iron regulatory protein (IRP). The noncoding IRE stem-loop (30-nt) structure control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. High cellular iron concentrations promote IRE RNA binding to ribosome and initiation factors, and allow synthesis of ferritin. Methods: In vitro translation assay was performed in depleted wheat germ lysate with supplementation of initiation factors. Fluorescence spectroscopy was used to characterize eIF4F/IRE binding. Results: Eukaryotic initiation factor eIF4G increases the translation of ferritin through binding to stem loop structure of iron responsive elements mRNA in the 5-untranslated region. Our translation experiment demonstrated that exogenous addition of eIF4G selectively enhanced the translation of ferritin IRE RNA in depleted WG lysate. However, eIF4G facilitates capped IRE RNA translation significantly higher than uncapped IRE RNA translation. Addition of iron with eIF4G to depleted WG lysate significantly enhanced translation for both IRE mRNA (capped and uncapped), confirming the contribution of eIF4G and iron as a potent enhancer of ferritin IRE mRNA translation. Fluorescence data revealed that ferritin IRE strongly interacts to eIF4G (Kd = 63 nM), but not eIF4E. Further equilibrium studies showed that iron enhanced (~4-fold) the ferritin IRE binding to eIF4G. The equilibrium binding effects of iron on ferritin IRE RNA/eIFs interaction and the temperature dependence of this reaction were measured and compared. The Kd values for the IRE binding to eIF4G ranging from 18.2 nM to 63.0 nM as temperature elevated from 5 °C to 25 °C, while the presence of iron showed much stronger affinity over the same range of temperatures. Thermodynamic parameter revealed that IRE RNA binds to eIF4G with ΔH = –42.6 ± 3.3 kJ. mole-1, ΔS = –11.5 ± 0.4 J. mole-1K-1, and ΔG = –39.2 ± 2.7 kJ. mole-1, respectively. Furthermore, addition of iron significantly changed the values of thermodynamic parameters, favoring stable complex formation, thus favoring efficient protein synthesis. This study first time demonstrate the participation of eIF4G in ferritin IRE mRNA translation. Conclusions: eIF4G specifically interacts with ferritin IRE RNA and promotes eIF4G-dependent translation.

eukaryotic initiation factor
in vitro translation
protein-RNA interaction
Fig. 1.
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