Ferritin is a protein that has a prominent physiological role of maintaining iron homeostasis through iron sequestration. The synthesis of this protein is regulated by the Iron Responsive Element (IRE) on the Ferritin mRNA and its interaction with an apo-aconitase protein or the Iron Regulatory Protein (IRP). The binding of the IRP to the untranslated stem loop region of the IRE will inhibit translation of Ferritin creating a mechanism that is a major controller to cellular iron metabolism. Current research has demonstrated that metal ions, Fe2+ and Mn2+, and other small molecule drugs weaken the IRE-IRP interaction. The model we are investigating indicates that the metal ions are directly influencing the structure of the IRE, disrupting the binding of the IRE-IRP complex and as a result, up regulating Ferritin translation. Our study involves three different methods for observing the binding of the IRE-IRP complex, and the effect of metal ion interference. The first, an electrophoretic mobility shift assay (EMSA) was run to confirm both the binding of the IRE-IRP complex, and the dissociation of the complex by Mn2+. Second, we used a technique called high throughput selective 2’-hydroxyl acylation analyzed by primer extension (hSHAPE) chemistry to determine the structure of the IRE. hSHAPE chemistry allows us to interrogate the structure of RNA at single nucleotide resolution. We are currently using this technique to determine the subsequent modifications to the IRE when bound to IRP using a complete ligand titration of Mn2+ and Fe2+ ions and small molecule drugs. Lastly we used Atomic Force Microscopy to present a physical representation on the molecular scale of the binding interaction of the IRE-IRP complex.

Identifying the direct effects of Mn2+, Fe2+ and small molecule drugs on the iron responsive element in the human FTH1 IRE/IRP complex

249th National Meeting (2015)

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