Fe2+ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA...

Area of Science:

• Molecular Biology
• RNA Biology
• Iron Metabolism

Background:

• Iron-responsive element (IRE)-mRNAs regulate protein synthesis crucial for iron homeostasis.
• Current models inadequately explain how iron influences IRE-RNA/protein interactions and subsequent translation.
• The role of IRE-RNA structure in mediating these regulatory events remains incompletely understood.

Purpose of the Study:

• To elucidate the molecular mechanism by which iron (Fe2+) modulates IRE-RNA binding to regulatory proteins.
• To investigate the conformational changes in IRE-RNA induced by metal ions.
• To identify the factors involved in iron-dependent translation of IRE-mRNAs.

Main Methods:

• Fe2+ binding to IRE-RNA was assessed using 2-aminopurine fluorescence and ethidium bromide displacement assays.
• In vitro translation assays were performed using IRE-mRNA and metal ions.
• Protein-RNA interactions were studied using binding assays with iron-regulatory proteins (IRP) and eukaryotic initiation factor (eIF)4F.

Main Results:

• Fe2+ directly binds to IRE-RNA, inducing conformational changes.
• Metal ions (Fe2+, Mn2+) were shown to increase IRE-RNA/IRP dissociation (K(D)) and enhance IRE-mRNA translation in vitro.
• Eukaryotic initiation factor (eIF)4F binds IRE-RNA with high affinity, and Fe2+ enhances this interaction, outcompeting IRP binding.
• Exogenous eIF4F rescued metal-dependent translation in depleted extracts.

Conclusions:

• IRE-RNA acts as a riboregulator, directly sensing metabolic iron (Fe2+).
• Iron binding to IRE-RNA promotes dissociation of inhibitory IRPs and facilitates binding of activating eIF4F.
• This mechanism explains how iron increases protein synthesis rates from IRE-mRNAs, highlighting a novel regulatory pathway.