Relevance of the iron-responsive element (IRE) pseudotriloop structure for IRP1/2 binding and validation of IRE-like structures using the yeast three-hybrid system

Abstract

Iron-responsive-elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5' or 3' untranslated regions (UTRs) of mRNAs, and mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5'-CAGWGH-3' (W = A or U and H= A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Mutational analysis of FTL IRE showed that deletion of the bulged-out U6 of the pseudotriloop does not significantly affect its binding to either IRP1 or 2, but substitution with C enhances binding of both IRPs. In addition, IRP1 was found more sensitive toward changes in the pseudotriloop-stabilizing C1-G5 base pair than IRP2, while mutation of the conserved G3 was lowering the binding of both IRPs. In comparison to FTL IRE other variant IREs, IRE of 5'-aminolevulinate synthase 2 (ALAS2), SLC40A1 (also known as Ferroportin-1), and endothelial PAS domain protein 1 (EPAS1) mRNA showed slightly higher, similar, and slightly weaker affinity for IRPs, respectively, while SLC11A2 IRE exhibited very weak binding to IRP1 and medium binding to IRP2, indicating the different binding modes of IRP1 and 2. Notably, -Synuclein IRE showed no detectable binding to either IRP1 or 2. Our results indicate that Y3H represents a bona fide system to characterize binding between IRPs and various IRE-like structures.