Iron responsive mRNAs: a family of Fe2+ sensitive riboregulators

Abstract

Messenger RNAs (mRNAs) are emerging as prime targets for small-molecule drugs. They afford an opportunity to assert control over an enormous range of biological processes: mRNAs regulate protein synthesis rates, have specific 3-D regulatory structures, and, in nucleated cells, are separated from DNA in space and time. All of the many steps between DNA copying (transcription) and ribosome binding (translation) represent potential control points. Messenger RNAs can fold into complex, 3-D shapes, such as tRNAs and rRNAs, providing an added dimension to the 2-D RNA structure (base pairing) targeted in many mRNA interference approaches. In this Account, we describe the structural and functional properties of the IRE (iron-responsive element) family, one of the few 3-D mRNA regulatory elements with known 3-D structure. This family of related base sequences regulates the mRNAs that encode proteins for iron metabolism. We begin by considering the IRE-RNA structure, which consists of a short (~30-nucleotide) RNA helix. Nature tuned the structure by combining a conserved AGU pseudotriloop, a closing C-G base pair, and a bulge C with various RNA helix base pairs. The result is a set of IRE-mRNAs with individual iron responses. The physiological iron signal is hexahydrated ferrous ion; in vivo iron responses vary over 10-fold depending on the individual IRE-RNA structure. We then discuss the interaction between the IRE-RNA structure and the proteins associated with it. IRE-RNA structures, which are usually noncoding, tightly bind specific proteins called IRPs. These repressor proteins are bound to IRE-RNA through C-bulge and AGU contacts that flip out a loop AG and a bulge C, bending the RNA helix. After binding, the exposed RNA surface then invites further interactions, such as with iron and other proteins. Binding of the IRE-RNA and the IRP also changes the IRP conformation. IRP binding stabilities vary 10-fold within the IRE family, reflecting individual IRE-RNA paired and unpaired bases. This variation contributes to the graded (hierarchical) iron responses in vivo. We also consider the mechanisms of IRE-mRNA control. The binding of Fe(2+) to IRE-RNA facilitates IRP release and the binding of eukaryotic initiation factors (eIFs), which are proteins that assemble mRNA, ribosomes, and tRNA for translation. IRE-RNAs are riboregulators for the inorganic metabolic signal, Fe(2+); they control protein synthesis rates by changing the distribution of the iron metabolic mRNAs between complexes with enhancing eIFs and inhibitory IRPs. The regulation of mRNA in the cytoplasm of eukaryotic cells is a burgeoning frontier in biomedicine. The evolutionarily refined IRE-RNAs, although absent in plants and bacteria, constitute a model system for 3-D mRNAs in all organisms. IRE-mRNAs have yielded "proof of principle" data for small-molecule targeting of mRNA structures, demonstrating tremendous potential for chemical manipulation of mRNA and protein synthesis in living systems.

Figure 1. IRP1 binding to FRT IRE-RNA

The RNA helix bends and contact bases C8 and triloop bases A15 and G16 are flipped out. Panel A: ■, hydrated Mg²⁺, determined by solution NMR; ▲, Cu¹⁺-1.10-phenanthroline, determined by RNA cleavage in O₂. The figure prepared from Protein Data Bank file 2IPY. (From ref. , with permission.) Panel B: IRE:IRP complex showing IRP domains (1 yellow; 2 green; 3 blue; 4 red) and contacts with IRE-RNA. Panel C: Electron density of IRE in IRE:IRP complex. (From Ref. , see for details).

Figure 2. IRP1/IRE-RNA solution binding compared for three IRE-RNAs

IRP1 preferentially binds to the FRT IRE-RNA compared with ACO2 IRE-RNA with nM affinity: binding curve (protein fluorescence quenching) on the left, conserved IRE secondary structures on the right (From ref. 9).

Figure 3. Crystal structure of IRP1 as c-aconitase and complexes with IRE-RNA

A. Differences in protein domain positions between c-aconitase and IRP1:IRE-RNA complex. Left: FeS-apo-IRP; Right: IRE-RNA/IRP. RNA-magenta; protein- blue, green, red, yellow. B. Close up of the protein–RNA contacts at the RNA triloop; C. Close-up of the protein–RNA contacts at the RNA bulge C8. (From ref. , with permission).