The influence of the base-paired flanking region on structure and function of the ferritin mRNA iron regulatory element.

Ferritin and transferrin receptors are co-ordinately regulated by the same RNA-protein interaction: the conserved iron regulatory element (IRE) in mRNA and the IRE-binding protein (IRE-BP/IRP/FRP/P-90). The 28 nucleotide IRE in ferritin mRNA is a single copy, with base-paired flanking regions (FL), located near the 5' cap. In the transferrin receptor mRNA, the IRE is located in the 3' untranslated region, as five variable copies and lacking predicted base-paired flanking regions; an alternate predicted structure without IREs has similar stability. When iron is scarce, ferritin mRNA does not form polyribosomes whereas the transferrin receptor mRNA is translated; when iron is abundant, ferritin mRNA forms polyribosomes and the transferrin receptor mRNA is degraded. To investigate structures which contribute to differences in the regulation of the two mRNAs, the effect of mutation of the ferritin FL was studied. Changes in structure (changes in reactivity with RNase V1 and RNase S1. Fe-bleomycin) and changes in function (translation in rabbit reticulocyte extracts) were compared for mutant and wild-type FL sequences in ferritin mRNA. The disruption of a triplet of base-pairs in the FL had diminished regulation; a second mutation to restore the triplet base-pairs conferred wild-type translational regulation. Conformation of the mutant RNA-IRE-BP complex was also different. We show that the triplet of base-pairs is conserved; the triplet is also the location of IRE-BP-dependent conformational changes in the FL structure previously observed. Increasing FL base-pairs had no effect on function. Structural changes associated with altered function included bleomycin sites in the IRE, suggesting an alternate conformation of the hairpin, and different base-stacking (V1 sensitivity) in the FL. The function of the FL, which is altered by mutation of phylogenetically conserved triplet base-pairs, may be enhancement of formation of a particular IRE stem-loop-protein interaction.