Structural requirements of iron-responsive elements for binding of the protein involved in both transferrin receptor and ferritin mRNA post-transcriptional regulation

Abstract

The synthesis of both transferrin receptor (TfR) and ferritin is regulated post-transcriptionally by iron. This is mediated by iron responsive elements (IREs) in the 5''- and 3''-untranslated regions, respectively, of TfR and ferritin mRNAs. Although these IREs have different sequences, they both form a chracteristic stem-loop. We used competition assays and partial peptide mapping of UV-crosslinked ferritin and TfR IRE-protein complexes to show that the cytosolic protein binding to the ferritin 5''-IRE, the iron-responsive element binding protein (IRE-BP), also binds to TfR 3''-IREs. To identify the structural requirements necessary for RNA-protein binding, ferritin IRE RNAs were synthesized which contained altered secondary structures and base substitutions. Affinities of thse RNAs for IRE-BP were assayed in RNA-protein binding gels. Substitutions disrupting base-pairing of the stem preventing the IRE-BP binding. Substitutions which restored base-pairing also restored IRE-BP binding. We conclude that teh IRE-BP binds to both ferritin and TfR IREs and recognizes a particular IRE conformation.