A Stem-Loop in the 3′-UTR Mediates Iron-Dependent Regulation of Alpha Hemoglobin Stabilizing Protein mRNA Stability.

Alpha-hemoglobin stabilizing protein (AHSP) acts as a molecular chaperone that binds and stabilizes alpha globin, minimizing the formation of cytotoxic alpha-globin precipitates in red blood cells. It has been proposed that variations in AHSP protein levels could influence disease severity in patients with beta-thalassemia. A number of genomic sequences that could be involved with AHSP transcriptional regulation have been studied. Multiple consensus GATA-1 binding motifs and a single CACCC-binding site have been identified and characterized. In vivo studies demonstrated that AHSP gene transcription is dependent on both EKLF and GATA-1 transcription factors. Here, we identify an IRE (iron response-like element) in the AHSP 3′ untranslated region that is predicted to regulate mRNA stability. IREs are regulatory elements that encode a phylogenetically defined sequence-structure motif that provide a specific recognization site for the IRE-binding proteins (IRE-BP1 or IRE-BP2). Iron starvation of cells induces high affinity binding of cytoplasmic IRE-BP to an IRE, which has at least two distinct known biological consequences. IRE in the 5′-UTR of an mRNA provides for iron-dependent regulation of translation, whereas an IRE in the 3′-UTR confers iron-dependent regulation of mRNA stability. Computer modeling identified a potential IRE stem-loop structure near the 3′-end of murine and human AHSP mRNAs. In vitro binding studies indicate that this structure recognizes an IRE-BP. Moreover, the affinity of this interaction was reduced by mutations that disrupt base pairing within the stem-loop. Iron supplementation, which is predicted to decrease IRE-BP affinity for the IRE, destabilizes AHSP in human and mouse erythroleukemia cells. These results indicate that AHSP expression is regulated by RNA stability in an iron-dependent fashion. Our findings have implications for the control of AHSP expression in beta-thalassemia.