TRANSLATIONAL REGULATION OF MESSENGER-RNAS WITH DISTINCT IRE SEQUENCES BY IRON REGULATORY PROTEIN-1 AND PROTEIN-2

Iron regulatory proteins 1 and 2 (IRP-1, IRP-2) interact with iron-res ponsive elements (IREs) present in the 5'- or 3'-untranslated regions (UTR) of several mRNAs coding for proteins in iron metabolism, Whereas binding of IRP-1 and -2 to an IRE in the 5'-UTR inhibits mRNA transla tion in vitro, it has remained unknown whether either endogenous prote in is sufficient to control translation in mammalian cells. We analyze d this question by taking advantage of published mutant IREs that are exclusively recognized by either IRP-1 or IRP-2 in vitro. These IREs w ere inserted into the 5'-UTR of a human growth hormone reporter mRNA, and translational regulation was measured in stably transfected mouse L cells, Cells cultured in iron-rich or -depleted medium were labeled with [S-35]methionine, and secreted growth hormone was immunoprecipita ted, IREs with loop sequences specific for IRP-1 (UAGUAC), IRP-2 (CCGA GC), or both proteins (GAGUCG and the wild-type CAGUGC sequence) all m ediated translational regulation, in contrast to a control sequence (G CUCCG) that binds neither IRP-1 nor IRP-2, Control experiments exclude d IRP-1 binding to the IRP-2-specific sequence in vivo, The present da ta demonstrate that IRP-1 and IRP-2 can independently function as tran slational repressors in living cells.,