Iron is an essential but potentially toxic nutrient for nearly all organisms. Iron deficiency is the most common human nutritional deficiency disease with 2 to 22% of Americans suffering from it depending on age and gender. At the same time excessive iron stores are associated with neurological disorders and certain cancers. Mammalian iron metabolism is modulated by two regulatory RNA binding proteins, iron regulatory protein 1 (IRP1) and IRP2. IRPs bind to iron responsive elements (IRE) in up to seven different mRNA encoding proteins critical for the maintenance of iron homeostasis or for other pathways needed during the adaptive response to iron deficiency. This includes proteins involved in the transport, use and storage of iron as well as the TCA cycle enzyme mitochondrial aconitase. It is clear that IRE-containing mRNA are differentially regulated by IRP in order to meet the physiological demands of various cell types yet relatively little is known as to how IRPs discriminate between different mRNA. Because IRPs are pivotal regulators of iron metabolism, and dysregulation of the expression of proteins encoded by IRE-containing mRNA contributes to neurodegenerative, iron overload and other diseases, it is important to elucidate the mechanisms through which IRE-containing mRNA are selectively regulated. Our overall goal is to understand how iron metabolism is controlled through the hierarchical regulation of IRE-containing mRNA. We demonstrate that mRNA with one IRE in their 5' untranslated region are differentially regulated by IRP; we propose several novel hypotheses to explain this hierarchical regulation and we propose to: 1) determine the structure of the 5' IRE region of mitochondrial aconitase (macon) mRNA and the role of IRP and specific translation factors as well as flanking sequences in the structure and/or thermodynamic stability of this region; 2) determine the role of specific translation factors, cellular protein synthetic capacity and individual IRP in the selective regulation of the use of IRE-containing mRNA; 3) elucidate the role of IRE and flanking sequences in the hierarchical regulation of the translation in vivo of mRNA containing functionally strong or weak IRE-regions in the 5' UTR. Our studies provide a comprehensive approach from the molecular to the cellular level that will: a) delineate the mechanisms that define the breadth of the IRP regulatory spectrum; b) demonstrate how target site diversity amongst RNA regulatory elements controls mRNA fate; and c) serve as a paradigm for understanding how combinatorial mRNA regulation controls fundamental biological processes.
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