Combinatorial mRNA Regulation: Iron Regulatory Proteins and Iso-iron-responsive Elements (Iso-IREs)

iron-responsive element iron regulatory protein internal loop/bulge protein kinase C transferrin receptor erythroid aminolevulinate synthase mitochondrial aconitase cytoplasmic aconitase Combinations of RNA elements (mRNA specific) with binding proteins give a wide range of responses to biological signals from iron, oxygen, NO, or growth factors. Combinatorial regulation of transcription to coordinate synthesis of groups of proteins is well known and is exemplified by steroid hormone-responsive genes (1Darimont B.D. Wagner R.L. Apriletti J.W. Stallcup M.R. Kushner P.J. Baxter J.D. Fletterick R.J. Yamamoto K.R. Genes Dev. 1998; 12: 3343-3356Crossref PubMed Scopus (824) Google Scholar). Combinatorial regulation of mRNA utilization to coordinate synthesis of groups of proteins is unique currently to iron and oxygen metabolism in animals (2–15) (see Fig. 1). The RNA elements are called iso-iron-responsive elements (iso-IREs),1 and the binding proteins, called iso-iron regulatory proteins (iso-IRPs), are aconitase homologues. Examples of iso-IRE mRNAs are ferritin to concentrate iron, TfR and DMT-1 for iron uptake, and ferroportin (Fpn1/IREG1/MTP1) for iron efflux. Several proteins for oxygen metabolism are also encoded in iso-IRE mRNAs, exemplified by aminolevulinate synthase (eALAS) in heme synthesis and mt-aconitase in the trichloroacetic acid cycle. Signals that control iso-IRE/iso-IRP binding include iron, oxygen, hydrogen peroxide, NO, and activators of protein kinase C.When IRE regulation of mRNA function was last described in a Minireview (1990) only two IRE-mRNAs (ferritin and TfR) were known (2Theil E.C. J. Biol. Chem. 1990; 265: 4771-4774Abstract Full Text PDF PubMed Google Scholar), in contrast to the many IRE mRNAs currently known. Iron was the only known signal, and knowledge of structure was limited to RNA sequence and secondary structure determined by prediction and enzymatic/chemical probes (2Theil E.C. J. Biol. Chem. 1990; 265: 4771-4774Abstract Full Text PDF PubMed Google Scholar). Annotation of the literature in the intervening period is in Refs. 4Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 5Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar, 6Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (565) Google Scholar, 7Theil E.C. Met. Ions Biol. Syst. 1998; 35: 403-434PubMed Google Scholar. Now much is known about IRE tertiary structure (7Theil E.C. Met. Ions Biol. Syst. 1998; 35: 403-434PubMed Google Scholar). Multiple signaling pathways are known to converge on the IRE/IRP interaction (4Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 6Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (565) Google Scholar, 8Ke Y. Wu J. Leibold E.A. Walden W.E. Theil E.C. J. Biol. Chem. 1998; 273: 23637-23640Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 9Ke Y.H. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar, 10Fleming M.D. Trenor III, C.C. Su M.A. Foernzler D. Beier D.R. Dietrich W.F. Andrews N.C. Nat. Genet. 1997; 16: 383-386Crossref PubMed Scopus (1015) Google Scholar, 11Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2632) Google Scholar). The combinatorial RNA/protein family and the effects of the RNA protein complex on protein synthesis are illustrated in Fig. 1. The result of the RNA-binding protein specificity, for the different iso-IRE-containing mRNAs, is quantitative differences in the expression of proteins that are finely tuned over a wide range. Such precise control over the synthesis of each of the proteins relates to the central role of the proteins in normal cell biology: iron trafficking, heme synthesis, and cellular ATP production.The flexibility of regulation using the IRE/IRP mRNA/protein interactions is illustrated by the liver where the same amount of iron induces ferritin synthesis up to 100-fold (2Theil E.C. J. Biol. Chem. 1990; 265: 4771-4774Abstract Full Text PDF PubMed Google Scholar), but mitochondrial aconitase is only induced 2–3-fold (3Eisenstein R.S. Blemings K.P. J. Nutr. 1998; 128: 2295-2298Crossref PubMed Scopus (141) Google Scholar); the difference likely relates to a narrow tolerance of cells to concentration changes in trichloroacetic acid cycle enzymes. Differences in the iso-IRE binding in each mRNA suggest a higher percentage of ferritin mRNA will be bound to IRPs than mt-aconitase mRNA (see Fig. 3), allowing quantitative variations in the response of protein synthesis to signals. An alternate mechanism for IRE/IRP control of protein synthesis is regulated mRNA turnover, illustrated by the TfR IRE.Figure 3IRE· IRP complexes: IRP1 phosphorylation and the Fe-S/apo cycle; IRP2 sensitivity to the internal loop/bulge.The differential behavior of IRP1 and IRP2 in IRE recognition is illustrated in two ways. Left, indirect activation of IRP1 by altering Fe-S cluster stability is enhanced by phosphorylation. For IRP2, in contrast, phosphorylation appears to modulate the redox state of the protein. Red, wild type; yellow, S138A. Phosphomimetic mutants S138D (orange) and S138E (blue) are shown. Data are taken from Refs. 37Schalinske K.L. Eisenstein R.S. J. Biol. Chem. 1996; 271: 7168-7176Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar and 39Brown N.M. Anderson S.A. Steffen D.W. Carpenter T.B. Kennedy M.C. Walden W.E. Eisenstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15235-15240Crossref PubMed Scopus (73) Google Scholar.Right, differential binding of iso-IRPs to iso-IREs. IRP1 binds all iso-IREs, whereas IRP2 binds well only when the internal loop/bulge is present (Fig. 2). Lanes 1–5 are iso-IREs of ferritin, Fer-ΔU6, TfR, eALAS, and mt-aconitase, respectively, from Ref. 8Ke Y. Wu J. Leibold E.A. Walden W.E. Theil E.C. J. Biol. Chem. 1998; 273: 23637-23640Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar.View Large Image Figure ViewerDownload (PPT)Iso-IRE StructureIRE-containing mRNAs have been identified in vertebrates, invertebrates, and bacteria. They encode proteins that function in iron uptake, storage, and export (mammals, birds, amphibia, insects, and bacteria) (4Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 10Fleming M.D. Trenor III, C.C. Su M.A. Foernzler D. Beier D.R. Dietrich W.F. Andrews N.C. Nat. Genet. 1997; 16: 383-386Crossref PubMed Scopus (1015) Google Scholar, 11Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2632) Google Scholar, 12Donovan A. Brownlie A. Zhou Y. Shepard J. Pratt S.J. Moynihan J. Paw B.H. Drejer A. Barut B. Zapata A. Law T.C. Brugnara C. Lux S.E. Pinkus G.S. Pinkus J.L. Kingsley P.D. Palis J. Fleming M.D. Andrews N.C. Zon L.I. Nature. 2000; 403: 779-781Crossref Scopus (1338) Google Scholar, 13McKie A.T. Marciani P. Rolfs A. Brennan K. Wehr K. Barrow D. Miret S. Bomford A. Peters T.J. Farzaneh F. Hediger M.A. Hentze M.W. Simpson R.J. Mol. Cell. 2000; 5: 299-309Abstract Full Text Full Text PDF PubMed Scopus (1183) Google Scholar, 15Alén C. Sonenshein A.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10412-10417Crossref PubMed Scopus (132) Google Scholar) and for heme synthesis or the trichloroacetic acid cycle/ATP production (mammals, amphibia, fish, insects, and bacteria). No IREs have been detected in plants, although an IRE-hybridizable, nonferritin sequence in soybean has been observed. 2M. Ragland and E. C. Theil, unpublished results. IREs present in the 5′ or 3′ noncoding regions of mRNA were originally thought to be structurally the same, based on the predicted secondary structure of the stem loop and the similarity of IRP1 binding. However, based on comparisons among larger numbers of IRE sequences from different mRNAs coupled with additional studies of structure and binding with purified IRPs it is now apparent that the mRNA-specific divergences in IRE sequence and structure define isoforms of the IREs. Variations in IRE structure selectively influence the interactions with iso-IRPs.PrimaryComparisons of animal IRE sequences reveal that the conservation of sequence identity is much higher (>90% identity) between species for the same mRNA than between different mRNAs in the same species (36–85% identity) (16Johansson H.E. Theil E.C. Templeton D. Metal Ion Gene Regulation. Marcel Dekker, Inc., New York2001Google Scholar) (Fig. 2). IREs have 26–30 nucleotides (based on sequence conservation and protein footprint), with a central CAGUG sequence and a C residue five bases upstream. Complementary base pairs flank the C and CAGUGX. In ferritin IREs an additional set of conserved bases, U/C G-C, upstream from C create a pocket related iso-IRP2 binding (Fig. 3). Conserved non-IRE sequences occur in both the ferritin and TfR-IRE regulatory elements and influence function (17Dix D.J. Lin P.-N. McKenzie A.R. Walden W.E. Theil E.C. J. Mol. Biol. 1993; 231: 230-240Crossref PubMed Scopus (58) Google Scholar, 18Schlegl J. Gegout V. Schlager B. Hentze M.W. Westhof E. Ehresmann C. Ehresmann B. Romby P. RNA. 1997; 3: 1159-1172PubMed Google Scholar).Figure 2Iso-IRE structure. Top,primary structure of an iso-IRE (TfR-IREb); there are five copies of IREs in the TfR turnover element (a, b, c, d, e)). Note the high interspecies conservation (>95%), which contrasts with the iso-IRE mRNA-specific variation in the same species (15–65%) (GenBankTM accession numbers M11507, X01060, M58040,X13753, X55348, and AW454691. Bottom left, hairpin secondary structure. IL/B (ferritin) and C-bulge (eALAS) IREs illustrate a conserved terminal loop with variable midstem distortion and helix base pairs. Bottom right, three-dimensional structure MC-SYM/NMR model of IL/B IRE (ferritin) viewed from the major groove (21Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (76) Google Scholar).Yellow, IL/B cavity; blue, docked metal (Co(III) hexamine). (See Ref. 20Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (167) Google Scholar to compare a C-bulge IRE structure.)View Large Image Figure ViewerDownload (PPT)SecondaryIso-IREs fold with the CAGUGX in a terminal hexaloop (Fig. 2) containing a C-G base pair required for IRP binding, because substitution of A for G prevents binding of either iso-IRP1 or iso-IRP2 (2Theil E.C. J. Biol. Chem. 1990; 265: 4771-4774Abstract Full Text PDF PubMed Google Scholar, 9Ke Y.H. Sierzputowska-Gracz H. Gdaniec Z. Theil E.C. Biochemistry. 2000; 39: 6235-6242Crossref PubMed Scopus (44) Google Scholar, 19Henderson B.R. Menotti E. Kuhn L.C. J. Biol. Chem. 1996; 271: 4900-4908Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Substitution of U-A for C-G selectively inhibits binding of iso-IRP2 (19Henderson B.R. Menotti E. Kuhn L.C. J. Biol. Chem. 1996; 271: 4900-4908Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). IRE stems have 9–10 base pairs and form an A-helix (20Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (167) Google Scholar, 21Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (76) Google Scholar) with a small distortion caused by the conserved, unpaired C or the internal loop bulge, created by the conserved G-C base pair. Structure appears to be modulated by the base pair closing the hexaloop (18Schlegl J. Gegout V. Schlager B. Hentze M.W. Westhof E. Ehresmann C. Ehresmann B. Romby P. RNA. 1997; 3: 1159-1172PubMed Google Scholar). In addition, the sequence of base pairs in the upper helix between the stem distortion and the hexaloop contributes to protein binding, exemplified by the 30-fold change in IRP1 binding for CAA/UUG → UUG/CAA (22Leibold E.A. Laudano A. Yu Y. Nucleic Acids Res. 1990; 18: 1819-1824Crossref PubMed Scopus (84) Google Scholar). Melting cooperativity of the entire IRE was also influenced by engineering the substitution of one natural helix sequence for another between the bulge/loop and the hexaloop 3Y. Ke and E. C. Theil, manuscript in preparation. or when natural iso-IREs were compared.TertiaryNMR spectroscopy and nuclease (protein/chemical) probing define IRE structures. The C-G base pair across the CAGUGX hexaloop pushes AGU into the solvent (18Schlegl J. Gegout V. Schlager B. Hentze M.W. Westhof E. Ehresmann C. Ehresmann B. Romby P. RNA. 1997; 3: 1159-1172PubMed Google Scholar, 20Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (167) Google Scholar, 23Cliftan S.A. Theil E.C. Thorp H.H. Chem. Biol. 1998; 5: 679-689Abstract Full Text PDF PubMed Scopus (15) Google Scholar). At the middle of the helix of the ferritin IRE, a G-C base pair folds the internal/loop bulge into a pocket of the large groove that selectively enhances IRP2 binding and binds metals (8Ke Y. Wu J. Leibold E.A. Walden W.E. Theil E.C. J. Biol. Chem. 1998; 273: 23637-23640Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Protonation in the physiological range alters the IRE structure at either the IL/B or C-bulge of IREs (20Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (167) Google Scholar, 21Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (76) Google Scholar), but the proton acceptor (cytosine? phosphate?) is yet not known. The large groove of the IRE stem is enlarged by distortions at the C-bulge or IL/B (20Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (167) Google Scholar, 21Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (76) Google Scholar) creating specific base and ribose contact sites for protein (18Schlegl J. Gegout V. Schlager B. Hentze M.W. Westhof E. Ehresmann C. Ehresmann B. Romby P. RNA. 1997; 3: 1159-1172PubMed Google Scholar).Taken together the studies show the impact of the IRE primary sequence and of the base pairs in the IRE hairpin on iso-IRE structure that is the foundation for the selective iso-IRP binding and regulation of the use of different iso-IRE mRNAs. trans-Factor participation in the translational regulation of ferritin mRNA was first suggested by data obtained in the 1970s and early 1980s (2Theil E.C. J. Biol. Chem. 1990; 265: 4771-4774Abstract Full Text PDF PubMed Google Scholar, 6Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (565) Google Scholar, 7Theil E.C. Met. Ions Biol. Syst. 1998; 35: 403-434PubMed Google Scholar). The two proteins identified since then, IRP1 and IRP2 (4Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 6Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (565) Google Scholar), specifically inhibit the translation or turnover of IRE-containing mRNAs. It has become clear that the IRPs also have distinct binding properties (8Ke Y. Wu J. Leibold E.A. Walden W.E. Theil E.C. J. Biol. Chem. 1998; 273: 23637-23640Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), sensitivity to environmental iron and oxygen signals, and mechanisms of response to the signals and to phosphorylation (6Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (565) Google Scholar, 24Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar).Iso-IRP StructureIso-IRPs are aconitase homologues and (as for iso-IREs) are each more highly conserved between species (>90% identity) than for IRP1 and IRP2 in the same species (61% identity). The first IRP identified, IRP1, cycles between the RNA binding form (apo-c-aconitase) and cytoplasmic aconitase (c-aconitase), which has an [4Fe-4S] iron-sulfur cofactor.Iso-IRP1When peptides from c-aconitase are compared with the sequence of IRP1 predicted from the cDNA, the identity is >98%. In crystals of mt-aconitase the Fe-S cluster is in a solvent-filled cleft (25Beinert H. Kiley P.J. Curr. Opin. Chem. Biol. 1999; 3: 152-157Crossref PubMed Scopus (176) Google Scholar). By analogy, assuming the IRP-specific insertions are only in the surface loops that do not affect folding (4Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 5Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar, 26Kaldy P. Menotti E. Moret R. Kuhn L.C. EMBO J. 1999; 18: 6073-6083Crossref PubMed Scopus (48) Google Scholar), the IRE binding site has been suggested to be close to or the same as the binding cleft of the Fe-S cluster. Some residues predicted to reside in the putative cleft of iso-IRP1/c-aconitase are required for both aconitase function and iron regulation of mRNA function/RNA binding, based on site-directed mutagenesis and cross-linking studies (4Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 5Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar, 26Kaldy P. Menotti E. Moret R. Kuhn L.C. EMBO J. 1999; 18: 6073-6083Crossref PubMed Scopus (48) Google Scholar). It has been hypothesized that the presence or absence of the Fe-S cluster modulates the extent to which the cleft is open and able to bind the IRE complex, and evidence supporting this model has been obtained (26Kaldy P. Menotti E. Moret R. Kuhn L.C. EMBO J. 1999; 18: 6073-6083Crossref PubMed Scopus (48) Google Scholar, 42Basilion J.P. Rouault T.A. Massinople C.M. Klausner R.D. Burgess W.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 574-578Crossref PubMed Scopus (108) Google Scholar, 43Schalinske K.L. Anderson S.A. Tuazon P.T. Chen O.S. Eisenstein R.S. Biochemistry. 1997; 36: 3950-3958Crossref PubMed Scopus (63) Google Scholar). However, the detailed structural analyses required to prove such a notion have yet to be completed.Iso-IRP2The high sequence identity between IRP1 and IRP2, 61%, excludes a 73-amino acid insertion unique to IRP2 near the amino terminus of the protein. IRP2 does not form an Fe-S cluster; iron regulates RNA binding by targeted proteasomal degradation (24Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar). The 73-amino acid loop is required for iron-induced, IRP2 degradation because a chimera of IRP1 with the IRP2-specific loop inserted at the analogous site displayed enhanced degradation in cells with excess iron. The redox state of cysteine residues in both IRPs and complexation with the Fe-S cluster in IRP1 can influence RNA binding and provide a potential site for regulation by oxygen, NO, and oxyradicals (26Kaldy P. Menotti E. Moret R. Kuhn L.C. EMBO J. 1999; 18: 6073-6083Crossref PubMed Scopus (48) Google Scholar).Multifactorial Regulation of IRP FunctionThe ratio of IRP1/IRP2 varies in a cell-specific fashion, although exploration of cell-specific control of the ratio has been studied only briefly (24Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar). Iron regulates IRP expression post-transcriptionally (27Guo B. Brown F.M. Phillips J.D., Yu, Y. Leibold E.A. J. Biol. Chem. 1995; 270: 11653-16529Google Scholar) and post-translationally (6Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (565) Google Scholar, 28Goessling L.S. Mascotti D.P. Thach R.E. J. Biol. Chem. 1998; 273: 12555-12557Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In addition to differences in steady state concentrations of iso-IRPs, the iron signal acts post-translationally on two different IRP activity parameters: direct RNA binding (IRP1) or protein turnover (IRP2) with differing sensitivities. When the iron signal is heme, protein turnover is enhanced for both IRP1 and IRP2 (28Goessling L.S. Mascotti D.P. Thach R.E. J. Biol. Chem. 1998; 273: 12555-12557Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The oxygen signals also have differential effects on the iso-IRPs. In sum, the relative contributions of IRP1 and IRP2 to IRE binding vary over a broad range including the complete absence of IRP1 (29Schalinske K.L. Blemings K.P. Steffen D.W. Chen O.S. Eisenstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10681-10686Crossref PubMed Scopus (57) Google Scholar). Ablation of the gene for IRP1 in mice has no detectable phenotype to but of the IRP2 gene has R.J. Iron New The Scholar). In addition to the differences in binding, selective regulation of IRP function by different biological signals the of this regulatory and required for iron is a protein. Iron regulates IRP1 by an Fe-S cluster on the which RNA binding The Fe-S form of IRP1 is which is the of the [4Fe-4S] iron-sulfur aconitase (4Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 5Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar). The Fe-S cluster is a in one of the two protein the detailed structural changes induced of the Fe-S cluster it is clear that the two of the protein are and that changes in cellular iron can the protein to (4Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 6Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (565) Google Scholar). The in IRP1 function with the Fe-S cluster the role of iron-sulfur proteins as (25Beinert H. Kiley P.J. Curr. Opin. Chem. Biol. 1999; 3: 152-157Crossref PubMed Scopus (176) Google a of gene will be required to the iron and based on studies of Fe-S cluster in and proteins in and (25Beinert H. Kiley P.J. Curr. Opin. Chem. Biol. 1999; 3: 152-157Crossref PubMed Scopus (176) Google Scholar, J. D.J. J. Nutr. 1999; PubMed Scopus Google Scholar). of Fe-S in in genes for of and of iron for cluster D.R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar); analogous genes occur in C. B. H. J. E.A. Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar, Rouault T.A. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the proteins and such as and Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus (44) Google Scholar, S. P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), which Fe-S cluster are for the the cytoplasmic of IRP2 protein by and proteasomal of or of IRP2 protein in cells although the which to be (6Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (565) Google Scholar, S. P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The specific sites and mechanisms of of IRP2 are currently variations in the activity of the protein and degradation pathways are likely to to quantitative differences in the of iron and oxygen signals on IRP2 in the literature about changes of IRP2 for be in to such cell-specific variations in two mechanisms that IRP function will be are cell-specific variations in the ratio of expression of and in the concentration of iso-IREs. When with the differential interaction of the iso-IRPs with iso-IREs (Fig. 3), an range of responses can be predicted and the sensitivity of the iso-IRE/iso-IRP response is an for IRP and oxygen are central to cell The regulation of which encode proteins of both iron and oxygen metabolism (2Theil E.C. J. Biol. Chem. 1990; 265: 4771-4774Abstract Full Text PDF PubMed Google Scholar, 7Theil E.C. Met. Ions Biol. Syst. 1998; 35: 403-434PubMed Google Scholar), and the phosphorylation of IRP1 or IRP2 that the regulatory is with more signal of both IRP1 and IRP2 has been in cells in which the RNA binding activity of IRP1 and IRP2 was also K.L. Eisenstein R.S. J. Biol. Chem. 1996; 271: 7168-7176Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). liver IRP1 is an for protein kinase C up to of of protein in Ref. R.S. Kennedy M.C. H. S. Walden Metal Ions and Gene Regulation. and New Scholar). and were for is near and for and is in the for high RNA binding. of the Fe-S cluster phosphorylation sensitivity of residues is also (Fig. (29Schalinske K.L. Blemings K.P. Steffen D.W. Chen O.S. Eisenstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10681-10686Crossref PubMed Scopus (57) Google Scholar). Such in suggest that phosphorylation the c-aconitase (IRP1) and the set at which iron regulates IRE/IRP binding. Phosphomimetic in IRP1, S138D and aconitase (Fig. (29Schalinske K.L. Blemings K.P. Steffen D.W. Chen O.S. Eisenstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10681-10686Crossref PubMed Scopus (57) Google Scholar) and to in contrast to wild IRP1 (29Schalinske K.L. Blemings K.P. Steffen D.W. Chen O.S. Eisenstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10681-10686Crossref PubMed Scopus (57) Google Scholar, J. R. Walden W.E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, 39Brown N.M. Anderson S.A. Steffen D.W. Carpenter T.B. Kennedy M.C. Walden W.E. Eisenstein R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15235-15240Crossref PubMed Scopus (73) Google Scholar). and the cells also the sensitivity of a Fe-S cluster in the protein K. H. E. Kiley P.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google phosphorylation has been much but the predicted phosphorylation sites are in the 73-amino acid insertion required for activation the high RNA binding (6Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (565) Google Scholar) form of IRP2 in cells K.L. Eisenstein R.S. J. Biol. Chem. 1996; 271: 7168-7176Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), a role for IRP2 phosphorylation in and differences in phosphorylation of IRP1, have a major impact on the control of iron IRP1 or IRP2 phosphorylation will the cell of expression and will a large to the range of responses of IRE-containing mRNAs to cellular iron regulation of mRNA proteins of iron was by variations in the of regulatory effects over a range of O.S.