STRUCTURAL AND FUNCTIONAL STUDIES OF RNA BINDING GENE REGULATORY AND IRON STORAGE PROTEINS
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In phylogenetically diverse bacteria, the conserved protein RapZ plays a central role in RNA-mediated regulation of amino-sugar metabolism. RapZ contributes to the control of glucosamine phosphate biogenesis by selectively presenting the regulatory small RNA GlmZ to the essential ribonuclease RNase E for inactivation. Here, we report the crystal structures of full length Escherichia coli RapZ at 3.40 Å and 3.25 Å, and its isolated C-terminal domain at 1.17 Å resolution. The structural data confirm that the N-terminal domain of RapZ possesses a kinase fold, whereas the C-terminal domain bears closest homology to a subdomain of 6-phosphofructokinase, an important enzyme in the glycolytic pathway. RapZ self-associates into a domain swapped dimer of dimers, and in vivo data support the importance of quaternary structure in RNA-mediated regulation of target gene expression. Based on biochemical, structural and genetic data, we suggest a mechanism for binding and presentation by RapZ of GlmZ and the closely related decoy sRNA, GlmY. We discuss a scenario for the molecular evolution of RapZ through re-purpose of enzyme components from central metabolism.
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1. INTRODUCTION 1952. THE PROTEIN SYNTHESIS SYSTEM 1992.1 Aminoacyl-tRNA synthetases 1992.1.1 Glutaminyl-tRNA synthetase 2022.1.2 Glutamyl-tRNA synthetase 2062.1.3 Tyrosyl- and tryptophanyl-tRNA synthetases 2072.1.4 Methionyl-tRNA synthetase 2082.1.5 Aspartyl-tRNA synthetase 2082.1.6 Lysyl-tRNA synthetase 2092.1.7 Seryl-tRNA synthetase 2102.1.8 Glycyl- and histidyl-tRNA synthetases 2102.1.9 Phenylalanyl-tRNA synthetase 2112.2 Ribosomal proteins 2112.2.1 L7/L12 2122.2.2 L30 2142.2.3 S5 2152.2.4 S17 2152.2.5 L6 2152.2.6 L9 2162.2.7 S6 2172.2.8 L1 2172.2.9 L14 2172.2.10 S8 2172.3 Elongation factors 2182.3.1 EF-Tu 2182.3.2 EF-G 2193. SPLICEOSOMAL PROTEINS 2213.1 U1 snRNP protein A 2224. PROTEINS FROM RNA VIRUSES 2234.1 Viral enzymes and regulatory proteins 2244.1.1 Reverse transcriptase 2244.1.2 Tat 2244.1.3 Rev 2254.2 Viral capsid proteins 2254.2.1 Tobacco Mosaic Virus 2264.2.2 Satellite Tobacco Mosaic Virus 2264.2.3 Bean-Pod Mottled Virus 2264.2.4 Black Beetle Virus and Flock House Virus 2284.2.5 Bacteriophage MS2 2285. OTHER RNA-BINDING PROTEINS 2305.1 tRNA-guanine transglycosylase 2305.2 Major cold-shock protein 2305.3 Rop 2315.4 Ricin 2316. CONCLUSION 2327. ACKNOWLEDGEMENTS 233
Amino Acyl-tRNA Synthetases
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Transferrin receptor
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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 date, but loss of the IRP2 gene has very severe consequences (30Rouault T. Badman D.G. Bergeron R.J. Brittenham G.M. Iron Chelators: New Development Strategies. The Saratoga Group, Ponte Verda Beach, FL2000: 133-144Google Scholar). In addition to the differences in IRP/IRE binding, selective regulation of IRP function by different biological signals expands the versatility of this regulatory and sensory network required for maintaining iron homeostasis.Iso-IRP1IRP1/c-aconitase is a bifunctional protein. Iron regulates IRP1 by building an Fe-S cluster on the apoprotein, which blocks RNA binding activity. The Fe-S form of IRP1 is c-aconitase, which is the cytosolic isoform of the mitochondrial, [4Fe-4S] iron-sulfur enzyme, aconitase (mt-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 key determinant in selecting one of the two possible protein functions. Although the detailed structural changes induced upon formation of the Fe-S cluster remain unknown, it is clear that the two functions of the protein are mutually exclusive and that changes in cellular iron status can drive the protein to its functional extremes (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 shifts in IRP1 function associated with the Fe-S cluster assembly emphasize the emerging role of iron-sulfur proteins as biosensors (25Beinert H. Kiley P.J. Curr. Opin. Chem. Biol. 1999; 3: 152-157Crossref PubMed Scopus (176) Google Scholar).Likely a number of gene products will be required to deliver the iron and sulfur, based on studies of Fe-S cluster assembly in nitrogenase and copper trafficking proteins in yeast and humans (25Beinert H. Kiley P.J. Curr. Opin. Chem. Biol. 1999; 3: 152-157Crossref PubMed Scopus (176) Google Scholar, 33Pena M.M.O. Lee J. Thiele D.J. J. Nutr. 1999; 129: 1251-1260Crossref PubMed Scopus (602) Google Scholar). Assembly of Fe-S clusters in nitrogenase in bacteria involves genes for localized generation of sulfide and delivery of iron for cluster formation (31Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar); analogous genes occur in eucaryotes (34Voisine C. Schilke B. Ohlson M. Beinert H. Marszalek J. Craig E.A. Mol. Cell. Biol. 2000; 20: 3677-3684Crossref PubMed Scopus (72) Google Scholar, 48Land T. Rouault T.A. Mol. Cell. 1998; 2: 807-815Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Understanding the accessory proteins and factors such as NO and H2O2 (35Oliveira L. Drapier J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6550-6555Crossref PubMed Scopus (44) Google Scholar, 36Kim S. Ponka P. J. Biol. Chem. 2000; 275: 6220-6226Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), which regulate Fe-S cluster formation, are problems for the future.Iso-IRP2Iron decreases the cytoplasmic pool of IRP2 through protein oxidation, followed by its ubiquitination and proteasomal degradation. Agents capable of generating ⋅NO or NO+promote loss of IRP2 protein in cultured cells although the exact details through which these agents act remain to be defined (6Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (565) Google Scholar, 36Kim S. Ponka P. J. Biol. Chem. 2000; 275: 6220-6226Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The specific sites and mechanisms of iron-induced oxidation of IRP2 are currently undefined. Cell-specific variations in the activity of the protein oxidation and ubiquitin degradation pathways are likely to contribute to quantitative differences in the effect of iron and oxygen signals on IRP2 activity. Apparent inconsistencies in the literature about hypoxia-induced changes of IRP2 activity, for example, may be attributed in part to such cell-specific variations in IRP2.The two mechanisms that IRP function will be superimposed upon are cell-specific variations in the ratio of expression of IRP1:IRP2 and in the concentration of iso-IREs. When combined with the differential interaction of the iso-IRPs with iso-IREs (Fig. 3), an enormous range of responses can be predicted and the exquisite sensitivity of the iso-IRE/iso-IRP response is illuminated.Phosphorylation, an Iron-independent Means for Modulating IRP FunctionIron and oxygen homeostasis are central to cell biology. The cytokine regulation of IRE-mRNAs, 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 indicate that the iso-IRE/IRP regulatory system is integrated with more general signal transduction pathways.Phosphorylation of both IRP1 and IRP2 has been demonstrated in phorbol 12-myristate 13-acetate-treated HL-60 cells in which the RNA binding activity of IRP1 and IRP2 was also stimulated (37Schalinske K.L. Eisenstein R.S. J. Biol. Chem. 1996; 271: 7168-7176Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Purified rat liver IRP1 is an efficient substrate for bovine brain protein kinase C (PKC) (Km ∼0.5 μm), accommodating up to 2 mol of phosphate/mol of protein (reviewed in Ref. 32Eisenstein R.S. Kennedy M.C. Beinert H. Silver S. Walden W. Metal Ions and Gene Regulation. Chapman and Hall, New York1997: 157-216Google Scholar). Ser-138 and Ser-711 were substrates for PKC. Ser-138 is near Asp-125 and His-126 (required for catalysis) and is in the region critical for high affinity RNA binding. Removal of the Fe-S cluster increases phosphorylation 5-fold; chymotrypsin sensitivity of adjacent residues is also increased (Fig. 3) (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 vitro observations suggest that phosphorylation controls the c-aconitase ⇔ apo-c-aconitase (IRP1) interconversion and influences the set point at which iron regulates IRE/IRP binding. Phosphomimetic mutations in IRP1, S138D and S138E, destabilized aconitase (Fig. 3) (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 failed to rescue aconitase-deficient yeast grown aerobically in contrast to wild type 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, 38Narahari J. Ma R. Wang M. Walden W.E. J. Biol. Chem. 2000; 275: 16227-16234Abstract Full Text Full Text PDF PubMed Scopus (53) 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). Both mutations and S138A rescued the cells grown anaerobically. Mutation also altered the sensitivity of a Fe-S cluster in the bacterial protein FNR (40Bates D.M. Popescu C.V. Khoroshilova N. Vogt K. Beinert H. Munck E. Kiley P.J. J. Biol. Chem. 2000; 275: 6234-6240Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar).IRP2 phosphorylation has been much less studied, but the predicted PKC phosphorylation sites are in the 73-amino acid insertion required for iron-induced degradation. PKC activation stabilized the high affinity, reduced RNA binding (6Eisenstein R.S. Annu. Rev. Nutr. 2000; 20: 627-662Crossref PubMed Scopus (565) Google Scholar) form of IRP2 in HL-60 cells (37Schalinske K.L. Eisenstein R.S. J. Biol. Chem. 1996; 271: 7168-7176Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), suggesting a role for IRP2 phosphorylation in regulation.Cell type- and tissue-specific differences in phosphorylation of IRP, particularly IRP1, may have a major impact on the interorgan control of mammalian iron metabolism. IRP1 or IRP2 phosphorylation will reflect the cell specificity of PKC expression and will add a large dimension to the range of responses of IRE-containing mRNAs to cellular signals.PerspectiveCombinatorial iron regulation of mRNA encoding proteins of iron homeostasis was obscured by variations in the magnitude of regulatory effects over a range of 50–100-fold (47Chen O.S. Schali
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Efficient and precise microRNA (miRNA) biogenesis in Arabidopsis is mediated by the RNaseIII-family enzyme DICER-LIKE 1 (DCL1), double-stranded RNA-binding protein HYPONASTIC LEAVES 1 and the zinc-finger (ZnF) domain-containing protein SERRATE (SE). In the present study, we examined primary miRNA precursor (pri-miRNA) processing by highly purified recombinant DCL1 and SE proteins and found that SE is integral to pri-miRNA processing by DCL1. SE stimulates DCL1 cleavage of the pri-miRNA in an ionic strength-dependent manner. SE uses its N-terminal domain to bind to RNA and requires both N-terminal and ZnF domains to bind to DCL1. However, when DCL1 is bound to RNA, the interaction with the ZnF domain of SE becomes indispensible and stimulates the activity of DCL1 without requiring SE binding to RNA. Our results suggest that the interactions among SE, DCL1 and RNA are a potential point for regulating pri-miRNA processing.
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