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• W2018906039 abstract "A family of noncoding mRNA sequences, iron-responsive elements (IREs), coordinately regulate several mRNAs through binding a family of mRNA-specific proteins, iron regulatory proteins (IRPs). IREs are hairpins with a constant terminal loop and base-paired stems interrupted by an internal loop/bulge (in ferritin mRNA) or a C-bulge (in m-aconitase, erythroid aminolevulinate synthase, and transferrin receptor mRNAs). IRP2 binding requires the conserved C-G base pair in the terminal loop, whereas IRP1 binding occurs with the C-G or engineered U-A. Here we show the contribution of the IRE internal loop/bulge to IRP2 binding by comparing natural and engineered IRE variants. Conversion of the internal loop/bulge in the ferritin-IRE to a C-bulge, by deletion of U, decreased IRP2 binding by >95%, whereas IRP1 binding changed only 13%. Moreover, IRP2 binding to natural IREs with the C-bulge was similar to the ΔU6 ferritin-IRE: >90% lower than the ferritin-IRE. The results predict mRNA-specific variation in IRE-dependent regulation in vivo and may relate to previously observed differences in iron-induced ferritin and m-aconitase synthesis in liver and cultured cells. Variations in IRE structure and cellular IRP1/IRP2 ratios can provide a range of finely tuned, mRNA-specific responses to the same (iron) signal. A family of noncoding mRNA sequences, iron-responsive elements (IREs), coordinately regulate several mRNAs through binding a family of mRNA-specific proteins, iron regulatory proteins (IRPs). IREs are hairpins with a constant terminal loop and base-paired stems interrupted by an internal loop/bulge (in ferritin mRNA) or a C-bulge (in m-aconitase, erythroid aminolevulinate synthase, and transferrin receptor mRNAs). IRP2 binding requires the conserved C-G base pair in the terminal loop, whereas IRP1 binding occurs with the C-G or engineered U-A. Here we show the contribution of the IRE internal loop/bulge to IRP2 binding by comparing natural and engineered IRE variants. Conversion of the internal loop/bulge in the ferritin-IRE to a C-bulge, by deletion of U, decreased IRP2 binding by >95%, whereas IRP1 binding changed only 13%. Moreover, IRP2 binding to natural IREs with the C-bulge was similar to the ΔU6 ferritin-IRE: >90% lower than the ferritin-IRE. The results predict mRNA-specific variation in IRE-dependent regulation in vivo and may relate to previously observed differences in iron-induced ferritin and m-aconitase synthesis in liver and cultured cells. Variations in IRE structure and cellular IRP1/IRP2 ratios can provide a range of finely tuned, mRNA-specific responses to the same (iron) signal. iron-responsive element(s) iron regulatory protein(s) mitochondrial aconitase transferrin receptor erythroid aminolevulinate synthase. The iron-responsive element (IRE),1 present in the 5′- or 3′-noncoding regions of animal mRNAs encoding proteins of iron and oxidative metabolism, regulates synthesis of the encoded proteins posttranscriptionally. Iron regulatory proteins (IRPs) bind to the IREs to inhibit ribosome binding or protect mRNA from ribonuclease cleavage (1Theil E.C. Sigel A. Signel H. Metal Ions in Biological Systems. Marcel Dekker, New York1998: 403-434Google Scholar, 2Theil E.C. Metal Ions in Gene Regulation.in: Silver S. Walden W. International Thomson Publishing, New York1997Google Scholar, 3Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 4Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1141) Google Scholar, 5Leibold E.A. Guo B. Annu. Rev. Nutr. 1992; 12: 345-368Crossref PubMed Scopus (120) Google Scholar). The predicted secondary structures of the IRE family are hairpins with a six-nucleotide terminal loop (CAGUGN*, N* = A, C, or U), interrupted by an internal loop/bulge (UGC/C) (ferritin-IRE) or a C-bulge (TfR, eALAS, and m-aconitase IREs), that is generally supported by enzymatic cleavage and chemical probing (6Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar, 7Wang Y.-H. Lin P.-N. Sczekan S.R. McKenzie R.A. Theil E.C. Biol. Metals. 1991; 4: 56-61Crossref PubMed Scopus (15) Google Scholar, 8Bettany A.J.E. Eisenstein R.S. Munro H.M. J. Biol. Chem. 1992; 267: 16531-16537Abstract Full Text PDF PubMed Google Scholar); NMR spectroscopy shows a G-C base pair in the hairpin loop and in the internal loop/bulge (9Sierzputowska-Gracz H. McKenzie R.A. Theil E.C. Nucleic Acids Res. 1995; 23: 145-152Crossref Scopus (48) Google Scholar, 10Liang L.G. Hall K.B. Biochemistry. 1996; 35: 13585-13596Google Scholar, 11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar, 12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar). Two IRE-binding proteins, IRP1 and IRP2, have a high sequence identity except for a 73-amino acid insertion unique to IRP2, and each of them has 30% sequence identity to m-aconitase; IRP1 can have aconitase activity (13Samaniego F. Chin J. Iwai K. Rouault T.A. Klausner R.D. J. Biol. Chem. 1994; 269: 30904-30910Abstract Full Text PDF PubMed Google Scholar, 14Iwai K. Klausner R.D. Rouault T.A. EMBO J. 1995; 14: 5350-5357Crossref PubMed Scopus (193) Google Scholar, 15Hentze W.H. Argos P. Nucleic Acids Res. 1991; 19: 1739-1740Crossref PubMed Scopus (132) Google Scholar, 16Hirling H. Henderson B.R. Kuhn L.C. EMBO J. 1994; 13: 453-461Crossref PubMed Scopus (153) Google Scholar, 17Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (301) Google Scholar). IRP1 and IRP2 binding to IREs in iron-depleted cells is abrogated when iron is in excess, with IRP1 forming an [4Fe-4S] cluster (16Hirling H. Henderson B.R. Kuhn L.C. EMBO J. 1994; 13: 453-461Crossref PubMed Scopus (153) Google Scholar, 17Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (301) Google Scholar, 18Haile D.J. Rouault R.A. Harford J.B. Kennedy M.C. Blondin G.A. Beinert H. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11735-11739Crossref PubMed Scopus (266) Google Scholar, 19Philpott C.C. Klausner R.D. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7321-7325Crossref PubMed Scopus (117) Google Scholar), and IRP2 being degraded (14Iwai K. Klausner R.D. Rouault T.A. EMBO J. 1995; 14: 5350-5357Crossref PubMed Scopus (193) Google Scholar, 20Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar, 21Guo B. Brown F.M. Phillips J.D., Yu, Y. Leibold E.A. J. Biol. Chem. 1995; 270: 16529-16535Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22Guo B. Phillips J.D., Yu, Y. Leibold E.A. J. Biol. Chem. 1995; 270: 21645-21651Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). IRP phosphorylation (23Eisenstein R.L. Tuazon P.T. Schalinske K.L. Anderson S.A. Traugh J.A. J. Biol. Chem. 1993; 268: 27363-27370Abstract Full Text PDF PubMed Google Scholar, 24Schalinske K.L. Eisenstein R.S. J. Biol. Chem. 1996; 271: 7168-7175Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), indicates that IRP functions may be integrated with more general metabolic signals. The significance of two IRPs, apparently equivalent in terms of RNA binding and posttranscriptional regulation, is a puzzle, since exclusivity of IRP1 or IRP2 binding for one or another natural IRE sequence has not yet been observed (25Henderson B.R. Menotti E. Bonnard C. Kuhn L.C. J. Biol. Chem. 1994; 269: 17481-17489Abstract Full Text PDF PubMed Google Scholar, 26Henderson B.R. Menotti E. Kuhn L.C. J. Biol. Chem. 1996; 271: 4900-4908Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 27Menotti E. Henderson B.R. Kuhn L.C. J. Biol. Chem. 1998; 273: 1821-1824Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 28Butt J. Kim H.-Y. Basilion J.P. Cohen S. Iwai K. Philpott C.C. Altschul S. Klausner R.D. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4345-4349Crossref PubMed Scopus (129) Google Scholar). IRP binding specificity for the internal loop/bulge and C-bulge of IREs examined in this study, showed that conversion of the ferritin-IRE internal loop/bulge to a C-bulge, by deletion of a single base U6, decreased IRP2 binding 20-fold, with only a small effect on IRP1 binding. Similarly, a C-bulge in the natural IREs (m-aconitase, erythroid ALAS (eALAS), and the transferrin receptor (TfR)), decreased IRP2 binding 10-fold, compared with the ferritin-IRE. Natural IRP1 and IRP2 in a cell extract produced results similar to those observed with recombinant IRPs. The results coincide with structural differences observed by NMR spectroscopy (11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar, 12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar) and Cu(phen)2 probing (6Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar). 2Y. Ke and E. C. Theil, manuscript in preparation. The differential sensitivity of IRP1 and IRP2 binding to natural variations in IREs at the junction of the two helices (internal loop/bulge or C-bulge) suggests that the presence of two IRPs broadens the regulatory range of IREs and emphasizes the importance of the internal loop/bulge region in RNA-protein interactions. RNA, transcribed using T7 RNA polymerase and a chemically synthesized DNA template (9Sierzputowska-Gracz H. McKenzie R.A. Theil E.C. Nucleic Acids Res. 1995; 23: 145-152Crossref Scopus (48) Google Scholar, 29Milligan J.F. Uhlenbeck U.C. Methods Enzymol. 1989; 180: 51-61Crossref PubMed Scopus (1026) Google Scholar), was purified on 12% polyacrylamide/urea gels; concentrated by ethanol precipitation, resuspended in water and stored at −80 °C until use. 5′-32P labeling of RNA was carried out as described previously (6Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar, 30Harrell C.M. McKenzie A.R. Patino M.M. Walden W.E. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1-6Crossref PubMed Scopus (78) Google Scholar), with purification through NENSORBTMcolumns (DuPont). 5′-32P-Labeled RNAs were heated to 85 °C for 5 min in 100 mm KCl, 40 mm Hepes, pH 7.2 and annealed to 25 °C before each use. In competition experiments, unlabeled RNAs were heated and annealed as described for labeled RNA before adding to the binding reaction. If RNA was only heated to 65 °C, the percentage that bound IRP1 was greatly decreased (50–60%). Binding of recombinant IRPs was accomplished by incubation of RNA (0.9 fmol, ∼1.5 × 105 cpm) and protein at 10 °C for 30 min in 20 μl of 60 mm KCl, 24 mm Hepes·Na, pH = 7.2, 4 mm MgCl2, 5% glycerol, 2% 2-mercaptoethanol; protein:RNA was 15:1. Almost all (80–90%) of the ferritin-IRE was bound by IRP1, but only 30–45% of the RNA was bound by IRP2, suggesting that inactive IRP2 was present in preparations of IRP2; 2% 2-mercaptoethanol does not decrease binding by IRP1 or IRP2 (14Iwai K. Klausner R.D. Rouault T.A. EMBO J. 1995; 14: 5350-5357Crossref PubMed Scopus (193) Google Scholar). RNA-protein complexes were separated from RNA in 4% nondenaturing acrylamide gels (acrylamide:bis = 19:1) in Tris borate-EDTA buffer (90 mm Tris borate, 2 mmEDTA, pH 8.0), 8 volts/cm for 1 h at 10 °C. Binding of IRPs in rabbit reticulocyte lysates (∼30 μg/20 μl reaction mixture), prepared as before (31Shull G.E. Theil E.C. J. Biol. Chem. 1982; 257: 14187-14191Abstract Full Text PDF PubMed Google Scholar), used the same binding buffer, but with tRNA (50 μg/ml). The IRP2·IRE complex was identified with anti-IRP2 serum, generated against the 73-amino acid insertion in IRP2 (20Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar); 2 μl of the serum were added after 20 min of incubation, followed by 10 min incubation, addition of heparin (7.5 mg/ml) (20Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar, 32Leibold E.A. Munro H.N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2171-2175Crossref PubMed Scopus (561) Google Scholar, 33Phillips J.D. Guo B., Yu, Y. Brown F.M. Leibold E.A. Biochemistry. 1996; 35: 15704-15714Crossref PubMed Scopus (42) Google Scholar) and electrophoresis in a 5% native acrylamide gel (acrylamide:bis = 19:1), 12 volts/cm at 4 °C. Order of antiserum addition had no significant effect on the results. Recombinant IRP1 was isolated from the cytosol of Saccharomyces cerevisiae BJ5465 (34Jones E.W. Methods Enzymol. 1991; 194: 428-453Crossref PubMed Scopus (367) Google Scholar) containing the rabbit liver IRP1 sequence (35Patino M.M. Walden W.E. J. Biol. Chem. 1992; 267: 19011-19016Abstract Full Text PDF PubMed Google Scholar) in plasmid pYES-His (Invitrogen, Inc.), grown in minimal medium without uracil, and with 3% glycerol, 2% galactose (36Rose M.D. Winston F.M. Heiter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990: 179-186Google Scholar). The IRP1 was purified as His-tagged protein with a nickel-chelate column (Amersham Pharmacia Biotech), followed by heparin-agarose chromatography in 20 mm Tris-Cl, pH 7.4, 50 mm KCl, 1 mm EDTA, 2 mm sodium citrate, 10% glycerol, 7 mm mercaptoethanol, and stored at −80 °C. Recombinant IRP2-His, which appears to be less stable than IRP1 (22Guo B. Phillips J.D., Yu, Y. Leibold E.A. J. Biol. Chem. 1995; 270: 21645-21651Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 37Iwai K. Drake S. Wehr N.B. Weissman A.M. LaVaute T. Minato N. Klausner R.D. Levine R.L. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; VOL: 4924-4928Crossref Scopus (265) Google Scholar), was prepared on nickel-chelate columns as described by Phillips et al. (33Phillips J.D. Guo B., Yu, Y. Brown F.M. Leibold E.A. Biochemistry. 1996; 35: 15704-15714Crossref PubMed Scopus (42) Google Scholar). Previous studies that compared IRP1 and IRP2 binding had shown that differential IRP binding occurred only with mutations in the hairpin loop (25Henderson B.R. Menotti E. Bonnard C. Kuhn L.C. J. Biol. Chem. 1994; 269: 17481-17489Abstract Full Text PDF PubMed Google Scholar, 26Henderson B.R. Menotti E. Kuhn L.C. J. Biol. Chem. 1996; 271: 4900-4908Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 27Menotti E. Henderson B.R. Kuhn L.C. J. Biol. Chem. 1998; 273: 1821-1824Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 28Butt J. Kim H.-Y. Basilion J.P. Cohen S. Iwai K. Philpott C.C. Altschul S. Klausner R.D. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4345-4349Crossref PubMed Scopus (129) Google Scholar), but not in natural IREs (20, 38-40). The hairpin loop is the most conserved part of the IREs; evolutionary divergence occurs in the stem and internal loop regions (1Theil E.C. Sigel A. Signel H. Metal Ions in Biological Systems. Marcel Dekker, New York1998: 403-434Google Scholar, 2Theil E.C. Metal Ions in Gene Regulation.in: Silver S. Walden W. International Thomson Publishing, New York1997Google Scholar, 3Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 4Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1141) Google Scholar, 5Leibold E.A. Guo B. Annu. Rev. Nutr. 1992; 12: 345-368Crossref PubMed Scopus (120) Google Scholar). Recent studies of IREs by NMR and other approaches, which showed significant structural differences in the internal loop/bulge and C-bulge IREs (11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar, 12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar, 41Ke Y. Theil E.C. FASEB J. 1998; 12: A1474Google Scholar), stimulated reexamination of whether IRP1 and IRP2 differentially bind to the internal loop/bulge and C-bulge IREs. To enhance RNA conformational homogeneity, we synthesized RNA of comparable size (28–30 nucleotides), purified the RNA using denaturing gel electrophoresis, heated the purified RNA to 85 °C, and annealed before each use (see “Experimental Procedures”). The influence of the internal loop/bulge characteristic of the ferritin IRE was investigated with recombinant IRPs, by examining the effect of the deletion of U6, which converted the internal loop/bulge into the C-bulge (Fig. 1, a and e). IRP2 recognizes the ferritin-IRE ΔU6 much more poorly than the ferritin-IRE (Fig. 2 Aand Table I) in contrast to IRP1. Mutated ferritin-IRE HL1, HL2, and C8A (Fig. 1, f–h and Fig. 2 A) were controls, to show that results under the conditions used were comparable to those previously observed (25Henderson B.R. Menotti E. Bonnard C. Kuhn L.C. J. Biol. Chem. 1994; 269: 17481-17489Abstract Full Text PDF PubMed Google Scholar, 26Henderson B.R. Menotti E. Kuhn L.C. J. Biol. Chem. 1996; 271: 4900-4908Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 28Butt J. Kim H.-Y. Basilion J.P. Cohen S. Iwai K. Philpott C.C. Altschul S. Klausner R.D. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4345-4349Crossref PubMed Scopus (129) Google Scholar, 42Leibold E.A. Laudano A. Yu Y. Nucleic Acids Res. 1990; 18: 1819-1824Crossref PubMed Scopus (86) Google Scholar).Figure 2Sensitivity of IRP1 and IRP2 binding to the structure of the IRE internal loop/bulge-recombinant proteins.5′-32P-IREs were incubated with or without purified, recombinant IRP1 or IRP2 at a ratio of 1:15, RNA:protein (A). In the competition experiments (B) 5′-32P-labeled ferritin-IRE was mixed with unlabeled RNA, at ratios of 1:10 or 1:50, before adding IRP. The results are from 3–5 experiments with at least two preparations of RNA and protein. Predicted structures of the RNAs are in Fig. 1. A,32P-RNA + IRP1 or IRP2; B, 32P-RNA + unlabeled competitor RNA + IRP2. ⇑ = IRP/RNA complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IQuantitation of recombinant IRP binding to the internal loop/bulge or the C bulge structures of natural and mutant IREIRE bindingIRP1 bindingIRP2 bindingMolar excess of unlabeled RNA required to prevent 32P-labeled RNAFer (IL/B)10010010xFer ΔU6 (B)87 ± 5<5>50xTfR (B)102 ± 310 ± 4>50xeALAS (B)100 ± 111 ± 2>50xm-Aconitase (B)80 ± 212 ± 4>50xFer C6A (IL/B)24 ± 7<2NDFer HL1 (IL/B)<1<1NDFer HL2 (IL/B)89 ± 4<5NDRecombinant IRP1 and IRP2 were incubated with 32P-labeled RNA, and the protein/RNA complexes were resolved by electrophoresis (see “Experimental Procedures”). The relative amount of the IRE bound by IRP was quantitated with a PhosphorImager (Molecular Dynamics) and Image Quant software. Data for IRE/IRP complexes were normalized to the Fer IRE·IRP complex. The data are the average of 2–4 experiments using 2 RNA preparations for each IRE; the error is presented as the S.D. Fer = ferritin.ND, = not determined. Open table in a new tab Recombinant IRP1 and IRP2 were incubated with 32P-labeled RNA, and the protein/RNA complexes were resolved by electrophoresis (see “Experimental Procedures”). The relative amount of the IRE bound by IRP was quantitated with a PhosphorImager (Molecular Dynamics) and Image Quant software. Data for IRE/IRP complexes were normalized to the Fer IRE·IRP complex. The data are the average of 2–4 experiments using 2 RNA preparations for each IRE; the error is presented as the S.D. Fer = ferritin. ND, = not determined. Natural IREs all have the same C-G base pair and the pentameric sequence, CAGUG in the terminal hexaloop, but vary in structure at the interhelix junction (internal loop/bulge or C-bulge). Conversion of the ferritin internal loop/bulge to a C-bulge by deletion (ΔU6) differentially altered IRP recognition (Fig. 2 A and Table I). Thus, IRP1 and IRP2 should also have different interactions with the natural C-bulge IREs (m-aconitase, TfR, and eALAS IREs) compared with the natural internal loop/bulge IRE (ferritin-IRE). The results proved our prediction. All natural C-bulge IREs bind IRP2 much more poorly than IRP1 (10–12%) when compared with ferritin-IRE binding (Fig. 2 A and Table I) and are similar to the ferritin-IRE ΔU6. The observation that the internal loop/bulge enhanced IRP2 binding was confirmed by competition experiments with unlabeled RNAs. A 10-fold molar excess of unlabeled ferritin-IRE (internal loop/bulge IRE) prevented binding of the labeled ferritin-IRE to IRP2, whereas >50-fold molar excess of unlabeled TfR, m-aconitase, or eALAS IREs (C-bulge IREs) were required (Fig. 2 B and Table I). To ensure that the differential binding of recombinant IRP2 to IREs with an internal loop/bulge or a C-bulge (Fig. 2 and Table I) was a property of IRPs, and independent of possible differences between natural and recombinant proteins, IRE-protein binding was examined with IRPs in rabbit reticulocyte lysates comparing of the ferritin-IRE to the TfR-IRE. IRPs in such cell extracts can regulate translation of IRE-containing mRNAs (43Dickey L.F. Wang Y.-H. Shull G.E. Wortman I.A. Theil E.C. J. Biol. Chem. 1988; 263: 3071-3074Abstract Full Text PDF PubMed Google Scholar, 44Walden W.E. Daniels-McQueen S. Brown P.H. Gaffield L. Russell D.A. Bielser D. Bailey L.C. Thach R.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9503-9507Crossref PubMed Scopus (81) Google Scholar, 45Dix D.J. Lin P.-N. Kimata Y. Theil E.C. Biochemistry. 1992; 31: 2818-2822Crossref PubMed Scopus (52) Google Scholar, 46Bhasker C.R. Burgiel G. Neupert B. Emery-Goodman A. Kuhn L.C. May B.K. J. Biol. Chem. 1993; 268: 12699-12705Abstract Full Text PDF PubMed Google Scholar). The ferritin-IRE formed two RNA-protein complexes in the red cell extracts, in relatively equal amounts (Fig. 3 A, lane 3), whereas the TfR-IRE formed only one RNA-protein complex (Fig. 3 A, lane 4). The complex in the lower band formed with the ferritin IRE was identified as an IRP2·RNA complex with IRP2 antibody (compare lanes 5, 6, and 3, 4 in Fig. 3 A). The binding of TfR-IRE to IRP1 is 0.52 ± 0.05 that of the ferritin-IRE (Fig. 3 A, lanes 5 and 6, upper bands). (Similar results were obtained with fresh preparations of purified natural IRP1, but with recombinant IRP1, ferritin-IRE and TfR-IRE binding was the same, either because of the His tag used or the absence of posttranslational modifications such as phosphorylation or both.) The binding of TfR-IRE to endogenous IRP2 in red cell extracts was 0.10 ± 0.03 that of the ferritin-IRE (Fig. 3 A), which was comparable with binding to recombinant IRP2 (Fig. 2 A). IRP isoforms IRP1 and IRP2 showed quantitative differences in binding to IREs from different mRNAs (Figs. 2 and 3 and Table I). The ferritin IRE is recognized best by both IRP1 and IRP2, compared with the m-aconitase, TfR, and eALAS IREs. Accordingly, a larger fraction of the ferritin IRE is likely to be complexed with IRPs than other IREs, which explains the observation that IRE-dependent regulation in vivo and in vitro has the greatest range for the ferritin IRE (38Kim H.-Y. LaVaute T. Iwai K. Klausner R.D. Rouault T.A. J. Biol. Chem. 1996; 271: 24226-24230Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 40Dandekar T. Stripecke R. Gray N.K. Goossen B. Constable A. Johansson H.E. Hentze M.W. EMBO J. 1991; 10: 1903-1909Crossref PubMed Scopus (281) Google Scholar,47Casey J.L. Hentze M.W. Koeller D.M. Caughman S.W. Rouault T.A. Klausner R.D. Harford J.B. Science. 1988; 240: 924-928Crossref PubMed Scopus (513) Google Scholar, 48Melefors O. Goossen B. Johansson H.E. Stripecke R. Gray N.K. Hentze M.W. J. Biol. Chem. 1993; 268: 5974-5978Abstract Full Text PDF PubMed Google Scholar, 49Chen O.S. Schalinske K.L. Eisenstein R.S. J. Nutr. 1997; 127: 238-248Crossref PubMed Scopus (102) Google Scholar). Among IRE isoforms, variations in the IRE/IRP interaction were greatest for IRP2, suggesting that IRP2 will make the major contribution to differential IRE-dependent regulationin vivo. IRP1/IRP2 ratios vary considerably in different cell types, exemplified by liver, kidney, and intestine: IRP1 > IRP2 (50Henderson B.R. Seiser C. Kuhn L.C. J. Biol. Chem. 1993; 268: 27327-27334Abstract Full Text PDF PubMed Google Scholar), in RRL: IRP1∼IRP2 (Fig. 3 A) and in a pro-B-lymphocyte cell line, which appears to have only IRP2 (51Schalinske 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 (58) Google Scholar). IRP2 is sensitive to engineered changes in the IRE hairpin loop and internal loop/bulge. Because the hairpin loop structure is conserved in all natural IREs, its contribution to IRP2 binding will be constant. However, the variation in structure of natural IREs, with C-bulge or the internal loop/bulge, will differentially influence IRP2 binding to natural IREs (Figs. 2 and 3 and Table I). The ferritin-IRE ΔU6 with the C-bulge was an even poorer competitor for IRP2 binding than natural IRE isoforms with a C-bulge (Fig. 2 B), suggesting context effects even within the group of IREs with a C-bulge. NMR studies suggest more conformational flexibility at the internal loop/bulge than at the C-bulge in IREs (11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar, 12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar). The IRE consensus sequence designed for NMR studies (11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar), contained a C-bulge, ΔU6, and 3 G-C base pairs next to the bulge in the lower stem, creating an analogue of the ΔU6 ferritin-IRE, which likely behaves similarly in IRP2 binding. Note that in addition to the C-bulge or internal loop/bulge, effects of IRE flanking regions have been observed on the predicted structure (m-aconitase IRE) (52Schalinske K.L. Chen O.S. Eisenstein R.S. J. Biol. Chem. 1998; 273: 3740-3746Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and on both solution structure and translation regulation (ferritin-IRE) (53Dix D.J. Lin P.-N. McKenzie A.R. Walden W.E. Theil E.C. J. Mol. Biol. 1993; 231: 230-240Crossref PubMed Scopus (59) Google Scholar). RNA conformational flexibility which is matched to differences in the binding proteins, as recently emphasized for BIV-TAT/tar interactions (54Frankel A.D. Smith C.A. Cell. 1998; 92: 149-151Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), may also explain the differential binding of IRP1 and IRP2 to the IRE isoforms (Figs. 2 and 3 and Table I). For example, the appropriate conformation around the C residue required to bind IRP2 may be more readily achieved by an IRE with an internal loop/bulge, whereas IRP1 may be able to lock onto the C in any IRE. The C residue was disordered in both C-bulge and internal loop/bulge IREs (11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar, 12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar), but the internal loop/bulge forms a flexible pocket in the major groove near the conserved C residue (12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar). IRP contacts the RNA surface on the minor groove (55Basilion 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 (110) Google Scholar), and the flexibility of the internal loop/bulge in the major groove may allow an “induced fit” needed for IRP2 binding on the minor groove surfaces. The question of IRE-dependent coordination of ferritin, m-aconitase, or TfR synthesis is raised by the tissue- and cell type-specific distribution of IRP1 and IRP2 and the differential binding of IRP2 to ferritin, m-aconitase, and TfR IREs in vitro (Figs. 2 and 3 and Table I). When IRP2 predominates in cells, IRE-dependent repression may be greater for ferritin than for other mRNAs, which can explain differential iron regulation of ferritin and m-aconitase mRNAs in rat liver and cultured cells (38Kim H.-Y. LaVaute T. Iwai K. Klausner R.D. Rouault T.A. J. Biol. Chem. 1996; 271: 24226-24230Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar,49Chen O.S. Schalinske K.L. Eisenstein R.S. J. Nutr. 1997; 127: 238-248Crossref PubMed Scopus (102) Google Scholar, 52Schalinske K.L. Chen O.S. Eisenstein R.S. J. Biol. Chem. 1998; 273: 3740-3746Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). On the other hand, the apparently equal regulation of TfR and ferritin by iron in B-lymphocyte cells lacking IRP1 (51Schalinske 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 (58) Google Scholar) could be attributed to other factors such as multiple IRE copies and/or to alternate structures (56Mullner E.W. Neupert B. Kuhn L.C. Cell. 1989; 58: 373-382Abstract Full Text PDF PubMed Scopus (404) Google Scholar, 57Theil E.C. Biochem. J. 1994; 304: 1-11Crossref PubMed Scopus (183) Google Scholar). Since both IRP2 and IRP1 can be phosphorylated by protein kinase C (24Schalinske K.L. Eisenstein R.S. J. Biol. Chem. 1996; 271: 7168-7175Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), phosphorylation of IRP2 may change IRE binding and could allow coordinate regulation of m-aconitase and TfR mRNAs with ferritin mRNA in IRP2-dominant cell types. Controlled coordination of IRE-dependent regulation through protein kinases would create a regulatory interface between the IRE-dependent regulatory pathways and other metabolic pathways. Differential binding among IRE isoforms, coupled with IRP responses to iron (17Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (301) Google Scholar, 18Haile D.J. Rouault R.A. Harford J.B. Kennedy M.C. Blondin G.A. Beinert H. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11735-11739Crossref PubMed Scopus (266) Google Scholar, 20Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar), and the potential for regulated phosphorylation and modulation of RNA/protein recognition, indicate the potential for high precision and fine-tuning of IRE-dependent mRNA regulation." @default.
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• W2018906039 title "Loops and Bulge/Loops in Iron-responsive Element Isoforms Influence Iron Regulatory Protein Binding" @default.
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