Generic placeholder image

Current Chemical Biology


ISSN (Print): 2212-7968
ISSN (Online): 1872-3136

Research Article

Analysis of Ion and pH Effects on Iron Response Element (IRE) and mRNA-Iron Regulatory Protein (IRP1) Interactions

Author(s): Mateen A. Khan*

Volume 14, Issue 2, 2020

Page: [88 - 99] Pages: 12

DOI: 10.2174/2212796814999200604121937

Price: $65


Background: Cellular iron uptake, utilization, and storage are tightly controlled through the action of iron regulatory proteins (IRPs). IRPs achieve this control by binding to IREs-mRNA in the 5'- or 3'-end of mRNAs that encode proteins involved in iron metabolism. The interaction of iron regulatory proteins with mRNAs containing an iron responsive element plays a central role in this regulation. The IRE RNA family of mRNA regulatory structures combines absolutely conserved protein binding sites with phylogenetically conserved base pairs that are specific to each IREs and influence RNA/protein stability. Our previous result revealed the binding and kinetics of IRE RNA with IRP1. The aim of the present study is to gain further insight into the differences in protein/RNA stability as a function of pH and ionic strength.

Objective: To determine the extent to which the binding affinity and stability of protein/RNA complex was affected by ionic strength and pH.

Methods: Fluorescence spectroscopy was used to characterize IRE RNA-IRP protein interaction.

Results: Scatchard analysis revealed that the IRP1 protein binds to a single IRE RNA molecule. The binding affinity of two IRE RNA/IRP was significantly changed with the change in pH. The data suggests that the optimum binding of RNA/IRP complex occurred at pH 7.6. Dissociation constant for two IRE RNA/IRP increased with an increase in ionic strength, with a larger effect for FRT IRE RNA. This suggests that numerous electrostatic interactions occur in the ferritin IRE RNA/IRP than ACO2 IRE RNA/IRP complex. Iodide quenching shows that the majority of the tryptophan residues in IRP1 are solvent-accessible, assuming that most of the tryptophan residues contribute to protein fluorescence.

Conclusion: The results obtained from this study clearly indicate that IRE RNA/IRP complex is destabilized by the change in pH and ionic strength. These observations suggest that both pH and ion are important for the assembly and stability of the IRE RNA/IRP complex formation.

Keywords: Fluorescence, Binding affinity, IRE RNA, IRP1, pH, ionic strength.

« Previous
Graphical Abstract
De Domenico I, McVey Ward D, Kaplan J. Regulation of iron acquisition and storage: consequences for iron-linked disorders. Nat Rev Mol Cell Biol 2008; 9(1): 72-81.
[] [PMID: 17987043]
Piccinelli P, Samuelsson T. Evolution of the iron-responsive element. RNA 2007; 13(7): 952-66.
[] [PMID: 17513696]
Khan MA, Walden WE, Goss DJ, Theil EC. Direct Fe2+ sensing by iron-responsive messenger RNA: repressor complexes weakens binding. J Biol Chem 2009; 284(44): 30122-8.
[] [PMID: 19720833]
Khan MA, Ma J, Walden WE, Merrick WC, Theil EC, Goss DJ. Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions. Nucleic Acids Res 2014; 42(10): 6567-77.
[] [PMID: 24728987]
Ma J, Haldar S, Khan MA, et al. Fe2+ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA repression and activation. Proc Natl Acad Sci USA 2012; 109(22): 8417-22.
[] [PMID: 22586079]
Walden WE, Selezneva AI, Dupuy J, et al. Structure of dual function iron regulatory protein 1 complexed with ferritin IRE-RNA. Science 2006; 314(5807): 1903-8.
[] [PMID: 17185597]
Selezneva AI, Walden WE, Volz KW. Nucleotide-specific recognition of iron-responsive elements by iron regulatory protein 1. J Mol Biol 2013; 425(18): 3301-10.
[] [PMID: 23806658]
Theil EC, Eisenstein RS. Combinatorial mRNA regulation: iron regulatory proteins and iso-iron-responsive elements (Iso-IREs). J Biol Chem 2000; 275(52): 40659-62.
[] [PMID: 11062250]
Hentze MW, Muckenthaler MU, Galy B, Camaschella C. Two to tango: regulation of Mammalian iron metabolism. Cell 2010; 142(1): 24-38.
[] [PMID: 20603012]
Wilkinson N, Pantopoulos K. The IRP/IRE system in vivo: insights from mouse models. Front Pharmacol 2014; 5: 176.
[] [PMID: 25120486]
Zhang DL, Ghosh MC, Rouault TA. The physiological functions of iron regulatory proteins in iron homeostasis - an update. Front Pharmacol 2014; 5: 124.
[] [PMID: 24982634]
Chen SC, Olsthoorn RCL. Relevance of the iron-responsive element (IRE) pseudotriloop structure for IRP1/2 binding and validation of IRE-like structures using the yeast three-hybrid system. Gene 2019; 710: 399-405.
[] [PMID: 31200088]
Zähringer J, Baliga BS, Munro HN. Novel mechanism for translational control in regulation of ferritin synthesis by iron. Proc Natl Acad Sci USA 1976; 73(3): 857-61.
[] [PMID: 1083028]
Theil EC, Goss DJ. Living with iron (and oxygen): questions and answers about iron homeostasis. Chem Rev 2009; 109(10): 4568-79.
[] [PMID: 19824701]
Muckenthaler M, Gray NK, Hentze MW. IRP-1 binding to ferritin mRNA prevents the recruitment of the small ribosomal subunit by the cap-binding complex eIF4F. Mol Cell 1998; 2(3): 383-8.
[] [PMID: 9774976]
Muckenthaler MU, Galy B, Hentze MW. Systemic iron homeostasis and the iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network. Annu Rev Nutr 2008; 28: 197-213.
[] [PMID: 18489257]
Guo B, Phillips JD, Yu Y, Leibold EA. Iron regulates the intracellular degradation of iron regulatory protein 2 by the proteasome. J Biol Chem 1995; 270(37): 21645-51.
[] [PMID: 7665579]
Fleming RE, Ponka P. Iron overload in human disease. N Engl J Med 2012; 366(4): 348-59.
[] [PMID: 22276824]
Leibold EA, Munro HN. Characterization and evolution of the expressed rat ferritin light subunit gene and its pseudogene family. Conservation of sequences within noncoding regions of ferritin genes. J Biol Chem 1987; 262(15): 7335-41.
[PMID: 3584116]
Cox TC, Bawden MJ, Martin A, May BK. Human erythroid 5-aminolevulinate synthase: promoter analysis and identification of an iron-responsive element in the mRNA. EMBO J 1991; 10(7): 1891-902.
[] [PMID: 2050125]
Dandekar T, Stripecke R, Gray NK, et al. Identification of a novel iron-responsive element in murine and human erythroid delta-aminolevulinic acid synthase mRNA. EMBO J 1991; 10(7): 1903-9.
[] [PMID: 2050126]
Zhou Z D, Tan E K. Iron regulatory protein (IRP)- iron responsive element (IRE) signaling pathway in human neurodegenerative diseases. Mol Neurodegener 2017; 12(1): 75,1-13.
Khan MA, Walden WE, Theil EC, Goss DJ. Thermodynamic and Kinetic Analyses of Iron Response Element (IRE)-mRNA Binding to Iron Regulatory Protein, IRP1. Sci Rep 2017; 7(1): 1-11.
Ke Y, Wu J, Leibold EA, Walden WE, Theil EC. Loops and bulge/loops in iron-responsive element isoforms influence iron regulatory protein binding. Fine-tuning of mRNA regulation? J Biol Chem 1998; 273(37): 23637-40.
[] [PMID: 9726965]
Chen OS, Schalinske KL, Eisenstein RS. Dietary iron intake modulates the activity of iron regulatory proteins and the abundance of ferritin and mitochondrial aconitase in rat liver. J Nutr 1997; 127(2): 238-48.
[] [PMID: 9039823]
Selezneva AI, Cavigiolio G, Theil EC, Walden WE, Volz K. Crystallization and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex with ferritin IRE RNA. Acta Crystallogr Sect F Struct Biol Cryst Commun 2006; 62(Pt 3): 249-52.
[] [PMID: 16511314]
Brazzolotto X, Timmins P, Dupont Y, Moulis JM. Structural changes associated with switching activities of human iron regulatory protein 1. J Biol Chem 2002; 277(14): 11995-2000.
[] [PMID: 11812787]
Gdaniec Z, Sierzputowska-Gracz H, Theil EC. Iron regulatory element and internal loop/bulge structure for ferritin mRNA studied by cobalt(III) hexammine binding, molecular modeling, and NMR spectroscopy. Biochemistry 1998; 37(6): 1505-12.
[] [PMID: 9484220]
Erlitzki R, Long JC, Theil EC. Multiple, conserved iron-responsive elements in the 3′-untranslated region of transferrin receptor mRNA enhance binding of iron regulatory protein 2. J Biol Chem 2002; 277(45): 42579-87.
[] [PMID: 12200453]
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-54.
[] [PMID: 942051]
Khan MA, Miyoshi H, Ray S, Natsuaki T, Suehiro N, Goss DJ. Interaction of genome-linked protein (VPg) of turnip mosaic virus with wheat germ translation initiation factors eIFiso4E and eIFiso4F. J Biol Chem 2006; 281(38): 28002-10.
[] [PMID: 16880203]
Scatchard G. The attraction of proteins for small molecules and ions. Ann N Y Acad Sci 1949; 51: 660-72.
Tayyab S, Khan NJ, Khan MA, Kumar Y. Behavior of various mammalian albumins towards bilirubin binding and photochemical properties of different bilirubin-albumin complexes. Int J Biol Macromol 2003; 31(4-5): 187-93.
[] [PMID: 12568927]
Levine RL. Fluorescence-quenching studies of the binding of bilirubin to albumin. Clin Chem 1977; 23(12): 2292-301.
[] [PMID: 923078]
Khan MA, Muzammil S, Musarrat J. Differential binding of tetracyclines with serum albumin and induced structural alterations in drug-bound protein. Int J Biol Macromol 2002; 30(5): 243-9.
[] [PMID: 12297231]
Sha M, Wang Y, Xiang T, van Heerden A, Browning KS, Goss DJ. Interaction of wheat germ protein synthesis initiation factor eIF-(iso)4F and its subunits p28 and p86 with m7GTP and mRNA analogues. J Biol Chem 1995; 270(50): 29904-9.
[] [PMID: 8530388]
Khan MA, Goss DJ. Poly (A) binding protein enhances the binding affinity of potyvirus VPg to eukaryotic initiation factor eIF4F and activates in vitro translation. Int J Biol Macromol 2019; 121: 947-55.
[] [PMID: 30342940]
Lehrer SS. Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry 1971; 10(17): 3254-63.
[] [PMID: 5119250]
Lakowicz JR. Principles of Fluorescence Spectroscopy. New York: Plenum Publishing 1983; pp. 279-84.
Carberry SE, Rhoads RE, Goss DJ. A spectroscopic study of the binding of m7GTP and m7GpppG to human protein synthesis initiation factor 4E. Biochemistry 1989; 28(20): 8078-83.
[] [PMID: 2605173]
Khan MA. Phosphorylation of translation initiation factor eIFiso4E promotes translation through enhanced binding to potyvirus VPg. J Biochem 2019; 165(2): 167-76.
[] [PMID: 30371907]
Sha M, Balasta ML, Goss DJ. An interaction of wheat germ initiation factor 4B with oligoribonucleotides. J Biol Chem 1994; 269(21): 14872-7.
[PMID: 8195117]
Walden WE, Selezneva A, Volz K. Accommodating variety in iron-responsive elements: Crystal structure of transferrin receptor 1 B IRE bound to iron regulatory protein 1. FEBS Lett 2012; 586(1): 32-5.
[] [PMID: 22119729]
Rhoads RE, Hellmann GM, Remy P, Ebel JP. Translational recognition of messenger ribonucleic acid caps as a function of pH. Biochemistry 1983; 22(26): 6084-8.
[] [PMID: 6661428]
Khan MA, Yumak H, Goss DJ. Kinetic mechanism for the binding of eIF4F and tobacco Etch virus internal ribosome entry site rna: effects of eIF4B and poly(A)-binding protein. J Biol Chem 2009; 284(51): 35461-70.
[] [PMID: 19858189]
Katsamba PS, Myszka DG, Laird-Offringa IA. Two functionally distinct steps mediate high affinity binding of U1A protein to U1 hairpin II RNA. J Biol Chem 2001; 276(24): 21476-81.
[] [PMID: 11297556]
Lipfert J, Das R, Chu VB, et al. Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae. J Mol Biol 2007; 365(5): 1393-406.
[] [PMID: 17118400]
Coppins RL, Hall KB, Groisman EA. The intricate world of riboswitches. Curr Opin Microbiol 2007; 10(2): 176-81.
[] [PMID: 17383225]
Brantl S. Metal sensing by RNA in bacteria: exception or rule? ACS Chem Biol 2007; 2(10): 656-60.
[] [PMID: 18041815]
Noeske J, Schwalbe H, Wöhnert J. Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain. Nucleic Acids Res 2007; 35(15): 5262-73.
[] [PMID: 17686787]

Rights & Permissions Print Cite
© 2024 Bentham Science Publishers | Privacy Policy