Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese
2020; Elsevier BV; Volume: 295; Issue: 50 Linguagem: Inglês
10.1074/jbc.ra120.015025
ISSN1083-351X
AutoresDaniel G.J. Smethurst, Nikolay Kovalev, Erica R. McKenzie, Dimitri G. Pestov, Natalia Shcherbik,
Tópico(s)Corrosion Behavior and Inhibition
ResumoProtein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg2+ for structure. Recent work has indicated that other metal cations can substitute for Mg2+, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe2+ and the ribosome and identify Mn2+ as a factor capable of attenuating oxidant-induced Fe2+-mediated degradation of rRNA. We report that Fe2+ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn2+ competes with Fe2+ for rRNA-binding sites and that protection of ribosomes from Fe2+-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress. Protein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg2+ for structure. Recent work has indicated that other metal cations can substitute for Mg2+, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe2+ and the ribosome and identify Mn2+ as a factor capable of attenuating oxidant-induced Fe2+-mediated degradation of rRNA. We report that Fe2+ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn2+ competes with Fe2+ for rRNA-binding sites and that protection of ribosomes from Fe2+-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress. All organisms require a number of metal elements in trace amounts, with manganese, iron, copper, zinc, selenium, cobalt, and molybdenum all considered essential for plants and animals, whereas larger amounts of magnesium, calcium, potassium, and sodium are also required (1Zoroddu M.A. Aaseth J. Crisponi G. Medici S. Peana M. Nurchi V.M. The essential metals for humans: a brief overview.J. Inorg. Biochem. 2019; 195 (30939379): 120-12910.1016/j.jinorgbio.2019.03.013Crossref PubMed Scopus (139) Google Scholar). Divalent metal cations have a long-established involvement in biomolecules including stabilizing structures and participating in the active sites of enzymes operating across a vast catalytic range (2Andreini C. Bertini I. Cavallaro G. Holliday G.L. Thornton J.M. Metal ions in biological catalysis: from enzyme databases to general principles.J. Biol. Inorg. Chem. 2008; 13 (18604568): 1205-121810.1007/s00775-008-0404-5Crossref PubMed Scopus (600) Google Scholar, 3McCall K.A. Huang C. Fierke C.A. Function and mechanism of zinc metalloenzymes.J. Nutr. 2000; 130 (10801957): 1437S-1446S10.1093/jn/130.5.1437SCrossref PubMed Google Scholar, 4Weston J. Biochemistry of magnesium.in: Rappoport Z. PATAI'S Chemistry of Functional Groups. John Wiley & Sons, Chichester, UK2009Crossref Google Scholar). Enzymes coordinating divalent metal ions act in important processes such as DNA replication (5Vashishtha A.K. Wang J. Konigsberg W.H. Different divalent cations alter the kinetics and fidelity of DNA polymerases.J. Biol. Chem. 2016; 291 (27462081): 20869-2087510.1074/jbc.R116.742494Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6Ricchetti M. Buc H. E. coli DNA polymerase I as a reverse transcriptase.EMBO J. 1993; 12 (7679988): 387-39610.1002/j.1460-2075.1993.tb05670.xCrossref PubMed Scopus (49) Google Scholar, 7Cowan J.A. Metal activation of enzymes in nucleic acid biochemistry.Chem. Rev. 1998; 98 (11848925): 1067-108810.1021/cr960436qCrossref PubMed Google Scholar), cellular metabolism and respiration (8Smith M.R. Fernandes J. Go Y.-M. Jones D.P. Redox dynamics of manganese as a mitochondrial life-death switch.Biochem. Biophys. Res. Commun. 2017; 482 (28212723): 388-39810.1016/j.bbrc.2016.10.126Crossref PubMed Scopus (60) Google Scholar, 9Poyner R.R. Cleland W.W. Reed G.H. Role of metal ions in catalysis by enolase: an ordered kinetic mechanism for a single substrate enzyme.Biochemistry. 2001; 40 (11434770): 8009-801710.1021/bi0103922Crossref PubMed Scopus (43) Google Scholar), the phosphorylation underpinning much of cellular signaling (10Knape M.J. Ballez M. Burghardt N.C. Zimmermann B. Bertinetti D. Kornev A.P. Herberg F.W. Divalent metal ions control activity and inhibition of protein kinases.Metallomics. 2017; 9 (29043344): 1576-158410.1039/c7mt00204aCrossref PubMed Google Scholar), the oxygen-evolving function of photosystem II (11Shen J.-R. The structure of photosystem II and the mechanism of water oxidation in photosynthesis.Annu. Rev. Plant Biol. 2015; 66 (25746448): 23-4810.1146/annurev-arplant-050312-120129Crossref PubMed Scopus (328) Google Scholar), and the oxygen-transporting function of hemoglobin (12Sánchez M. Sabio L. Gálvez N. Capdevila M. Dominguez-Vera J.M. Iron chemistry at the service of life: iron chemistry at the service of life.IUBMB Life. 2017; 69 (28150902): 382-38810.1002/iub.1602Crossref PubMed Scopus (44) Google Scholar). Additionally, metal cations have essential stabilizing roles in structures including phospholipid bilayers (13Martín-Molina A. Rodríguez-Beas C. Faraudo J. Effect of calcium and magnesium on phosphatidylserine membranes: experiments and all-atomic simulations.Biophys. J. 2012; 102 (22824273): 2095-210310.1016/j.bpj.2012.03.009Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and the ribosome (14Sigel R.K.O. Pyle A.M. Alternative roles for metal ions in enzyme catalysis and the implications for ribozyme chemistry.Chem. Rev. 2007; 107 (17212472): 97-11310.1021/cr0502605Crossref PubMed Scopus (231) Google Scholar). Although a given binding site may exhibit a preference for a given ion, competition between and substitution of ions occurs throughout metal-coordinating biomolecules with a variety of effects (15Anjem A. Varghese S. Imlay J.A. Manganese import is a key element of the OxyR response to hydrogen peroxide in Escherichia coli.Mol. Microbiol. 2009; 72 (19400769): 844-85810.1111/j.1365-2958.2009.06699.xCrossref PubMed Scopus (221) Google Scholar, 16Dudev T. Lim C. Competition among metal ions for protein binding sites: determinants of metal ion selectivity in proteins.Chem. Rev. 2014; 114 (24040963): 538-55610.1021/cr4004665Crossref PubMed Scopus (219) Google Scholar, 17Beyer W.F. Fridovich I. In vivo competition between iron and manganese for occupancy of the active site region of the manganese-superoxide dismutase of Escherichia coli.J. Biol. Chem. 1991; 266 (1985901): 303-308Abstract Full Text PDF PubMed Google Scholar, 18Foster A.W. Osman D. Robinson N.J. Metal preferences and metallation.J. Biol. Chem. 2014; 289 (25160626): 28095-2810310.1074/jbc.R114.588145Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Many questions remain unresolved regarding how a preferred metal cofactor is selected and whether promiscuous coordination of ions is advantageous (19Cotruvo Jr., J.A. Stubbe J. Metallation and mismetallation of iron and manganese proteins in vitroin vivo: the class I ribonucleotide reductases as a case study.Metallomics. 2012; 4 (22991063): 1020-103610.1039/c2mt20142aCrossref PubMed Scopus (81) Google Scholar). Among biological molecules that utilize divalent metal cations, magnesium is the most common (20Wacker W.E.C. The biochemistry of magnesium.Ann. N.Y. Acad. Sci. 1969; 162 (4242156): 717-72610.1111/j.1749-6632.1969.tb13003.xCrossref PubMed Scopus (70) Google Scholar, 21Nierhaus K.H. Mg2+, K+, and the ribosome.J. Bacteriol. 2014; 196 (25225274): 3817-381910.1128/JB.02297-14Crossref PubMed Scopus (49) Google Scholar, 22Sissi C. Palumbo M. Effects of magnesium and related divalent metal ions in topoisomerase structure and function.Nucleic Acids Res. 2009; 37 (19188255): 702-71110.1093/nar/gkp024Crossref PubMed Scopus (102) Google Scholar), because of a combination of its abundance and amenable chemical properties such as its small radius and lack of redox activity (7Cowan J.A. Metal activation of enzymes in nucleic acid biochemistry.Chem. Rev. 1998; 98 (11848925): 1067-108810.1021/cr960436qCrossref PubMed Google Scholar, 23Klein D.J. Moore P.B. Steitz T.A. The contribution of metal ions to the structural stability of the large ribosomal subunit.RNA. 2004; 10 (15317974): 1366-137910.1261/rna.7390804Crossref PubMed Scopus (205) Google Scholar). Although other divalent cations may be similar enough to be coordinated in place of Mg2+, the differing properties can impact the biomolecule to which they are bound. For example, kinases that usually utilize Mg2+ can associate with other trace metal ions but suffer a loss in efficiency (10Knape M.J. Ballez M. Burghardt N.C. Zimmermann B. Bertinetti D. Kornev A.P. Herberg F.W. Divalent metal ions control activity and inhibition of protein kinases.Metallomics. 2017; 9 (29043344): 1576-158410.1039/c7mt00204aCrossref PubMed Google Scholar). DNA polymerases require divalent metal cations, and most often employ Mg2+ in this role. Coordinating other metal cofactors including Mn2+ and Co2+ can increase activity, but negatively affect fidelity, and can be carcinogenic as a result (5Vashishtha A.K. Wang J. Konigsberg W.H. Different divalent cations alter the kinetics and fidelity of DNA polymerases.J. Biol. Chem. 2016; 291 (27462081): 20869-2087510.1074/jbc.R116.742494Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 6Ricchetti M. Buc H. E. coli DNA polymerase I as a reverse transcriptase.EMBO J. 1993; 12 (7679988): 387-39610.1002/j.1460-2075.1993.tb05670.xCrossref PubMed Scopus (49) Google Scholar, 7Cowan J.A. Metal activation of enzymes in nucleic acid biochemistry.Chem. Rev. 1998; 98 (11848925): 1067-108810.1021/cr960436qCrossref PubMed Google Scholar). The ribosome is a massive ribonucleoprotein complex that is particularly dependent on Mg2+ (14Sigel R.K.O. Pyle A.M. Alternative roles for metal ions in enzyme catalysis and the implications for ribozyme chemistry.Chem. Rev. 2007; 107 (17212472): 97-11310.1021/cr0502605Crossref PubMed Scopus (231) Google Scholar) to fold and maintain stability because of the negative charge of the RNA backbone, and as many as 200 Mg2+ ions can be associated with just the large subunit, coordinated in six distinct geometries (23Klein D.J. Moore P.B. Steitz T.A. The contribution of metal ions to the structural stability of the large ribosomal subunit.RNA. 2004; 10 (15317974): 1366-137910.1261/rna.7390804Crossref PubMed Scopus (205) Google Scholar, 24Petrov A.S. Bowman J.C. Harvey S.C. Williams L.D. Bidentate RNA–magnesium clamps: on the origin of the special role of magnesium in RNA folding.RNA. 2011; 17 (21173199): 291-29710.1261/rna.2390311Crossref PubMed Scopus (55) Google Scholar, 25Guth-Metzler R. Bray M.S. Frenkel-Pinter M. Suttapitugsakul S. Montllor-Albalate C. Bowman J.C. Wu R. Reddi A.R. Okafor C.D. Glass J.B. Williams L.D. Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage.Nucleic Acids Res. 2020; 48 (32663277): 8663-867410.1093/nar/gkaa586Crossref PubMed Scopus (2) Google Scholar). How other ions with different properties are able to promote RNA folding is not well-understood (26Nguyen H.T. Hori N. Thirumalai D. Theory and simulations for RNA folding in mixtures of monovalent and divalent cations.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (31570624): 21022-2103010.1073/pnas.1911632116Crossref PubMed Scopus (14) Google Scholar). Ribosomal Mg2+ can be substituted with other divalent cations, including Fe2+ or Mn2+ (27Athavale S.S. Petrov A.S. Hsiao C. Watkins D. Prickett C.D. Gossett J.J. Lie L. Bowman J.C. O'Neill E. Bernier C.R. Hud N.V. Wartell R.M. Harvey S.C. Williams L.D. RNA folding and catalysis mediated by iron (II).PLoS One. 2012; 7 (22701543)e3802410.1371/journal.pone.0038024Crossref PubMed Scopus (59) Google Scholar, 28Grosshans C.A. Cech T.R. Metal ion requirements for sequence-specific endoribonuclease activity of the Tetrahymena ribozyme.Biochemistry. 1989; 28 (2684268): 6888-689410.1021/bi00443a017Crossref PubMed Google Scholar, 29Pyle A.M. Metal ions in the structure and function of RNA.J. Biol. Inorg. Chem. 2002; 7 (12203005): 679-69010.1007/s00775-002-0387-6Crossref PubMed Scopus (277) Google Scholar, 30Bray M.S. Lenz T.K. Haynes J.W. Bowman J.C. Petrov A.S. Reddi A.R. Hud N.V. Williams L.D. Glass J.B. Multiple prebiotic metals mediate translation.Proc. Natl. Acad. Sci. U.S.A. 2018; 115 (30413624): 12164-1216910.1073/pnas.1803636115Crossref PubMed Scopus (20) Google Scholar), and the ribosome can competently mediate translation in this state (30Bray M.S. Lenz T.K. Haynes J.W. Bowman J.C. Petrov A.S. Reddi A.R. Hud N.V. Williams L.D. Glass J.B. Multiple prebiotic metals mediate translation.Proc. Natl. Acad. Sci. U.S.A. 2018; 115 (30413624): 12164-1216910.1073/pnas.1803636115Crossref PubMed Scopus (20) Google Scholar). The intricately folded rRNA (rRNA) of the functional ribozyme core has remained largely the same since it evolved 3–4 billion years ago (31Petrov A.S. Bernier C.R. Hsiao C. Norris A.M. Kovacs N.A. Waterbury C.C. Stepanov V.G. Harvey S.C. Fox G.E. Wartell R.M. Hud N.V. Williams L.D. Evolution of the ribosome at atomic resolution.Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (24982194): 10251-1025610.1073/pnas.1407205111Crossref PubMed Scopus (109) Google Scholar, 32Fox G.E. Origin and evolution of the ribosome.Cold Spring Harb. Perspect. Biol. 2010; 2 (a003483, 20534711): a00348310.1101/cshperspect.a003483Crossref PubMed Google Scholar). Dozens of ribosomal proteins (r-proteins) interact with rRNA residues to assemble compact, flexible, and stable subunits (33Melnikov S. Ben-Shem A. Garreau de Loubresse N. Jenner L. Yusupova G. Yusupov M. One core, two shells: bacterial and eukaryotic ribosomes.Nat. Struct. Mol. Biol. 2012; 19 (22664983): 560-56710.1038/nsmb.2313Crossref PubMed Scopus (217) Google Scholar), which are abundant and long-lived in a cytoplasmic environment populated with RNases. However, in physiologically challenging environments such as nutrient scarcity or oxidative stress, the ribosome can lose stability and undergo degradation (34Kraft C. Deplazes A. Sohrmann M. Peter M. Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease.Nat. Cell Biol. 2008; 10 (18391941): 602-61010.1038/ncb1723Crossref PubMed Scopus (496) Google Scholar, 35Mroczek S. Kufel J. Apoptotic signals induce specific degradation of ribosomal RNA in yeast.Nucleic Acids Res. 2008; 36 (18385160): 2874-288810.1093/nar/gkm1100Crossref PubMed Scopus (53) Google Scholar), but the mechanisms initiating this process are still not well-understood. Recent work has drawn on the interesting observation that although evolution has conserved the ribosomal core through billions of years, the earth environment has changed dramatically over this time and with it the bioavailability of the metals on which the ribosome structure depends. Metal levels in the aqueous environment prior to abiogenesis included high concentrations of Fe2+ (36Bengtson S. Early Life on Earth: Nobel Symposium, No. 84. Columbia University Press, New York1994Google Scholar), whereas Mg2+ was less abundant than today (30Bray M.S. Lenz T.K. Haynes J.W. Bowman J.C. Petrov A.S. Reddi A.R. Hud N.V. Williams L.D. Glass J.B. Multiple prebiotic metals mediate translation.Proc. Natl. Acad. Sci. U.S.A. 2018; 115 (30413624): 12164-1216910.1073/pnas.1803636115Crossref PubMed Scopus (20) Google Scholar, 37Jones C. Nomosatryo S. Crowe S.A. Bjerrum C.J. Canfield D.E. Iron oxides, divalent cations, silica, and the early earth phosphorus crisis.Geology. 2015; 43: 135-13810.1130/G36044.1Crossref Scopus (96) Google Scholar). With the evolution of photosynthesis and the increase in molecular oxygen, much of the iron was lost from the aqueous environment through precipitation (38Feig A.L. Uhlenbeck O.C. The role of metal ions in RNA biochemistry.Cold Spring Harb. Monograph Arch. 1999; 37: 287-319Google Scholar). Similarly, abundant Mn2+ was oxidized and precipitated out, resulting in vast amounts of both transition metals in sedimentary rocks (39Johnson J.E. Webb S.M. Ma C. Fischer W.W. Manganese mineralogy and diagenesis in the sedimentary rock record.Geochim. Cosmochim. Acta. 2016; 173: 210-23110.1016/j.gca.2015.10.027Crossref Scopus (88) Google Scholar). Therefore, the interaction of Fe2+ with rRNAs presents a potential stabilizing interaction used by ancient ribosomes in the differing conditions of the earth when the core structure evolved, a role that was then filled by Mg2+ as Fe2+ availability decreased. Although this establishes Fe2+ and Mn2+ interactions with the ribosome as evolutionarily relevant, little is known about these associations in the present-day oxidative environment and how ribosomal stability is impacted. As well as the drop in available iron, the oxygenation of the atmosphere produced a further reason for the translation machinery to select another cofactor. The proclivity for Fe2+ to participate in the Fenton reaction with H2O2 (which produces hydroxyl radicals and Fe3+) is of consequence in cells under oxidative stress conditions because it can enhance oxidant-induced damage to biomolecules. In an increasingly oxygen-rich environment, continuous close associations with Fe2+ would have become a dangerous proposition (30Bray M.S. Lenz T.K. Haynes J.W. Bowman J.C. Petrov A.S. Reddi A.R. Hud N.V. Williams L.D. Glass J.B. Multiple prebiotic metals mediate translation.Proc. Natl. Acad. Sci. U.S.A. 2018; 115 (30413624): 12164-1216910.1073/pnas.1803636115Crossref PubMed Scopus (20) Google Scholar). Conversely, the higher reduction potential of manganese prevents its participation in Fenton chemistry (40Aguirre J.D. Culotta V.C. Battles with iron: manganese in oxidative stress protection.J. Biol. Chem. 2012; 287 (22247543): 13541-1354810.1074/jbc.R111.312181Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Aside from this difference, the biochemistry of these transition metal ions is linked by several similar parameters (including oxidation states, atomic radius, and preferred coordination geometries), as well as their abundant bioavailability in the environment (16Dudev T. Lim C. Competition among metal ions for protein binding sites: determinants of metal ion selectivity in proteins.Chem. Rev. 2014; 114 (24040963): 538-55610.1021/cr4004665Crossref PubMed Scopus (219) Google Scholar, 18Foster A.W. Osman D. Robinson N.J. Metal preferences and metallation.J. Biol. Chem. 2014; 289 (25160626): 28095-2810310.1074/jbc.R114.588145Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 30Bray M.S. Lenz T.K. Haynes J.W. Bowman J.C. Petrov A.S. Reddi A.R. Hud N.V. Williams L.D. Glass J.B. Multiple prebiotic metals mediate translation.Proc. Natl. Acad. Sci. U.S.A. 2018; 115 (30413624): 12164-1216910.1073/pnas.1803636115Crossref PubMed Scopus (20) Google Scholar). In our previous work, we have demonstrated that Fe2+ is a factor that contributes to destabilization of ribosomes and provided evidence that it promotes chemical hydrolysis of rRNA. Based on the structural predictions of the ES7L region of the 60S subunit (41Zinskie J.A. Ghosh A. Trainor B.M. Shedlovskiy D. Pestov D.G. Shcherbik N. Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.J. Biol. Chem. 2018; 293 (30021840): 14237-1424810.1074/jbc.RA118.004174Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 42Ben-Shem A. Garreau de Loubresse N. Melnikov S. Jenner L. Yusupova G. Yusupov M. The structure of the eukaryotic ribosome at 3.0 Å resolution.Science. 2011; 334 (22096102): 1524-152910.1126/science.1212642Crossref PubMed Scopus (710) Google Scholar), we proposed that Fe2+ replaces Mg2+ at specific binding sites on a ribosome (41Zinskie J.A. Ghosh A. Trainor B.M. Shedlovskiy D. Pestov D.G. Shcherbik N. Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.J. Biol. Chem. 2018; 293 (30021840): 14237-1424810.1074/jbc.RA118.004174Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) and that these sites become primed to undergo rapid hydrolysis upon exposure to ROS. In agreement with this model, cellular systems that limit the amounts of either cellular oxidant species or ribosome-associated Fe2+ act to protect ribosomal integrity (35Mroczek S. Kufel J. Apoptotic signals induce specific degradation of ribosomal RNA in yeast.Nucleic Acids Res. 2008; 36 (18385160): 2874-288810.1093/nar/gkm1100Crossref PubMed Scopus (53) Google Scholar, 41Zinskie J.A. Ghosh A. Trainor B.M. Shedlovskiy D. Pestov D.G. Shcherbik N. Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.J. Biol. Chem. 2018; 293 (30021840): 14237-1424810.1074/jbc.RA118.004174Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 43Shedlovskiy D. Zinskie J.A. Gardner E. Pestov D.G. Shcherbik N. Endonucleolytic cleavage in the expansion segment 7 of 25S rRNA is an early marker of low-level oxidative stress in yeast.J. Biol. Chem. 2017; 292 (28939771): 18469-1848510.1074/jbc.M117.800003Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Here we add detail to the understanding of divalent cation interactions with the ribosome by demonstrating that Fe2+ interacts directly with rRNA and in the presence of oxidants is sufficient to degrade all yeast rRNA molecules in reproducible patterns. Moreover, we identify Mn2+ as a factor that can protect the rRNA from these cleavage events by substituting Fe2+ at the ribosomal binding sites, which correlates with improved cell viability under oxidative stress. We have demonstrated in a previous study (41Zinskie J.A. Ghosh A. Trainor B.M. Shedlovskiy D. Pestov D.G. Shcherbik N. Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.J. Biol. Chem. 2018; 293 (30021840): 14237-1424810.1074/jbc.RA118.004174Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) that Fe2+ has a direct role in cleaving the sugar-phosphate backbone of rRNA under oxidative stress conditions. Specifically, one prominent cleavage site was observed within the ES7L region of the 25S rRNA in the large (60S) ribosomal subunit. To further understand the roles of Fe2+ in rRNA degradation, we chose to investigate whether an iron-dependent oxidative mechanism is responsible for the fragmentation of the 5S and 5.8S rRNAs of the 60S subunit or the 18S rRNA of the 40S subunit. For these experiments, we employed a strain deleted for the gene encoding the mitochondrial glutaredoxin Grx5, loss of which results in increased endogenous levels of labile iron in the cytoplasm (44Gomez M. Pérez-Gallardo R.V. Sánchez L.A. Díaz-Pérez A.L. Cortés-Rojo C. Meza Carmen V. Saavedra-Molina A. Lara-Romero J. Jiménez-Sandoval S. Rodríguez F. Rodríguez-Zavala J.S. Campos-García J. Malfunctioning of the iron–sulfur cluster assembly machinery in Saccharomyces cerevisiae produces oxidative stress via an iron-dependent mechanism, causing dysfunction in respiratory complexes.PLoS One. 2014; 9 (25356756)e11158510.1371/journal.pone.0111585Crossref PubMed Scopus (28) Google Scholar, 45Rodríguez-Manzaneque M.T. Tamarit J. Bellí G. Ros J. Herrero E. Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes.Mol. Biol. Cell. 2002; 13 (11950925): 1109-112110.1091/mbc.01-10-0517Crossref PubMed Scopus (373) Google Scholar) and has proved to be a useful system for modeling the effects of increased Fe2+ availability in cells. In our previous work, we observed that all rRNAs are unstable in a grx5Δ mutant (41Zinskie J.A. Ghosh A. Trainor B.M. Shedlovskiy D. Pestov D.G. Shcherbik N. Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.J. Biol. Chem. 2018; 293 (30021840): 14237-1424810.1074/jbc.RA118.004174Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), but it remained unclear whether the detected cleavages resulted primarily from enzymatic digestion by ribonucleases or Fe2+-dependent nonenzymatic reaction mechanisms. To investigate the extent of fragmentation of different rRNA species by a Fe2+-dependent mechanism, we utilized an in vitro Fe2+/ascorbic acid assay that was developed as part of our previous study (41Zinskie J.A. Ghosh A. Trainor B.M. Shedlovskiy D. Pestov D.G. Shcherbik N. Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.J. Biol. Chem. 2018; 293 (30021840): 14237-1424810.1074/jbc.RA118.004174Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), further validation of which is shown in Fig. S1. In this assay, we use purified ribosomes as a substrate, ammonium iron(II) sulfate hexahydrate (Fe(NH4)2(SO4)2) to supply extra Fe2+ ions, whereas ascorbic acid is used for its ability to generate H2O2 by inducing redox cycling at metal-binding sites (46Samuni A. Aronovitch J. Godinger D. Chevion M. Czapski G. On the cytotoxicity of vitamin C and metal ions: a site-specific Fenton mechanism.Eur. J. Biochem. 1983; 137 (6317379): 119-12410.1111/j.1432-1033.1983.tb07804.xCrossref PubMed Google Scholar, 47Khan M.M. Martell A.E. Metal ion and metal chelate catalyzed oxidation of ascorbic acid by molecular oxygen: I. Cupric and ferric ion catalyzed oxidation.J. Am. Chem. Soc. 1967; 89 (6045609): 4176-418510.1021/ja00992a036Crossref PubMed Google Scholar). Cellular lysates from exponentially growing WT cells were centrifuged through a sucrose cushion to obtain purified ribosomes, which were treated with ascorbic acid alone, ascorbic acid in combination with Fe(NH4)2(SO4)2, or ascorbic acid with both Fe(NH4)2(SO4)2 and the iron chelator deferoxamine (DFO). In a complementary experiment, yeast grx5Δ cells were subjected to oxidative stress by treatment with menadione before RNA extraction. RNA samples from the in vitro Fe2+/ascorbic acid assay and those from menadione-treated grx5Δ cells were resolved adjacent to one another on the same gel and analyzed by Northern hybridization with probes for each of the four rRNAs (Fig. 1, A–D and probe hybridization schematic in E). Consistent with our previous findings (41Zinskie J.A. Ghosh A. Trainor B.M. Shedlovskiy D. Pestov D.G. Shcherbik N. Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae.J. Biol. Chem. 2018; 293 (30021840): 14237-1424810.1074/jbc.RA118.004174Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), purified ribosomes treated with ascorbic acid alone produced distinct degradation fragments detected using a probe complementary to the 5′ end of the 25S rRNA (y540) (Fig. 1A). Addition of Fe2+ greatly increased the degradation, resulting in well-defined rRNA fragments and disappearance of full-length 25S rRNA. The inclusion of the iron chelator DFO in the reaction completely prevented degradation. Similarly, an increase in rRNA fragmentation was observed for the 18S rRNA (Fig. 1B), 5.8S rRNA (Fig. 1C), and 5S rRNA (Fig. 1D) when treated with ascorbic acid and Fe2+, and the degradation was again prevented by the inclusion of the chelator DFO. Interestingly, when rRNAs degradation products generated in vitro were compared side by side with those in menadione-treated grx5Δ cells, we noticed a clear resemblance between the rRNAs' fragmentation patterns, regardless of a probe used (Fig. 1, compare third and fifth lanes on each blot). The similarities were seen most clearly when using probes that detect well-resolved and defined rRNA fragments, such as probe y540 for 25S rRNA (Fig. 1A), probe y532 for 18S rRNA (Fig. 1B), probe y534 for 5.8S rRNA (Fig. 1C), and probe y506 for 5S rRNA (Fig. 1D). Further probes spanning the length of both the 25S and 18S rRNAs were also used to assess the fragmentation across the length of the molecules (Fig. S2C). In all cases, the addition of Fe2+ increased the degradation observed with ascorbic acid alone, and this degradation was prevented by inclusion of DFO. However, for both the 25S and 18S rRNAs, the degradation products appeared as a smear of low molecular weight fragments as probes moved away from the 5′ ends (Fig. S2). Restoration of integrity of rRNAs by an iron chelator in the presence of Fe2+ and ascorbic acid (Fig. 1, A–D) supports the hypothesis that Fe2+ is responsible for inducing rRNA cleavage in vitro. Additionally, probes targeting regions throughout the 18S and 25S rRNAs were able to hybridize with degradation products, suggesting that Fe2+-mediated degradation occurs at multiple sites in these molecules (Fig. S2). The similarity of rRNA degradation patterns observed between menadione-treated grx5Δ cells and those from the in vitro Fe2+/ascorbic acid assay indicates that cellular RNases do not substantially contribute to these cleavage events (Fig. 1, A–D) and that a Fe2+-mediated mechanism is primarily responsible for cleavages initiated in the 5.8S, 5S, and at the 5′ end of the 25S and 18S rRNAs when cellular redox state is perturbed. Thus, iron play
Referência(s)