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. 2022;2533:149–166. doi: 10.1007/978-1-0716-2501-9_9

Chemical Modifications of Ribosomal RNA.

Sunny Sharma, Karl-Dieter Entian
PMCID: PMC9761533  PMID: 35796987

Abstract

Cellular RNAs in all three kingdoms of life are modified with diverse chemical modifications. These chemical modifications expand the topological repertoire of RNAs, and fine-tune their functions. Ribosomal RNA in yeast contains more than 100 chemically modified residues in the functionally crucial and evolutionary conserved regions. The chemical modifications in the rRNA are of three types-methylation of the ribose sugars at the C2-positionAbstract (Nm), isomerization of uridines to pseudouridines (Ψ), and base modifications such as (methylation (mN), acetylation (acN), and aminocarboxypropylation (acpN)). The modifications profile of the yeast rRNA has been recently completed, providing an excellent platform to analyze the function of these modifications in RNA metabolism and in cellular physiology. Remarkably, majority of the rRNA modifications and the enzymatic machineries discovered in yeast are highly conserved in eukaryotes including humans. Mutations in factors involved in rRNA modification are linked to several rare severe human diseases (e.g., X-linked Dyskeratosis congenita, the Bowen-Conradi syndrome and the William-Beuren disease). In this chapter, we summarize all rRNA modifications and the corresponding enzymatic machineries of the budding yeast.

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References

  1. Boccaletto P, Machnicka MA, Purta E, Piatkowski P, Baginski B, Wirecki TK, de Crecy-Lagard V, Ross R, Limbach PA, Kotter A et al (2018) MODOMICS: a database of RNA modification pathways. 2017 update. Nucleic Acids Res 46:D303–D307 doi: 10.1093/nar/gkx1030. [DOI] [PMC free article] [PubMed]
  2. Woolford JL Jr, Baserga SJ (2013) Ribosome biogenesis in the yeast Saccharomyces cerevisiae. Genetics 195:643–681 doi: 10.1534/genetics.113.153197. [DOI] [PMC free article] [PubMed]
  3. Cech TR (2000) Structural biology. The ribosome is a ribozyme. Science 289:878–879 doi: 10.1126/science.289.5481.878. [DOI] [PubMed]
  4. Klootwijk J, Planta RJ (1973) Analysis of the methylation sites in yeast ribosomal RNA. Eur J Biochem 39:325–333 doi: 10.1111/j.1432-1033.1973.tb03130.x. [DOI] [PubMed]
  5. Motorin Y, Helm M (2011) RNA nucleotide methylation. Wiley Interdiscip Rev RNA 2:611–631 doi: 10.1002/wrna.79. [DOI] [PubMed]
  6. Sharma S, Lafontaine DLJ (2015) ‘View from a Bridge’: a new perspective on eukaryotic rRNA base modification. Trends Biochem Sci 40:560–575 doi: 10.1016/j.tibs.2015.07.008. [DOI] [PubMed]
  7. Armistead J, Khatkar S, Meyer B, Mark BL, Patel N, Coghlan G, Lamont RE, Liu S, Wiechert J, Cattini PA et al (2009) Mutation of a gene essential for ribosome biogenesis, EMG1, causes Bowen-Conradi syndrome. Am J Hum Genet 84:728–739 doi: 10.1016/j.ajhg.2009.04.017. [DOI] [PMC free article] [PubMed]
  8. Meyer B, Wurm JP, Kotter P, Leisegang MS, Schilling V, Buchhaupt M, Held M, Bahr U, Karas M, Heckel A et al (2011) The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Psi1191 in yeast 18S rRNA. Nucleic Acids Res 39:1526–1537 doi: 10.1093/nar/gkq931. [DOI] [PMC free article] [PubMed]
  9. Wurm JP, Meyer B, Bahr U, Held M, Frolow O, Kotter P, Engels JW, Heckel A, Karas M, Entian KD et al (2010) The ribosome assembly factor Nep1 responsible for Bowen-Conradi syndrome is a pseudouridine-N1-specific methyltransferase. Nucleic Acids Res 38:2387–2398 doi: 10.1093/nar/gkp1189. [DOI] [PMC free article] [PubMed]
  10. Doll A, Grzeschik KH (2001) Characterization of two novel genes, WBSCR20 and WBSCR22, deleted in Williams-Beuren syndrome. Cytogenet Cell Genet 95:20–27 doi: 10.1159/000057012. [DOI] [PubMed]
  11. Knight SW, Heiss NS, Vulliamy TJ, Greschner S, Stavrides G, Pai GS, Lestringant G, Varma N, Mason PJ, Dokal I et al (1999) X-linked dyskeratosis congenita is predominantly caused by missense mutations in the DKC1 gene. Am J Hum Genet 65:50–58 doi: 10.1086/302446. [DOI] [PMC free article] [PubMed]
  12. Nachmani D, Bothmer AH, Grisendi S, Mele A, Bothmer D, Lee JD, Monteleone E, Cheng K, Zhang Y, Bester AC et al (2019) Germline NPM1 mutations lead to altered rRNA 2′-O-methylation and cause dyskeratosis congenita. Nat Genet 51:1518–1529 doi: 10.1038/s41588-019-0502-z. [DOI] [PMC free article] [PubMed]
  13. Venturi G, Montanaro L (2020) How altered ribosome production can cause or contribute to human disease: the spectrum of ribosomopathies. Cell 9:2300 doi: 10.3390/cells9102300. [DOI] [PMC free article] [PubMed]
  14. Buchhaupt M, Sharma S, Kellner S, Oswald S, Paetzold M, Peifer C, Watzinger P, Schrader J, Helm M, Entian KD (2014) Partial methylation at Am100 in 18S rRNA of baker's yeast reveals ribosome heterogeneity on the level of eukaryotic rRNA modification. PLoS One 9:e89640 doi: 10.1371/journal.pone.0089640. [DOI] [PMC free article] [PubMed]
  15. Sloan KE, Warda AS, Sharma S, Entian KD, Lafontaine DLJ, Bohnsack MT (2017) Tuning the ribosome: the influence of rRNA modification on eukaryotic ribosome biogenesis and function. RNA Biol 14:1138–1152 doi: 10.1080/15476286.2016.1259781. [DOI] [PMC free article] [PubMed]
  16. Taoka M, Nobe Y, Yamaki Y, Yamauchi Y, Ishikawa H, Takahashi N, Nakayama H, Isobe T (2016) The complete chemical structure of Saccharomyces cerevisiae rRNA: partial pseudouridylation of U2345 in 25S rRNA by snoRNA snR9. Nucleic Acids Res 44:8951–8961 doi: 10.1093/nar/gkw564. [DOI] [PMC free article] [PubMed]
  17. Vlachos A (2017) Acquired ribosomopathies in leukemia and solid tumors. Hematology Am Soc Hematol Educ Program 2017:716–719 doi: 10.1182/asheducation-2017.1.716. [DOI] [PMC free article] [PubMed]
  18. Aspesi A, Ellis SR (2019) Rare ribosomopathies: insights into mechanisms of cancer. Nat Rev Cancer 19:228–238 doi: 10.1038/s41568-019-0105-0. [DOI] [PubMed]
  19. Barbieri I, Kouzarides T (2020) Role of RNA modifications in cancer. Nat Rev Cancer 20:303–322 doi: 10.1038/s41568-020-0253-2. [DOI] [PubMed]
  20. Bakin A, Lane BG, Ofengand J (1994) Clustering of pseudouridine residues around the peptidyltransferase center of yeast cytoplasmic and mitochondrial ribosomes. Biochemistry 33:13475–13483 doi: 10.1021/bi00249a036. [DOI] [PubMed]
  21. Maden BE (1988) Locations of methyl groups in 28 S rRNA of Xenopus laevis and man. Clustering in the conserved core of molecule. J Mol Biol 201:289–314 doi: 10.1016/0022-2836(88)90139-8. [DOI] [PubMed]
  22. Yang J, Sharma S, Watzinger P, Hartmann JD, Kotter P, Entian KD (2016) Mapping of complete set of ribose and base modifications of yeast rRNA by RP-HPLC and mung bean nuclease assay. PLoS One 11:e0168873 doi: 10.1371/journal.pone.0168873. [DOI] [PMC free article] [PubMed]
  23. Yang J, Sharma S, Kotter P, Entian KD (2015) Identification of a new ribose methylation in the 18S rRNA of S. cerevisiae. Nucleic Acids Res 43:2342–2352 doi: 10.1093/nar/gkv058. [DOI] [PMC free article] [PubMed]
  24. Sharma S, Marchand V, Motorin Y, Lafontaine DLJ (2017) Identification of sites of 2′-O-methylation vulnerability in human ribosomal RNAs by systematic mapping. Sci Rep 7:11490 doi: 10.1038/s41598-017-09734-9. [DOI] [PMC free article] [PubMed]
  25. Shi Z, Barna M (2015) Translating the genome in time and space: specialized ribosomes, RNA regulons, and RNA-binding proteins. Annu Rev Cell Dev Biol 31:31–54 doi: 10.1146/annurev-cellbio-100814-125346. [DOI] [PubMed]
  26. Kiss L (1996) Site-specific ribose methylation of preribosomal RNA: a novel function for small nucleolar RNAs. Cell 85:1077–1088 doi: 10.1016/s0092-8674(00)81308-2. [DOI] [PubMed]
  27. Watkins NJ, Bohnsack MT (2012) The box C/D and H/ACA snoRNPs: key players in the modification, processing and the dynamic folding of ribosomal RNA. Wiley Interdiscip Rev RNA 3:397–414 doi: 10.1002/wrna.117. [DOI] [PubMed]
  28. Sharma S, Yang J, van Nues R, Watzinger P, Kotter P, Lafontaine DLJ, Granneman S, Entian KD (2017) Specialized box C/D snoRNPs act as antisense guides to target RNA base acetylation. PLoS Genet 13:e1006804 doi: 10.1371/journal.pgen.1006804. [DOI] [PMC free article] [PubMed]
  29. Noon KR, Guymon R, Crain PF, McCloskey JA, Thomm M, Lim J, Cavicchioli R (2003) Influence of temperature on tRNA modification in archaea: Methanococcoides burtonii (optimum growth temperature [Topt], 23 degrees C) and Stetteria hydrogenophila (Topt, 95 degrees C). J Bacteriol 185:5483–5490 doi: 10.1128/JB.185.18.5483-5490.2003. [DOI] [PMC free article] [PubMed]
  30. Ganot P, Bortolin ML, Kiss T (1997) Site-specific pseudouridine formation in preribosomal RNA is guided by small nucleolar RNAs. Cell 89:799–809 doi: 10.1016/s0092-8674(00)80263-9. [DOI] [PubMed]
  31. Duan J, Li L, Lu J, Wang W, Ye K (2009) Structural mechanism of substrate RNA recruitment in H/ACA RNA-guided pseudouridine synthase. Mol Cell 34:427–439 doi: 10.1016/j.molcel.2009.05.005. [DOI] [PubMed]
  32. Kozbial PZ, Mushegian AR (2005) Natural history of S-adenosylmethionine-binding proteins. BMC Struct Biol 5:19 doi: 10.1186/1472-6807-5-19. [DOI] [PMC free article] [PubMed]
  33. Schubert HL, Blumenthal RM, Cheng X (2003) Many paths to methyltransfer: a chronicle of convergence. Trends Biochem Sci 28:329–335 doi: 10.1016/S0968-0004(03)00090-2. [DOI] [PMC free article] [PubMed]
  34. Anantharaman V, Koonin EV, Aravind L (2002) SPOUT: a class of methyltransferases that includes spoU and trmD RNA methylase superfamilies, and novel superfamilies of predicted prokaryotic RNA methylases. J Mol Microbiol Biotechnol 4:71–75 [PubMed]
  35. Thomas G, Gordon J, Rogg H (1978) N4-Acetylcytidine. A previously unidentified labile component of the small subunit of eukaryotic ribosomes. J Biol Chem 253:1101–1105 [PubMed]
  36. Johansson MJ, Bystrom AS (2004) The Saccharomyces cerevisiae TAN1 gene is required for N4-acetylcytidine formation in tRNA. RNA 10:712–719 doi: 10.1261/rna.5198204. [DOI] [PMC free article] [PubMed]
  37. Kawai G, Hashizume T, Miyazawa T, McCloskey JA, Yokoyama S (1989) Conformational characteristics of 4-acetylcytidine found in tRNA. Nucleic Acids Symp Ser (21):61–62 [PubMed]
  38. Kumbhar BV, Kamble AD, Sonawane KD (2013) Conformational preferences of modified nucleoside N(4)-acetylcytidine, ac4C occur at “wobble” 34th position in the anticodon loop of tRNA. Cell Biochem Biophys 66:797–816 doi: 10.1007/s12013-013-9525-8. [DOI] [PubMed]
  39. Stern L, Schulman LH (1978) The role of the minor base N4-acetylcytidine in the function of the Escherichia coli noninitiator methionine transfer RNA. J Biol Chem 253:6132–6139 [PubMed]
  40. Ben-Shem A, Garreau de Loubresse N, Melnikov S, Jenner L, Yusupova G, Yusupov M (2011) The structure of the eukaryotic ribosome at 3.0 A resolution. Science 334:1524–1529 doi: 10.1126/science.1212642. [DOI] [PubMed]
  41. Ikeuchi Y, Kitahara K, Suzuki T (2008) The RNA acetyltransferase driven by ATP hydrolysis synthesizes N4-acetylcytidine of tRNA anticodon. EMBO J 27:2194–2203 doi: 10.1038/emboj.2008.154. [DOI] [PMC free article] [PubMed]
  42. Chimnaronk S, Suzuki T, Manita T, Ikeuchi Y, Yao M, Suzuki T, Tanaka I (2009) RNA helicase module in an acetyltransferase that modifies a specific tRNA anticodon. EMBO J 28:1362–1373 doi: 10.1038/emboj.2009.69. [DOI] [PMC free article] [PubMed]
  43. Ito S, Akamatsu Y, Noma A, Kimura S, Miyauchi K, Ikeuchi Y, Suzuki T, Suzuki T (2014) A single acetylation of 18 S rRNA is essential for biogenesis of the small ribosomal subunit in Saccharomyces cerevisiae. J Biol Chem 289:26201–26212 doi: 10.1074/jbc.M114.593996. [DOI] [PMC free article] [PubMed]
  44. Sharma S, Langhendries JL, Watzinger P, Kotter P, Entian KD, Lafontaine DL (2015) Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1. Nucleic Acids Res 43:2242–2258 doi: 10.1093/nar/gkv075. [DOI] [PMC free article] [PubMed]
  45. Ohashi Z, Maeda M, McCloskey JA, Nishimura S (1974) 3-(3-Amino-3-carboxypropyl)uridine: a novel modified nucleoside isolated from Escherichia coli phenylalanine transfer ribonucleic acid. Biochemistry 13:2620–2625 doi: 10.1021/bi00709a023. [DOI] [PubMed]
  46. Meyer B, Immer C, Kaiser S, Sharma S, Yang J, Watzinger P, Weiss L, Kotter A, Helm M, Seitz HM et al (2020) Identification of the 3-amino-3-carboxypropyl (acp) transferase enzyme responsible for acp3U formation at position 47 in Escherichia coli tRNAs. Nucleic Acids Res 48:1435–1450 doi: 10.1093/nar/gkz1191. [DOI] [PMC free article] [PubMed]
  47. Meyer B, Wurm JP, Sharma S, Immer C, Pogoryelov D, Kotter P, Lafontaine DL, Wohnert J, Entian KD (2016) Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans. Nucleic Acids Res 44:4304–4316 doi: 10.1093/nar/gkw244. [DOI] [PMC free article] [PubMed]
  48. Lafontaine DL, Preiss T, Tollervey D (1998) Yeast 18S rRNA dimethylase Dim1p: a quality control mechanism in ribosome synthesis? Mol Cell Biol 18:2360–2370 doi: 10.1128/mcb.18.4.2360. [DOI] [PMC free article] [PubMed]
  49. Lafontaine D, Delcour J, Glasser AL, Desgres J, Vandenhaute J (1994) The DIM1 gene responsible for the conserved m6(2)Am6(2)A dimethylation in the 3′-terminal loop of 18 S rRNA is essential in yeast. J Mol Biol 241:492–497 doi: 10.1006/jmbi.1994.1525. [DOI] [PubMed]
  50. White J, Li Z, Sardana R, Bujnicki JM, Marcotte EM, Johnson AW (2008) Bud23 methylates G1575 of 18S rRNA and is required for efficient nuclear export of pre-40S subunits. Mol Cell Biol 28:3151–3161 doi: 10.1128/MCB.01674-07. [DOI] [PMC free article] [PubMed]
  51. Figaro S, Wacheul L, Schillewaert S, Graille M, Huvelle E, Mongeard R, Zorbas C, Lafontaine DL, Heurgue-Hamard V (2012) Trm112 is required for Bud23-mediated methylation of the 18S rRNA at position G1575. Mol Cell Biol 32:2254–2267 doi: 10.1128/MCB.06623-11. [DOI] [PMC free article] [PubMed]
  52. Sardana R, White JP, Johnson AW (2013) The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP. RNA 19:828–840 doi: 10.1261/rna.037671.112. [DOI] [PMC free article] [PubMed]
  53. Liang XH, Liu Q, Fournier MJ (2009) Loss of rRNA modifications in the decoding center of the ribosome impairs translation and strongly delays pre-rRNA processing. RNA 15:1716–1728 doi: 10.1261/rna.1724409. [DOI] [PMC free article] [PubMed]
  54. Taylor AB, Meyer B, Leal BZ, Kotter P, Schirf V, Demeler B, Hart PJ, Entian KD, Wohnert J (2008) The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site. Nucleic Acids Res 36:1542–1554 doi: 10.1093/nar/gkm1172. [DOI] [PMC free article] [PubMed]
  55. Wurm JP, Duchardt E, Meyer B, Leal BZ, Kotter P, Entian KD, Wohnert J (2009) Backbone resonance assignments of the 48 kDa dimeric putative 18S rRNAmethyltransferase Nep1 from Methanocaldococcus jannaschii. Biomol NMR Assign 3:251-254. [Epub 2009, Sep 25, https://doi.org/10.1007/s12104-009-9187-z] doi: 10.1007/s12104-009-9187-z. [DOI] [PubMed]
  56. Buchhaupt M, Kotter P, Entian KD (2007) Mutations in the nucleolar proteins Tma23 and Nop6 suppress the malfunction of the Nep1 protein. FEMS Yeast Res 7:771–781 doi: 10.1111/j.1567-1364.2007.00230.x. [DOI] [PubMed]
  57. Eschrich D, Buchhaupt M, Kotter P, Entian KD (2002) Nep1p (Emg1p), a novel protein conserved in eukaryotes and archaea, is involved in ribosome biogenesis. Curr Genet 40:326–338 doi: 10.1007/s00294-001-0269-4. [DOI] [PubMed]
  58. Buchhaupt M, Meyer B, Kotter P, Entian KD (2006) Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p. Mol Gen Genomics 276:273–284 doi: 10.1007/s00438-006-0132-x. [DOI] [PubMed]
  59. Strunk BS, Karbstein K (2009) Powering through ribosome assembly. RNA 15:2083–2104 doi: 10.1261/rna.1792109. [DOI] [PMC free article] [PubMed]
  60. Bousquet-Antonelli C, Vanrobays E, Gelugne JP, Caizergues-Ferrer M, Henry Y (2000) Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2. RNA 6:826–843 doi: 10.1017/s1355838200992288. [DOI] [PMC free article] [PubMed]
  61. Peifer C, Sharma S, Watzinger P, Lamberth S, Kotter P, Entian KD (2013) Yeast Rrp8p, a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA. Nucleic Acids Res 41:1151–1163 doi: 10.1093/nar/gks1102. [DOI] [PMC free article] [PubMed]
  62. Sharma S, Hartmann JD, Watzinger P, Klepper A, Peifer C, Kotter P, Lafontaine DLJ, Entian KD (2018) A single N(1)-methyladenosine on the large ribosomal subunit rRNA impacts locally its structure and the translation of key metabolic enzymes. Sci Rep 8:11904 doi: 10.1038/s41598-018-30383-z. [DOI] [PMC free article] [PubMed]
  63. Sharma S, Watzinger P, Kotter P, Entian KD (2013) Identification of a novel methyltransferase, Bmt2, responsible for the N-1-methyl-adenosine base modification of 25S rRNA in Saccharomyces cerevisiae. Nucleic Acids Res 41:5428–5443 doi: 10.1093/nar/gkt195. [DOI] [PMC free article] [PubMed]
  64. Postma L, Lehrach H, Ralser M (2009) Surviving in the cold: yeast mutants with extended hibernating lifespan are oxidant sensitive. Aging (Albany NY) 1:957–960 doi: 10.18632/aging.100104. [DOI] [PMC free article] [PubMed]
  65. Motorin Y, Lyko F, Helm M (2010) 5-methylcytosine in RNA: detection, enzymatic formation and biological functions. Nucleic Acids Res 38:1415–1430 doi: 10.1093/nar/gkp1117. [DOI] [PMC free article] [PubMed]
  66. Sharma S, Yang J, Watzinger P, Kotter P, Entian KD (2013) Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively. Nucleic Acids Res 41:9062–9076 doi: 10.1093/nar/gkt679. [DOI] [PMC free article] [PubMed]
  67. Foster PG, Nunes CR, Greene P, Moustakas D, Stroud RM (2003) The first structure of an RNA m5C methyltransferase, Fmu, provides insight into catalytic mechanism and specific binding of RNA substrate. Structure 11:1609–1620 doi: 10.1016/j.str.2003.10.014. [DOI] [PubMed]
  68. Hong B, Brockenbrough JS, Wu P, Aris JP (1997) Nop2p is required for pre-rRNA processing and 60S ribosome subunit synthesis in yeast. Mol Cell Biol 17:378–388 doi: 10.1128/mcb.17.1.378. [DOI] [PMC free article] [PubMed]
  69. Schosserer M, Minois N, Angerer TB, Amring M, Dellago H, Harreither E, Calle-Perez A, Pircher A, Gerstl MP, Pfeifenberger S et al (2015) Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan. Nat Commun 6:6158 doi: 10.1038/ncomms7158. [DOI] [PMC free article] [PubMed]
  70. Sharma S, Yang J, Duttmann S, Watzinger P, Kotter P, Entian KD (2014) Identification of novel methyltransferases, Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae. Nucleic Acids Res 42:3246–3260 doi: 10.1093/nar/gkt1281. [DOI] [PMC free article] [PubMed]
  71. Basturea GN, Rudd KE, Deutscher MP (2006) Identification and characterization of RsmE, the founding member of a new RNA base methyltransferase family. RNA 12:426–434 doi: 10.1261/rna.2283106. [DOI] [PMC free article] [PubMed]
  72. Nordlund ME, Johansson JO, von Pawel-Rammingen U, Bystrom AS (2000) Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae. RNA 6:844–860 doi: 10.1017/s1355838200992422. [DOI] [PMC free article] [PubMed]
  73. Taoka M, Nobe Y, Hori M, Takeuchi A, Masaki S, Yamauchi Y, Nakayama H, Takahashi N, Isobe T (2015) A mass spectrometry-based method for comprehensive quantitative determination of post-transcriptional RNA modifications: the complete chemical structure of Schizosaccharomyces pombe ribosomal RNAs. Nucleic Acids Res 43:e115 doi: 10.1093/nar/gkv560. [DOI] [PMC free article] [PubMed]

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