Amino Acids

, Volume 47, Issue 2, pp 345–356 | Cite as

Polyamine stimulation of eEF1A synthesis based on the unusual position of a complementary sequence to 18S rRNA in eEF1A mRNA

  • Yusuke Terui
  • Akihiko Sakamoto
  • Taketo Yoshida
  • Takuma Kasahara
  • Hideyuki Tomitori
  • Kyohei Higashi
  • Kazuei Igarashi
  • Keiko KashiwagiEmail author
Original Article


It is thought that Shine–Dalgarno-like sequences, which exhibit complementarity to the nucleotide sequences at the 3′-end of 18S rRNA, are not present in eukaryotic mRNAs. However, complementary sequences consisting of more than 5 nucleotides to the 3′-end of 18S rRNA, i.e., a CR sequence, are present at −17 to −32 upstream from the initiation codon AUG in 18 mRNAs involved in protein synthesis except eEF1A mRNA. Thus, effects of the CR sequence in mRNAs and polyamines on protein synthesis were examined using control and polyamine-reduced FM3A and NIH3T3 cells. Polyamines did not stimulate protein synthesis encoded by 18 mRNAs possessing a normal CR sequence. When the CR sequence was deleted, protein synthetic activities decreased to less than 70 % of intact mRNAs. In eEF1A mRNA, the CR sequence was located at −33 to −39 upstream from the initiation codon AUG, and polyamines stimulated eEF1A synthesis about threefold. When the CR sequence was shifted to −22 to −28 upstream from the AUG, eEF1A synthesis increased in polyamine-reduced cells and the degree of polyamine stimulation decreased greatly. The results indicate that the CR sequence exists in many eukaryotic mRNAs, and the location of a CR sequence in mRNAs influences polyamine stimulation of protein synthesis.


Polyamines Protein synthesis mRNA 5′-UTR CR sequence 



N 1-(3-aminopropyl)-cyclohexylamine


Circular dichroism

CR sequence

Complementary sequence to 18S rRNA




N 1-guanyl-1,7-diaminoheptane


Phosphate-buffered saline


5′-Untranslated region of mRNA



We thank Drs. A. J. Michael and K. Williams for their help in preparing the manuscript. We also thank Dr. R. G. Mirmira for his kind gift of antibody against hypusinated eIF5A. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

726_2014_1867_MOESM1_ESM.pptx (68 kb)
Fig. S1CR sequences in various mRNAs. The nucleotide sequences in the 5′-UTR of mRNAs were quoted from The CR sequences in the 5′-UTR of various mRNAs are shown in red together with the numbers of the complementary nucleotide sequence of the 3′-end of 18S rRNA(Holmberg et al. 1994). Non-complementary base in CR sequences is shown in black. Consensus Kozak sequences (Kozak 1991) were shown in blue. Number of nucleotides of the 5′-UTR of specified mRNAs is shown in the parentheses (PPTX 68 kb)
726_2014_1867_MOESM2_ESM.docx (28 kb)
Supplementary material 2 (DOCX 28 kb)


  1. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  2. Chappell SA, Dresios J, Edelman GM, Mauro VP (2006) Ribosomal shunting mediated by a translational enhancer element that base pairs to 18S rRNA. Proc Natl Acad Sci USA 103:9488–9493PubMedCentralPubMedCrossRefGoogle Scholar
  3. Chen H, Bjerknes M, Kumar R, Jay E (1994) Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. Nucleic Acids Res 22:4953–4957PubMedCentralPubMedCrossRefGoogle Scholar
  4. Cunningham-Rundles S, Maas WK (1975) Isolation, characterization, and mapping of Escherichia coli mutants blocked in the synthesis of ornithine decarboxylase. J Bacteriol 124:791–799PubMedCentralPubMedGoogle Scholar
  5. Dresios J, Chappell SA, Zhou W, Mauro VP (2006) An mRNA-rRNA base-pairing mechanism for translation initiation in eukaryotes. Nat Struct Mol Biol 13:30–34PubMedCrossRefGoogle Scholar
  6. Echandi G, Algranati ID (1975) Defective 30S ribosomal particles in a polyamine auxotroph of Escherichia coli. Biochem Biophys Res Commun 67:1185–1191PubMedCrossRefGoogle Scholar
  7. Higashi K, Terui Y, Suganami A, Tamura Y, Nishimura K, Kashiwagi K, Igarashi K (2008) Selective structural change by spermidine in the bulged-out region of double-stranded RNA and its effect on RNA function. J Biol Chem 283:32989–32994PubMedCrossRefGoogle Scholar
  8. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59PubMedCrossRefGoogle Scholar
  9. Holmberg L, Melander Y, Nygard O (1994) Probing the structure of mouse Ehrlich ascites cell 5.8S, 18S and 28S ribosomal RNA in situ. Nucleic Acids Res 22:1374–1382PubMedCentralPubMedCrossRefGoogle Scholar
  10. Igarashi K, Kashiwagi K (2006) Polyamine Modulon in Escherichia coli: genes involved in the stimulation of cell growth by polyamines. J Biochem 139:11–16PubMedCrossRefGoogle Scholar
  11. Igarashi K, Kashiwagi K (2010) Modulation of cellular function by polyamines. Int J Biochem Cell Biol 42:39–51PubMedCrossRefGoogle Scholar
  12. Igarashi K, Morris DR (1984) Physiological effects in bovine lymphocytes of inhibiting polyamine synthesis with ethylglyoxal bis(guanylhydrazone). Cancer Res 44:5332–5337PubMedGoogle Scholar
  13. Igarashi K, Kashiwagi K, Aoki R, Kojima M, Hirose S (1979a) Comparative studies on the increase by polyamines of fidelity of protein synthesis in Escherichia coli and wheat germ cell-free systems. Biochem Biophys Res Commun 91:440–448PubMedCrossRefGoogle Scholar
  14. Igarashi K, Kashiwagi K, Kishida K, Watanabe Y, Kogo A, Hirose S (1979b) Defect in the split proteins of 30-S ribosomal subunits and under-methylation of 16-S ribosomal RNA in a polyamine-requiring mutant of Escherichia coli grown in the absence of polyamines. Eur J Biochem 93:345–353PubMedCrossRefGoogle Scholar
  15. Igarashi K, Saisho T, Yuguchi M, Kashiwagi K (1997) Molecular mechanism of polyamine stimulation of the synthesis of oligopeptide-binding protein. J Biol Chem 272:4058–4064PubMedCrossRefGoogle Scholar
  16. Jakus J, Wolff EC, Park MH, Folk JE (1993) Features of the spermidine-binding site of deoxyhypusine synthase as derived from inhibition studies. Effective inhibition by bis- and mono-guanylated diamines and polyamines. J Biol Chem 268:13151–13159PubMedGoogle Scholar
  17. Jelenc PC, Kurland CG (1979) Nucleoside triphosphate regeneration decreases the frequency of translation errors. Proc Natl Acad Sci USA 76:3174–3178PubMedCentralPubMedCrossRefGoogle Scholar
  18. Kakinuma Y, Hoshino K, Igarashi K (1988) Characterization of the inducible polyamine transporter in bovine lymphocytes. Eur J Biochem 176:409–414PubMedCrossRefGoogle Scholar
  19. Kozak M (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 266:19867–19870PubMedGoogle Scholar
  20. Mandal S, Mandal A, Johansson HE, Orjalo AV, Park MH (2013) Depletion of cellular polyamines, spermidine and spermine, causes a total arrest in translation and growth in mammalian cells. Proc Natl Acad Sci USA 110:2169–2174PubMedCentralPubMedCrossRefGoogle Scholar
  21. Meng Z, Jackson NL, Shcherbakov OD, Choi H, Blume SW (2010) The human IGF1R IRES likely operates through a Shine-Dalgarno-like interaction with the G961 loop (E-site) of the 18S rRNA and is kinetically modulated by a naturally polymorphic polyU loop. J Cell Biochem 110:531–544PubMedCentralPubMedGoogle Scholar
  22. Miyamoto S, Kashiwagi K, Ito K, Watanabe S, Igarashi K (1993) Estimation of polyamine distribution and polyamine stimulation of protein synthesis in Escherichia coli. Arch Biochem Biophys 300:63–68PubMedCrossRefGoogle Scholar
  23. Nakano S, Kanzaki T, Sugimoto N (2004) Influences of ribonucleotide on a duplex conformation and its thermal stability: study with the chimeric RNA-DNA strands. J Am Chem Soc 126:1088–1095PubMedCrossRefGoogle Scholar
  24. Nielsen PJ, Manchester KL, Towbin H, Gordon J, Thomas G (1982) The phosphorylation of ribosomal protein S6 in rat tissues following cycloheximide injection, in diabetes, and after denervation of diaphragm. A simple immunological determination of the extent of S6 phosphorylation on protein blots. J Biol Chem 257:12316–12321PubMedGoogle Scholar
  25. Nishiki Y, Farb TB, Friedrich J, Bokvist K, Mirmira RG, Maier B (2013) Characterization of a novel polyclonal anti-hypusine antibody. Springerplus 2:421PubMedCentralPubMedCrossRefGoogle Scholar
  26. Nishimura K, Murozumi K, Shirahata A, Park MH, Kashiwagi K, Igarashi K (2005) Independent roles of eIF5A and polyamines in cell proliferation. Biochem J 385:779–785PubMedCentralPubMedCrossRefGoogle Scholar
  27. Nishimura K, Okudaira H, Ochiai E, Higashi K, Kaneko M, Ishii I, Nishimura T, Dohmae N, Kashiwagi K, Igarashi K (2009) Identification of proteins whose synthesis is preferentially enhanced by polyamines at the level of translation in mammalian cells. Int J Biochem Cell Biol 41:2251–2261PubMedCrossRefGoogle Scholar
  28. Ogasawara T, Ito K, Igarashi K (1989) Effect of polyamines on globin synthesis in a rabbit reticulocyte polyamine-free protein synthetic system. J Biochem 105:164–167PubMedGoogle Scholar
  29. Pegg AE (2009) Mammalian polyamine metabolism and function. IUBMB Life 61:880–894PubMedCentralPubMedCrossRefGoogle Scholar
  30. Pisarev AV, Kolupaeva VG, Yusupov MM, Hellen CU, Pestova TV (2008) Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes. EMBO J 27:1609–1621PubMedCentralPubMedCrossRefGoogle Scholar
  31. Sakamoto A, Terui Y, Yamamoto T, Kasahara T, Nakamura M, Tomitori H, Yamamoto K, Ishihama A, Michael AJ, Igarashi K, Kashiwagi K (2012) Enhanced biofilm formation and/or cell viability by polyamines through stimulation of response regulators UvrY and CpxR in the two-component signal transducing systems, and ribosome recycling factor. Int J Biochem Cell Biol 44:1877–1886PubMedCrossRefGoogle Scholar
  32. Sambrook J, Fritsch EF, Maniatis T (2001) Extraction, purification, and analysis of mRNA from eukaryotic cells. In: Sambrook J, Russell DW (eds) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 1.32–1.34Google Scholar
  33. Scheper GC, Voorma HO, Thomas AA (1994) Basepairing with 18S ribosomal RNA in internal initiation of translation. FEBS Lett 352:271–275PubMedCrossRefGoogle Scholar
  34. Shine J, Dalgarno L (1974) The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346PubMedCentralPubMedCrossRefGoogle Scholar
  35. Turner DH, Sugimoto N, Freier SM (1988) RNA structure prediction. Annu Rev Biophys Biophys Chem 17:167–192PubMedCrossRefGoogle Scholar
  36. Uemura T, Higashi K, Takigawa M, Toida T, Kashiwagi K, Igarashi K (2009) Polyamine modulon in yeast-Stimulation of COX4 synthesis by spermidine at the level of translation. Int J Biochem Cell Biol 41:2538–2545PubMedCrossRefGoogle Scholar
  37. Watanabe S, Kusama-Eguchi K, Kobayashi H, Igarashi K (1991) Estimation of polyamine binding to macromolecules and ATP in bovine lymphocytes and rat liver. J Biol Chem 266:20803–20809PubMedGoogle Scholar
  38. Yoshida M, Meksuriyen D, Kashiwagi K, Kawai G, Igarashi K (1999) Polyamine stimulation of the synthesis of oligopeptide-binding protein (OppA). Involvement of a structural change of the Shine-Dalgarno sequence and the initiation codon AUG in OppA mRNA. J Biol Chem 274:22723–22728PubMedCrossRefGoogle Scholar
  39. Yueh A, Schneider RJ (1996) Selective translation initiation by ribosome jumping in adenovirus-infected and heat-shocked cells. Genes Dev 10:1557–1567PubMedCrossRefGoogle Scholar
  40. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Yusuke Terui
    • 1
  • Akihiko Sakamoto
    • 1
  • Taketo Yoshida
    • 1
  • Takuma Kasahara
    • 1
  • Hideyuki Tomitori
    • 1
  • Kyohei Higashi
    • 2
  • Kazuei Igarashi
    • 2
    • 3
  • Keiko Kashiwagi
    • 1
    Email author
  1. 1.Faculty of PharmacyChiba Institute of ScienceChoshiJapan
  2. 2.Graduate School of Pharmaceutical SciencesChiba UniversityChibaJapan
  3. 3.Amine Pharma Research InstituteInnovation Plaza at Chiba UniversityChibaJapan

Personalised recommendations