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. 1996 Aug;16(8):4535–4543. doi: 10.1128/mcb.16.8.4535

Translation of a testis-specific Cu/Zn superoxide dismutase (SOD-1) mRNA is regulated by a 65-kilodalton protein which binds to its 5' untranslated region.

W Gu 1, N R Hecht 1
PMCID: PMC231452  PMID: 8754854

Abstract

Mouse testes contain two distinct superoxide dismutase (SOD-1) transcripts which differ by 114 nucleotides in their 5' untranslated regions (UTRs) (W. Gu, C. Morales, and N. B. Hecht, J. Biol. Chem. 270:236-243, 1995). The shorter SOD-1 mRNA, a somatic type SOD-I mRNA (SSOD-1), is ubiquitously expressed in all somatic tissues as well as in testes. The larger SOD-1 mRNA, a testis-specific SOD-1 mRNA (TSOD-1), derived from an alternative upstream start site, is transcribed solely in postmeiotic germ cells and is translationally regulated during spermiogenesis. Since the two mRNAs have identical nucleotides except that TSOD-1 has an additional sequence at its 5' terminus, we have proposed that the extra 5' UTR sequence may be involved in the translational control of the TSOD-1 mRNA during spermiogenesis. Here we show that, when assayed in a cell-free system, TSOD-1 is translated only slightly less efficiently than SSOD-1. RNA gel retardation and UV cross-linking assays reveal that a testicular cytoplasmic protein (Cu/Zn superoxide dismutase RNA-binding protein [SOD-RBP]) of about 65 kDa specifically binds to the extended 5' UTR of TSOD-1. After purification of SOD-RBP by RNA affinity chromatography, we demonstrate that SOD-RBP can repress the in vitro translation of TSOD-1 mRNA but not SSOD-1 mRNA or cotranslated luciferase mRNA. We conclude that SOD-RBP serves as a repressor in the translation of TSOD-1 mRNA during spermiogenesis and thereby fine-tunes the level of Cu/Zn superoxide dismutase produced in maturing germ cells.

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Selected References

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  1. Aharon T., Schneider R. J. Selective destabilization of short-lived mRNAs with the granulocyte-macrophage colony-stimulating factor AU-rich 3' noncoding region is mediated by a cotranslational mechanism. Mol Cell Biol. 1993 Mar;13(3):1971–1980. doi: 10.1128/mcb.13.3.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aitken R. J., Clarkson J. S. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil. 1987 Nov;81(2):459–469. doi: 10.1530/jrf.0.0810459. [DOI] [PubMed] [Google Scholar]
  3. Alvarez J. G., Storey B. T. Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation. J Androl. 1992 May-Jun;13(3):232–241. [PubMed] [Google Scholar]
  4. Alvarez J. G., Touchstone J. C., Blasco L., Storey B. T. Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as major enzyme protectant against oxygen toxicity. J Androl. 1987 Sep-Oct;8(5):338–348. doi: 10.1002/j.1939-4640.1987.tb00973.x. [DOI] [PubMed] [Google Scholar]
  5. Arrick B. A., Lee A. L., Grendell R. L., Derynck R. Inhibition of translation of transforming growth factor-beta 3 mRNA by its 5' untranslated region. Mol Cell Biol. 1991 Sep;11(9):4306–4313. doi: 10.1128/mcb.11.9.4306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Avraham K. B., Schickler M., Sapoznikov D., Yarom R., Groner Y. Down's syndrome: abnormal neuromuscular junction in tongue of transgenic mice with elevated levels of human Cu/Zn-superoxide dismutase. Cell. 1988 Sep 9;54(6):823–829. doi: 10.1016/s0092-8674(88)91153-1. [DOI] [PubMed] [Google Scholar]
  7. Bauché F., Fouchard M. H., Jégou B. Antioxidant system in rat testicular cells. FEBS Lett. 1994 Aug 8;349(3):392–396. doi: 10.1016/0014-5793(94)00709-8. [DOI] [PubMed] [Google Scholar]
  8. Benedetto M. T., Anzai Y., Gordon J. W. Isolation and analysis of the mouse genomic sequence encoding Cu(2+)-Zn2+ superoxide dismutase. Gene. 1991 Mar 15;99(2):191–195. doi: 10.1016/0378-1119(91)90126-v. [DOI] [PubMed] [Google Scholar]
  9. Bernstein P., Ross J. Poly(A), poly(A) binding protein and the regulation of mRNA stability. Trends Biochem Sci. 1989 Sep;14(9):373–377. doi: 10.1016/0968-0004(89)90011-x. [DOI] [PubMed] [Google Scholar]
  10. Braun R. E., Peschon J. J., Behringer R. R., Brinster R. L., Palmiter R. D. Protamine 3'-untranslated sequences regulate temporal translational control and subcellular localization of growth hormone in spermatids of transgenic mice. Genes Dev. 1989 Jun;3(6):793–802. doi: 10.1101/gad.3.6.793. [DOI] [PubMed] [Google Scholar]
  11. Chang E. C., Crawford B. F., Hong Z., Bilinski T., Kosman D. J. Genetic and biochemical characterization of Cu,Zn superoxide dismutase mutants in Saccharomyces cerevisiae. J Biol Chem. 1991 Mar 5;266(7):4417–4424. [PubMed] [Google Scholar]
  12. Clerch L. B. A 3' untranslated region of catalase mRNA composed of a stem-loop and dinucleotide repeat elements binds a 69-kDa redox-sensitive protein. Arch Biochem Biophys. 1995 Feb 20;317(1):267–274. doi: 10.1006/abbi.1995.1162. [DOI] [PubMed] [Google Scholar]
  13. Crapo J. D., Oury T., Rabouille C., Slot J. W., Chang L. Y. Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10405–10409. doi: 10.1073/pnas.89.21.10405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cummings A., Sommerville J. Protein kinase activity associated with stored messenger ribonucleoprotein particles of Xenopus oocytes. J Cell Biol. 1988 Jul;107(1):45–56. doi: 10.1083/jcb.107.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Deng H. X., Hentati A., Tainer J. A., Iqbal Z., Cayabyab A., Hung W. Y., Getzoff E. D., Hu P., Herzfeldt B., Roos R. P. Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase. Science. 1993 Aug 20;261(5124):1047–1051. doi: 10.1126/science.8351519. [DOI] [PubMed] [Google Scholar]
  16. Didier D. K., Schiffenbauer J., Woulfe S. L., Zacheis M., Schwartz B. D. Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7322–7326. doi: 10.1073/pnas.85.19.7322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Elroy-Stein O., Bernstein Y., Groner Y. Overproduction of human Cu/Zn-superoxide dismutase in transfected cells: extenuation of paraquat-mediated cytotoxicity and enhancement of lipid peroxidation. EMBO J. 1986 Mar;5(3):615–622. doi: 10.1002/j.1460-2075.1986.tb04255.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fajardo M. A., Butner K. A., Lee K., Braun R. E. Germ cell-specific proteins interact with the 3' untranslated regions of Prm-1 and Prm-2 mRNA. Dev Biol. 1994 Dec;166(2):643–653. doi: 10.1006/dbio.1994.1344. [DOI] [PubMed] [Google Scholar]
  19. Gold B., Stern L., Bradley F. M., Hecht N. B. Gene expression during mammalian spermatogenesis. II. Evidence for stage-specific differences in mRNA populations. J Exp Zool. 1983 Jan;225(1):123–134. doi: 10.1002/jez.1402250115. [DOI] [PubMed] [Google Scholar]
  20. Goossen B., Caughman S. W., Harford J. B., Klausner R. D., Hentze M. W. Translational repression by a complex between the iron-responsive element of ferritin mRNA and its specific cytoplasmic binding protein is position-dependent in vivo. EMBO J. 1990 Dec;9(12):4127–4133. doi: 10.1002/j.1460-2075.1990.tb07635.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goossen B., Hentze M. W. Position is the critical determinant for function of iron-responsive elements as translational regulators. Mol Cell Biol. 1992 May;12(5):1959–1966. doi: 10.1128/mcb.12.5.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Grens A., Scheffler I. E. The 5'- and 3'-untranslated regions of ornithine decarboxylase mRNA affect the translational efficiency. J Biol Chem. 1990 Jul 15;265(20):11810–11816. [PubMed] [Google Scholar]
  23. Gu W., Hecht N. B. Developmental expression of glutathione peroxidase, catalase, and manganese superoxide dismutase mRNAs during spermatogenesis in the mouse. J Androl. 1996 May-Jun;17(3):256–262. [PubMed] [Google Scholar]
  24. Gu W., Morales C., Hecht N. B. In male mouse germ cells, copper-zinc superoxide dismutase utilizes alternative promoters that produce multiple transcripts with different translation potential. J Biol Chem. 1995 Jan 6;270(1):236–243. doi: 10.1074/jbc.270.1.236. [DOI] [PubMed] [Google Scholar]
  25. Hake L. E., Richter J. D. CPEB is a specificity factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation. Cell. 1994 Nov 18;79(4):617–627. doi: 10.1016/0092-8674(94)90547-9. [DOI] [PubMed] [Google Scholar]
  26. Han J. R., Yiu G. K., Hecht N. B. Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9550–9554. doi: 10.1073/pnas.92.21.9550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Harrell C. M., McKenzie A. R., Patino M. M., Walden W. E., Theil E. C. Ferritin mRNA: interactions of iron regulatory element with translational regulator protein P-90 and the effect on base-paired flanking regions. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4166–4170. doi: 10.1073/pnas.88.10.4166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Henderson B. R., Seiser C., Kühn L. C. Characterization of a second RNA-binding protein in rodents with specificity for iron-responsive elements. J Biol Chem. 1993 Dec 25;268(36):27327–27334. [PubMed] [Google Scholar]
  29. Hentze M. W., Caughman S. W., Rouault T. A., Barriocanal J. G., Dancis A., Harford J. B., Klausner R. D. Identification of the iron-responsive element for the translational regulation of human ferritin mRNA. Science. 1987 Dec 11;238(4833):1570–1573. doi: 10.1126/science.3685996. [DOI] [PubMed] [Google Scholar]
  30. Holland M. K., Alvarez J. G., Storey B. T. Production of superoxide and activity of superoxide dismutase in rabbit epididymal spermatozoa. Biol Reprod. 1982 Dec;27(5):1109–1118. doi: 10.1095/biolreprod27.5.1109. [DOI] [PubMed] [Google Scholar]
  31. Houman F., Diaz-Torres M. R., Wright A. Transcriptional antitermination in the bgl operon of E. coli is modulated by a specific RNA binding protein. Cell. 1990 Sep 21;62(6):1153–1163. doi: 10.1016/0092-8674(90)90392-r. [DOI] [PubMed] [Google Scholar]
  32. Jow W. W., Schlegel P. N., Cichon Z., Phillips D., Goldstein M., Bardin C. W. Identification and localization of copper-zinc superoxide dismutase gene expression in rat testicular development. J Androl. 1993 Nov-Dec;14(6):439–447. [PubMed] [Google Scholar]
  33. Kennedy M. C., Mende-Mueller L., Blondin G. A., Beinert H. Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11730–11734. doi: 10.1073/pnas.89.24.11730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kilk A., Laan M., Torp A. Human CuZn superoxide dismutase enzymatic activity in cells is regulated by the length of the mRNA. FEBS Lett. 1995 Apr 10;362(3):323–327. doi: 10.1016/0014-5793(95)00266-c. [DOI] [PubMed] [Google Scholar]
  35. Kim S. J., Park K., Koeller D., Kim K. Y., Wakefield L. M., Sporn M. B., Roberts A. B. Post-transcriptional regulation of the human transforming growth factor-beta 1 gene. J Biol Chem. 1992 Jul 5;267(19):13702–13707. [PubMed] [Google Scholar]
  36. Koeller D. M., Casey J. L., Hentze M. W., Gerhardt E. M., Chan L. N., Klausner R. D., Harford J. B. A cytosolic protein binds to structural elements within the iron regulatory region of the transferrin receptor mRNA. Proc Natl Acad Sci U S A. 1989 May;86(10):3574–3578. doi: 10.1073/pnas.86.10.3574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
  38. Kwon Y. K., Hecht N. B. Binding of a phosphoprotein to the 3' untranslated region of the mouse protamine 2 mRNA temporally represses its translation. Mol Cell Biol. 1993 Oct;13(10):6547–6557. doi: 10.1128/mcb.13.10.6547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kwon Y. K., Hecht N. B. Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3584–3588. doi: 10.1073/pnas.88.9.3584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kwon Y. K., Murray M. T., Hecht N. B. Proteins homologous to the Xenopus germ cell-specific RNA-binding proteins p54/p56 are temporally expressed in mouse male germ cells. Dev Biol. 1993 Jul;158(1):99–100. doi: 10.1006/dbio.1993.1170. [DOI] [PubMed] [Google Scholar]
  41. Lasso J. L., Noiles E. E., Alvarez J. G., Storey B. T. Mechanism of superoxide dismutase loss from human sperm cells during cryopreservation. J Androl. 1994 May-Jun;15(3):255–265. [PubMed] [Google Scholar]
  42. Levanon D., Lieman-Hurwitz J., Dafni N., Wigderson M., Sherman L., Bernstein Y., Laver-Rudich Z., Danciger E., Stein O., Groner Y. Architecture and anatomy of the chromosomal locus in human chromosome 21 encoding the Cu/Zn superoxide dismutase. EMBO J. 1985 Jan;4(1):77–84. doi: 10.1002/j.1460-2075.1985.tb02320.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Liu X. F., Elashvili I., Gralla E. B., Valentine J. S., Lapinskas P., Culotta V. C. Yeast lacking superoxide dismutase. Isolation of genetic suppressors. J Biol Chem. 1992 Sep 15;267(26):18298–18302. [PubMed] [Google Scholar]
  44. Manzella J. M., Blackshear P. J. Specific protein binding to a conserved region of the ornithine decarboxylase mRNA 5'-untranslated region. J Biol Chem. 1992 Apr 5;267(10):7077–7082. [PubMed] [Google Scholar]
  45. Marello K., LaRovere J., Sommerville J. Binding of Xenopus oocyte masking proteins to mRNA sequences. Nucleic Acids Res. 1992 Nov 11;20(21):5593–5600. doi: 10.1093/nar/20.21.5593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Murray M. T., Krohne G., Franke W. W. Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos. J Cell Biol. 1991 Jan;112(1):1–11. doi: 10.1083/jcb.112.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Murray M. T., Schiller D. L., Franke W. W. Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):11–15. doi: 10.1073/pnas.89.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Neupert B., Thompson N. A., Meyer C., Kühn L. C. A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta. Nucleic Acids Res. 1990 Jan 11;18(1):51–55. doi: 10.1093/nar/18.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Nikolajczyk B. S., Murray M. T., Hecht N. B. A mouse homologue of the Xenopus germ cell-specific ribonucleic acid/deoxyribonucleic acid-binding proteins p54/p56 interacts with the protamine 2 promoter. Biol Reprod. 1995 Mar;52(3):524–530. doi: 10.1095/biolreprod52.3.524. [DOI] [PubMed] [Google Scholar]
  50. Odom T. A., Chipman D. M., Betts G., Bernhard S. A. Transient and steady-state kinetic studies of sodium-potassium adenosine triphosphatase using beta-(2-furyl)acryloyl phosphate as chromophoric substrate assay. Biochemistry. 1981 Feb 3;20(3):480–486. doi: 10.1021/bi00506a006. [DOI] [PubMed] [Google Scholar]
  51. Paris J., Swenson K., Piwnica-Worms H., Richter J. D. Maturation-specific polyadenylation: in vitro activation by p34cdc2 and phosphorylation of a 58-kD CPE-binding protein. Genes Dev. 1991 Sep;5(9):1697–1708. doi: 10.1101/gad.5.9.1697. [DOI] [PubMed] [Google Scholar]
  52. Parkin N. T., Sonenberg N. Identification of a protein that binds specifically to RNA from the first exon of c-myc. Oncogene. 1989 Jul;4(7):815–822. [PubMed] [Google Scholar]
  53. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Pearson W. R. Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol. 1990;183:63–98. doi: 10.1016/0076-6879(90)83007-v. [DOI] [PubMed] [Google Scholar]
  55. Pelletier J., Sonenberg N. Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell. 1985 Mar;40(3):515–526. doi: 10.1016/0092-8674(85)90200-4. [DOI] [PubMed] [Google Scholar]
  56. Ray B. K., Lawson T. G., Kramer J. C., Cladaras M. H., Grifo J. A., Abramson R. D., Merrick W. C., Thach R. E. ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors. J Biol Chem. 1985 Jun 25;260(12):7651–7658. [PubMed] [Google Scholar]
  57. Richter J. D., Smith L. D. Reversible inhibition of translation by Xenopus oocyte-specific proteins. Nature. 1984 May 24;309(5966):378–380. doi: 10.1038/309378a0. [DOI] [PubMed] [Google Scholar]
  58. Sachs A., Wahle E. Poly(A) tail metabolism and function in eucaryotes. J Biol Chem. 1993 Nov 5;268(31):22955–22958. [PubMed] [Google Scholar]
  59. Sherman L., Dafni N., Lieman-Hurwitz J., Groner Y. Nucleotide sequence and expression of human chromosome 21-encoded superoxide dismutase mRNA. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5465–5469. doi: 10.1073/pnas.80.18.5465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Sherman L., Levanon D., Lieman-Hurwitz J., Dafni N., Groner Y. Human Cu/Zn superoxide dismutase gene: molecular characterization of its two mRNA species. Nucleic Acids Res. 1984 Dec 21;12(24):9349–9365. doi: 10.1093/nar/12.24.9349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Sonenberg N. Picornavirus RNA translation continues to surprise. Trends Genet. 1991 Apr;7(4):105–106. doi: 10.1016/0168-9525(91)90440-2. [DOI] [PubMed] [Google Scholar]
  62. Tafuri S. R., Familari M., Wolffe A. P. A mouse Y box protein, MSY1, is associated with paternal mRNA in spermatocytes. J Biol Chem. 1993 Jun 5;268(16):12213–12220. [PubMed] [Google Scholar]
  63. Van Loon A. A., Den Boer P. J., Van der Schans G. P., Mackenbach P., Grootegoed J. A., Baan R. A., Lohman P. H. Immunochemical detection of DNA damage induction and repair at different cellular stages of spermatogenesis of the hamster after in vitro or in vivo exposure to ionizing radiation. Exp Cell Res. 1991 Apr;193(2):303–309. doi: 10.1016/0014-4827(91)90101-y. [DOI] [PubMed] [Google Scholar]
  64. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  65. Yu Y., Radisky E., Leibold E. A. The iron-responsive element binding protein. Purification, cloning, and regulation in rat liver. J Biol Chem. 1992 Sep 15;267(26):19005–19010. [PubMed] [Google Scholar]

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