Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Jun;16(6):3023–3034. doi: 10.1128/mcb.16.6.3023

A testis cytoplasmic RNA-binding protein that has the properties of a translational repressor.

K Lee 1, M A Fajardo 1, R E Braun 1
PMCID: PMC231297  PMID: 8649414

Abstract

Translation of the mouse protamine 1 (Prm-1) mRNA is repressed for several days during male germ cell differentiation. With the hope of cloning genes that regulate the translational repression of Prm-1, we screened male germ cell cDNA expression libraries with the 3' untranslated region of the Prm-1 RNA. From this screen we obtained two independent clones that encode Prbp, a Prm-1 RNA-binding protein. Prbp contains two copies of a double-stranded-RNA-binding domain. In vitro, the protein binds to a portion of the Prm-1 3' untranslated region previously shown to be sufficient for translational repression in transgenic mice, as well as to poly(I). poly(C). Prbp protein is present in multiple forms in cytoplasmic extracts prepared from wild-type mouse testes and is absent from testes of germ cell-deficient mouse mutants, suggesting that Prbp is restricted to the germ cells of the testis. Immunocytochemical localization confirmed that Prbp is present in the cytoplasmic compartment of late-stage meiotic cells and haploid round spermatids. Recombinant Prbp protein inhibits the translation of multiple mRNAs in a wheat germ lysate, suggesting that Prbp acts to repress translation in round spermatids. While this protein lacks complete specificity for Prm-1-containing RNAs in vitro, the properties of Prbp are consistent with it acting as a general repressor of translation.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Balhorn R., Weston S., Thomas C., Wyrobek A. J. DNA packaging in mouse spermatids. Synthesis of protamine variants and four transition proteins. Exp Cell Res. 1984 Feb;150(2):298–308. doi: 10.1016/0014-4827(84)90572-x. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Braun R. E. Temporal translational regulation of the protamine 1 gene during mouse spermatogenesis. Enzyme. 1990;44(1-4):120–128. doi: 10.1159/000468752. [DOI] [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Cole J. L., Gehman J. D., Shafer J. A., Kuo L. C. Solution oligomerization of the rev protein of HIV-1: implications for function. Biochemistry. 1993 Nov 9;32(44):11769–11775. doi: 10.1021/bi00095a004. [DOI] [PubMed] [Google Scholar]
  6. Cosentino G. P., Venkatesan S., Serluca F. C., Green S. R., Mathews M. B., Sonenberg N. Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9445–9449. doi: 10.1073/pnas.92.21.9445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Curtis D., Lehmann R., Zamore P. D. Translational regulation in development. Cell. 1995 Apr 21;81(2):171–178. doi: 10.1016/0092-8674(95)90325-9. [DOI] [PubMed] [Google Scholar]
  8. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eckner R., Ellmeier W., Birnstiel M. L. Mature mRNA 3' end formation stimulates RNA export from the nucleus. EMBO J. 1991 Nov;10(11):3513–3522. doi: 10.1002/j.1460-2075.1991.tb04915.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ephrussi A., Dickinson L. K., Lehmann R. Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell. 1991 Jul 12;66(1):37–50. doi: 10.1016/0092-8674(91)90137-n. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Ferrandon D., Elphick L., Nüsslein-Volhard C., St Johnston D. Staufen protein associates with the 3'UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner. Cell. 1994 Dec 30;79(7):1221–1232. doi: 10.1016/0092-8674(94)90013-2. [DOI] [PubMed] [Google Scholar]
  13. Gatignol A., Buckler-White A., Berkhout B., Jeang K. T. Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science. 1991 Mar 29;251(5001):1597–1600. doi: 10.1126/science.2011739. [DOI] [PubMed] [Google Scholar]
  14. Gatignol A., Buckler C., Jeang K. T. Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen. Mol Cell Biol. 1993 Apr;13(4):2193–2202. doi: 10.1128/mcb.13.4.2193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Green S. R., Mathews M. B. Two RNA-binding motifs in the double-stranded RNA-activated protein kinase, DAI. Genes Dev. 1992 Dec;6(12B):2478–2490. doi: 10.1101/gad.6.12b.2478. [DOI] [PubMed] [Google Scholar]
  16. Heaphy S., Finch J. T., Gait M. J., Karn J., Singh M. Human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich "bubble" located within the rev-responsive region of viral mRNAs. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7366–7370. doi: 10.1073/pnas.88.16.7366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jackson R. J., Standart N. Do the poly(A) tail and 3' untranslated region control mRNA translation? Cell. 1990 Jul 13;62(1):15–24. doi: 10.1016/0092-8674(90)90235-7. [DOI] [PubMed] [Google Scholar]
  18. Johnson P. A., Peschon J. J., Yelick P. C., Palmiter R. D., Hecht N. B. Sequence homologies in the mouse protamine 1 and 2 genes. Biochim Biophys Acta. 1988 May 6;950(1):45–53. doi: 10.1016/0167-4781(88)90071-1. [DOI] [PubMed] [Google Scholar]
  19. Kierszenbaum A. L., Tres L. L. Structural and transcriptional features of the mouse spermatid genome. J Cell Biol. 1975 May;65(2):258–270. doi: 10.1083/jcb.65.2.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kim-Ha J., Smith J. L., Macdonald P. M. oskar mRNA is localized to the posterior pole of the Drosophila oocyte. Cell. 1991 Jul 12;66(1):23–35. doi: 10.1016/0092-8674(91)90136-m. [DOI] [PubMed] [Google Scholar]
  21. Kleene K. C., Distel R. J., Hecht N. B. Translational regulation and deadenylation of a protamine mRNA during spermiogenesis in the mouse. Dev Biol. 1984 Sep;105(1):71–79. doi: 10.1016/0012-1606(84)90262-8. [DOI] [PubMed] [Google Scholar]
  22. Kleene K. C., Distel R. J., Hecht N. B. cDNA clones encoding cytoplasmic poly(A)+ RNAs which first appear at detectable levels in haploid phases of spermatogenesis in the mouse. Dev Biol. 1983 Aug;98(2):455–464. doi: 10.1016/0012-1606(83)90375-5. [DOI] [PubMed] [Google Scholar]
  23. Kleene K. C., Flynn J. Translation of mouse testis poly(A)+ mRNAs for testis-specific protein, protamine 1, and the precursor for protamine 2. Dev Biol. 1987 Sep;123(1):125–135. doi: 10.1016/0012-1606(87)90434-9. [DOI] [PubMed] [Google Scholar]
  24. Kozak C. A., Gatignol A., Graham K., Jeang K. T., McBride O. W. Genetic mapping in human and mouse of the locus encoding TRBP, a protein that binds the TAR region of the human immunodeficiency virus (HIV-1). Genomics. 1995 Jan 1;25(1):66–72. doi: 10.1016/0888-7543(95)80110-8. [DOI] [PubMed] [Google Scholar]
  25. Krawetz S. A., Connor W., Dixon G. H. Cloning of bovine P1 protamine cDNA and the evolution of vertebrate P1 protamines. DNA. 1987 Feb;6(1):47–57. doi: 10.1089/dna.1987.6.47. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Lee C. H., Hoyer-Fender S., Engel W. The nucleotide sequence of a human protamine 1 cDNA. Nucleic Acids Res. 1987 Sep 25;15(18):7639–7639. doi: 10.1093/nar/15.18.7639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lee C. H., Mansouri A., Hecht W., Hecht N. B., Engel W. Nucleotide sequence of a bovine protamine cDNA. Biol Chem Hoppe Seyler. 1987 Feb;368(2):131–135. doi: 10.1515/bchm3.1987.368.1.131. [DOI] [PubMed] [Google Scholar]
  30. Lee K., Haugen H. S., Clegg C. H., Braun R. E. Premature translation of protamine 1 mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12451–12455. doi: 10.1073/pnas.92.26.12451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lee R. C., Feinbaum R. L., Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993 Dec 3;75(5):843–854. doi: 10.1016/0092-8674(93)90529-y. [DOI] [PubMed] [Google Scholar]
  32. Leibold E. A., Munro H. N. Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated region of ferritin heavy- and light-subunit mRNAs. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2171–2175. doi: 10.1073/pnas.85.7.2171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lyon M. F., Hawkes S. G. X-linked gene for testicular feminization in the mouse. Nature. 1970 Sep 19;227(5264):1217–1219. doi: 10.1038/2271217a0. [DOI] [PubMed] [Google Scholar]
  34. MINTZ B., RUSSELL E. S. Gene-induced embryological modifications of primordial germ cells in the mouse. J Exp Zool. 1957 Mar;134(2):207–237. doi: 10.1002/jez.1401340202. [DOI] [PubMed] [Google Scholar]
  35. MONESI V. RIBONUCLEIC ACID SYNTHESIS DURING MITOSIS AND MEIOSIS IN THE MOUSE TESTIS. J Cell Biol. 1964 Sep;22:521–532. doi: 10.1083/jcb.22.3.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mali P., Kaipia A., Kangasniemi M., Toppari J., Sandberg M., Hecht N. B., Parvinen M. Stage-specific expression of nucleoprotein mRNAs during rat and mouse spermiogenesis. Reprod Fertil Dev. 1989;1(4):369–382. doi: 10.1071/rd9890369. [DOI] [PubMed] [Google Scholar]
  37. Malim M. H., Cullen B. R. HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency. Cell. 1991 Apr 19;65(2):241–248. doi: 10.1016/0092-8674(91)90158-u. [DOI] [PubMed] [Google Scholar]
  38. Manche L., Green S. R., Schmedt C., Mathews M. B. Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol Cell Biol. 1992 Nov;12(11):5238–5248. doi: 10.1128/mcb.12.11.5238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. OAKBERG E. F. A description of spermiogenesis in the mouse and its use in analysis of the cycle of the seminiferous epithelium and germ cell renewal. Am J Anat. 1956 Nov;99(3):391–413. doi: 10.1002/aja.1000990303. [DOI] [PubMed] [Google Scholar]
  40. Park H., Davies M. V., Langland J. O., Chang H. W., Nam Y. S., Tartaglia J., Paoletti E., Jacobs B. L., Kaufman R. J., Venkatesan S. TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4713–4717. doi: 10.1073/pnas.91.11.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Richter J. D. Translational control during early development. Bioessays. 1991 Apr;13(4):179–183. doi: 10.1002/bies.950130406. [DOI] [PubMed] [Google Scholar]
  42. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schneppenheim R., Rautenberg P. A luminescence Western blot with enhanced sensitivity for antibodies to human immunodeficiency virus. Eur J Clin Microbiol. 1987 Feb;6(1):49–51. doi: 10.1007/BF02097190. [DOI] [PubMed] [Google Scholar]
  44. Schumacher J. M., Lee K., Edelhoff S., Braun R. E. Distribution of Tenr, an RNA-binding protein, in a lattice-like network within the spermatid nucleus in the mouse. Biol Reprod. 1995 Jun;52(6):1274–1283. doi: 10.1095/biolreprod52.6.1274. [DOI] [PubMed] [Google Scholar]
  45. Schumacher J. M., Lee K., Edelhoff S., Braun R. E. Spnr, a murine RNA-binding protein that is localized to cytoplasmic microtubules. J Cell Biol. 1995 May;129(4):1023–1032. doi: 10.1083/jcb.129.4.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Singh H., Clerc R. G., LeBowitz J. H. Molecular cloning of sequence-specific DNA binding proteins using recognition site probes. Biotechniques. 1989 Mar;7(3):252–261. [PubMed] [Google Scholar]
  47. St Johnston D., Beuchle D., Nüsslein-Volhard C. Staufen, a gene required to localize maternal RNAs in the Drosophila egg. Cell. 1991 Jul 12;66(1):51–63. doi: 10.1016/0092-8674(91)90138-o. [DOI] [PubMed] [Google Scholar]
  48. St Johnston D., Brown N. H., Gall J. G., Jantsch M. A conserved double-stranded RNA-binding domain. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10979–10983. doi: 10.1073/pnas.89.22.10979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tafuri S. R., Wolffe A. P. DNA binding, multimerization, and transcription stimulation by the Xenopus Y box proteins in vitro. New Biol. 1992 Apr;4(4):349–359. [PubMed] [Google Scholar]
  50. Vinson C. R., LaMarco K. L., Johnson P. F., Landschulz W. H., McKnight S. L. In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage. Genes Dev. 1988 Jul;2(7):801–806. doi: 10.1101/gad.2.7.801. [DOI] [PubMed] [Google Scholar]
  51. Wightman B., Ha I., Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell. 1993 Dec 3;75(5):855–862. doi: 10.1016/0092-8674(93)90530-4. [DOI] [PubMed] [Google Scholar]
  52. Yelick P. C., Kwon Y. H., Flynn J. F., Borzorgzadeh A., Kleene K. C., Hecht N. B. Mouse transition protein 1 is translationally regulated during the postmeiotic stages of spermatogenesis. Mol Reprod Dev. 1989;1(3):193–200. doi: 10.1002/mrd.1080010307. [DOI] [PubMed] [Google Scholar]
  53. Zapp M. L., Hope T. J., Parslow T. G., Green M. R. Oligomerization and RNA binding domains of the type 1 human immunodeficiency virus Rev protein: a dual function for an arginine-rich binding motif. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7734–7738. doi: 10.1073/pnas.88.17.7734. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES