Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Mar 1;106(3):575–584. doi: 10.1083/jcb.106.3.575

Translocation of RNA-coated gold particles through the nuclear pores of oocytes

PMCID: PMC2115071  PMID: 2450095

Abstract

In the present study, various sized gold particles coated with tRNA, 5S RNA, or poly(A) were used to localize and characterize the pathways for RNA translocation to the cytoplasm. RNA-coated gold particles were microinjected into the nucleus of Xenopus oocytes. The cells were fixed after 15, 60 min, or 6 h, and the particle distribution was later observed by electron microscopy. Similar results were obtained with all classes of RNA used. After nuclear injection, particles ranging from 20- 230 A in diameter were observed within central channels of the nuclear pores and in the cytoplasm immediately adjacent to the pores. Particles of this size would not be expected to diffuse through the pores, suggesting that some form of mediated transport occurred. In addition, it was found that the translocation process is saturable. At least 97% of the pores analyzed appeared to be involved in the translocation process. Gold coated with nonphysiological polynucleotides (poly[I] or poly[dA]) were also translocated. When nuclei were injected with either BSA-, ovalbumin-, polyglutamic acid-, or PVP-coated gold, the particles were essentially excluded from the pores. These results indicate that the accumulation of RNA-gold within the pores and adjacent cytoplasm was not due to non-specific effects. We conclude that the translocation sites for gold particles coated with different classes of RNA are located in the centers of the nuclear pores and that particles at least 230 A in diameter can cross the envelope. Tracer particles injected into the cytoplasm were observed within the nuclear pores in areas near the site of injection. However, only a small percentage of the particles actually entered the nucleus. It was also determined, by performing double injection experiments, that individual pores are bifunctional, that is, capable of transporting both proteins and RNA.

Full Text

The Full Text of this article is available as a PDF (2.7 MB).

Selected References

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

  1. Agutter P. S., McCaldin B., McArdle H. J. Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins. Biochem J. 1979 Sep 15;182(3):811–819. doi: 10.1042/bj1820811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baer B. W., Kornberg R. D. The protein responsible for the repeating structure of cytoplasmic poly(A)-ribonucleoprotein. J Cell Biol. 1983 Mar;96(3):717–721. doi: 10.1083/jcb.96.3.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernd A., Schröder H. C., Zahn R. K., Müller W. E. Modulation of the nuclear-envelope nucleoside triphosphatase by poly(A)-rich mRNA and by microtubule protein. Eur J Biochem. 1982 Dec;129(1):43–49. doi: 10.1111/j.1432-1033.1982.tb07018.x. [DOI] [PubMed] [Google Scholar]
  4. Clawson G. A., Feldherr C. M., Smuckler E. A. Nucleocytoplasmic RNA transport. Mol Cell Biochem. 1985 Jul;67(2):87–99. doi: 10.1007/BF02370167. [DOI] [PubMed] [Google Scholar]
  5. Clawson G. A., James J., Woo C. H., Friend D. S., Moody D., Smuckler E. A. Pertinence of nuclear envelope nucleoside triphosphatase activity to ribonucleic acid transport. Biochemistry. 1980 Jun 10;19(12):2748–2756. doi: 10.1021/bi00553a033. [DOI] [PubMed] [Google Scholar]
  6. Clawson G. A., Koplitz M., Castler-Schechter B., Smuckler E. A. Energy utilization and RNA transport: their interdependence. Biochemistry. 1978 Sep 5;17(18):3747–3752. doi: 10.1021/bi00611a012. [DOI] [PubMed] [Google Scholar]
  7. Clawson G. A., Woo C. H., Button J., Smuckler E. A. Photoaffinity labeling of the major nucleosidetriphosphatase of rat liver nuclear envelope. Biochemistry. 1984 Jul 17;23(15):3501–3507. doi: 10.1021/bi00310a018. [DOI] [PubMed] [Google Scholar]
  8. De Robertis E. M., Lienhard S., Parisot R. F. Intracellular transport of microinjected 5S and small nuclear RNAs. Nature. 1982 Feb 18;295(5850):572–577. doi: 10.1038/295572a0. [DOI] [PubMed] [Google Scholar]
  9. Dingwall C., Laskey R. A. Protein import into the cell nucleus. Annu Rev Cell Biol. 1986;2:367–390. doi: 10.1146/annurev.cb.02.110186.002055. [DOI] [PubMed] [Google Scholar]
  10. Dingwall C., Sharnick S. V., Laskey R. A. A polypeptide domain that specifies migration of nucleoplasmin into the nucleus. Cell. 1982 Sep;30(2):449–458. doi: 10.1016/0092-8674(82)90242-2. [DOI] [PubMed] [Google Scholar]
  11. Dumont J. N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J Morphol. 1972 Feb;136(2):153–179. doi: 10.1002/jmor.1051360203. [DOI] [PubMed] [Google Scholar]
  12. FELDHERR C. M. THE EFFECT OF THE ELECTRON-OPAQUE PORE MATERIAL ON EXCHANGES THROUGH THE NUCLEAR ANNULI. J Cell Biol. 1965 Apr;25:43–53. doi: 10.1083/jcb.25.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feldherr C. M., Cohen R. J., Ogburn J. A. Evidence for mediated protein uptake by amphibian oocyte nuclei. J Cell Biol. 1983 May;96(5):1486–1490. doi: 10.1083/jcb.96.5.1486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feldherr C. M., Kallenbach E., Schultz N. Movement of a karyophilic protein through the nuclear pores of oocytes. J Cell Biol. 1984 Dec;99(6):2216–2222. doi: 10.1083/jcb.99.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Feldherr C. M. Ribosomal RNA synthesis and transport following disruption of the nuclear envelope. Cell Tissue Res. 1980;205(1):157–162. doi: 10.1007/BF00234451. [DOI] [PubMed] [Google Scholar]
  16. Feldherr C. M. The uptake of endogenous proteins by oocyte nuclei. Exp Cell Res. 1975 Jul;93(2):411–419. doi: 10.1016/0014-4827(75)90467-x. [DOI] [PubMed] [Google Scholar]
  17. Fey E. G., Ornelles D. A., Penman S. Association of RNA with the cytoskeleton and the nuclear matrix. J Cell Sci Suppl. 1986;5:99–119. doi: 10.1242/jcs.1986.supplement_5.6. [DOI] [PubMed] [Google Scholar]
  18. Franke W. W., Scheer U. Pathways of nucleocytoplasmic translocation of ribonucleoproteins. Symp Soc Exp Biol. 1974;(28):249–282. [PubMed] [Google Scholar]
  19. Kalt M. R., Tandler B. A study of fixation of early amphibian embryos for electron microscopy. J Ultrastruct Res. 1971 Sep;36(5):633–645. doi: 10.1016/s0022-5320(71)90020-7. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Paine P. L., Moore L. C., Horowitz S. B. Nuclear envelope permeability. Nature. 1975 Mar 13;254(5496):109–114. doi: 10.1038/254109a0. [DOI] [PubMed] [Google Scholar]
  22. Paine P. L. Nucleocytoplasmic movement of fluorescent tracers microinjected into living salivary gland cells. J Cell Biol. 1975 Sep;66(3):652–657. doi: 10.1083/jcb.66.3.652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sachs A. B., Kornberg R. D. Nuclear polyadenylate-binding protein. Mol Cell Biol. 1985 Aug;5(8):1993–1996. doi: 10.1128/mcb.5.8.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Setyono B., Greenberg J. R. Proteins associated with poly(A) and other regions of mRNA and hnRNA molecules as investigated by crosslinking. Cell. 1981 Jun;24(3):775–783. doi: 10.1016/0092-8674(81)90103-3. [DOI] [PubMed] [Google Scholar]
  25. Stevens B. J., Swift H. RNA transport from nucleus to cytoplasm in Chironomus salivary glands. J Cell Biol. 1966 Oct;31(1):55–77. doi: 10.1083/jcb.31.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tobian J. A., Drinkard L., Zasloff M. tRNA nuclear transport: defining the critical regions of human tRNAimet by point mutagenesis. Cell. 1985 Dec;43(2 Pt 1):415–422. doi: 10.1016/0092-8674(85)90171-0. [DOI] [PubMed] [Google Scholar]
  27. Zasloff M. tRNA transport from the nucleus in a eukaryotic cell: carrier-mediated translocation process. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6436–6440. doi: 10.1073/pnas.80.21.6436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. van Eekelen C. A., van Venrooij W. J. hnRNA and its attachment to a nuclear protein matrix. J Cell Biol. 1981 Mar;88(3):554–563. doi: 10.1083/jcb.88.3.554. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES