Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Feb 1;329(Pt 3):615–621. doi: 10.1042/bj3290615

tRNA is entrapped in similar, but distinct, nuclear and cytoplasmic ribonucleoprotein complexes, both of which contain vigilin and elongation factor 1 alpha.

C Kruse 1, A Grünweller 1, D K Willkomm 1, T Pfeiffer 1, R K Hartmann 1, P K Müller 1
PMCID: PMC1219084  PMID: 9445390

Abstract

Vigilin, which is found predominantly in cells and tissues with high levels of protein biosynthesis, was isolated in its native form from human HEp-2 cells (A.T.C.C. CCL23) by immunoaffinity chromatography. Here we demonstrate that vigilin is part of a novel large tRNA-binding ribonucleoprotein complex (tRNP), found not only in the cytoplasm, but also in the nuclei of human cells. Compositional differences in the protein pattern were detected between the nuclear and cytoplasmic tRNPs, although some properties of the purified nuclear tRNP, such as tRNA protection against nuclease attack, were identical with those of the cytoplasmic tRNP. By using either a pool of total human nuclear RNA or radioactively labelled yeast tRNAAsp in rebinding experiments, we could show that tRNA is specifically recaptured by the RNA-depleted, vigilin-containing nuclear complex. We could also show that vigilin is capable of binding tRNA in vitro. Another tRNA-binding protein is elongation factor 1 alpha, which appears to be enriched in the cytoplasmic and nuclear tRNP complexes. This suggests that the cytoplasmic tRNP may be involved in the channelled tRNA cycle in the cytoplasm of eukaryotic cells. Our results also suggest that the nuclear vigilin-containing tRNP may be related to the nuclear export of tRNA.

Full Text

The Full Text of this article is available as a PDF (342.6 KB).

Selected References

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

  1. Billaut-Mulot O., Fernandez-Gomez R., Loyens M., Ouaissi A. Trypanosoma cruzi elongation factor 1-alpha: nuclear localization in parasites undergoing apoptosis. Gene. 1996 Sep 26;174(1):19–26. doi: 10.1016/0378-1119(96)00254-5. [DOI] [PubMed] [Google Scholar]
  2. Boelens W. C., Jansen E. J., van Venrooij W. J., Stripecke R., Mattaj I. W., Gunderson S. I. The human U1 snRNP-specific U1A protein inhibits polyadenylation of its own pre-mRNA. Cell. 1993 Mar 26;72(6):881–892. doi: 10.1016/0092-8674(93)90577-d. [DOI] [PubMed] [Google Scholar]
  3. Boelens W. C., Palacios I., Mattaj I. W. Nuclear retention of RNA as a mechanism for localization. RNA. 1995 May;1(3):273–283. [PMC free article] [PubMed] [Google Scholar]
  4. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  5. Cheng Y., Dahlberg J. E., Lund E. Diverse effects of the guanine nucleotide exchange factor RCC1 on RNA transport. Science. 1995 Mar 24;267(5205):1807–1810. doi: 10.1126/science.7534442. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Condeelis J. Elongation factor 1 alpha, translation and the cytoskeleton. Trends Biochem Sci. 1995 May;20(5):169–170. doi: 10.1016/s0968-0004(00)88998-7. [DOI] [PubMed] [Google Scholar]
  8. Dejgaard K., Leffers H. Characterisation of the nucleic-acid-binding activity of KH domains. Different properties of different domains. Eur J Biochem. 1996 Oct 15;241(2):425–431. doi: 10.1111/j.1432-1033.1996.00425.x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Guddat U., Bakken A. H., Pieler T. Protein-mediated nuclear export of RNA: 5S rRNA containing small RNPs in xenopus oocytes. Cell. 1990 Feb 23;60(4):619–628. doi: 10.1016/0092-8674(90)90665-2. [DOI] [PubMed] [Google Scholar]
  11. Görlich D., Kraft R., Kostka S., Vogel F., Hartmann E., Laskey R. A., Mattaj I. W., Izaurralde E. Importin provides a link between nuclear protein import and U snRNA export. Cell. 1996 Oct 4;87(1):21–32. doi: 10.1016/s0092-8674(00)81319-7. [DOI] [PubMed] [Google Scholar]
  12. Henkel B., Schmidt C., Zorbas H., Pöschl E., Gloe T. R., Purschke W. G., Müller P. K. Chicken vigilin gene organization and expression pattern. The domain structure of the protein is reflected by the exon structure. Eur J Biochem. 1992 Oct 1;209(1):321–328. doi: 10.1111/j.1432-1033.1992.tb17292.x. [DOI] [PubMed] [Google Scholar]
  13. Izaurralde E., Lewis J., McGuigan C., Jankowska M., Darzynkiewicz E., Mattaj I. W. A nuclear cap binding protein complex involved in pre-mRNA splicing. Cell. 1994 Aug 26;78(4):657–668. doi: 10.1016/0092-8674(94)90530-4. [DOI] [PubMed] [Google Scholar]
  14. Janssen G. M., van Damme H. T., Kriek J., Amons R., Möller W. The subunit structure of elongation factor 1 from Artemia. Why two alpha-chains in this complex? J Biol Chem. 1994 Dec 16;269(50):31410–31417. [PubMed] [Google Scholar]
  15. Jarmolowski A., Boelens W. C., Izaurralde E., Mattaj I. W. Nuclear export of different classes of RNA is mediated by specific factors. J Cell Biol. 1994 Mar;124(5):627–635. doi: 10.1083/jcb.124.5.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kiseleva E., Goldberg M. W., Daneholt B., Allen T. D. RNP export is mediated by structural reorganization of the nuclear pore basket. J Mol Biol. 1996 Jul 19;260(3):304–311. doi: 10.1006/jmbi.1996.0401. [DOI] [PubMed] [Google Scholar]
  17. Klinger M. H., Kruse C. Immunocytochemical localization of vigilin, a tRNA-binding protein, after cell fractionation and within the exocrine pancreatic cell of the rat. Ann Anat. 1996 Aug;178(4):331–335. doi: 10.1016/S0940-9602(96)80086-0. [DOI] [PubMed] [Google Scholar]
  18. Kruse C., Grünweller A., Notbohm H., Kügler S., Purschke W. G., Müller P. K. Evidence for a novel cytoplasmic tRNA-protein complex containing the KH-multidomain protein vigilin. Biochem J. 1996 Nov 15;320(Pt 1):247–252. doi: 10.1042/bj3200247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kügler S., Grünweller A., Probst C., Klinger M., Müller P. K., Kruse C. Vigilin contains a functional nuclear localisation sequence and is present in both the cytoplasm and the nucleus. FEBS Lett. 1996 Mar 18;382(3):330–334. doi: 10.1016/0014-5793(96)00204-9. [DOI] [PubMed] [Google Scholar]
  20. McKnight G. L., Reasoner J., Gilbert T., Sundquist K. O., Hokland B., McKernan P. A., Champagne J., Johnson C. J., Bailey M. C., Holly R. Cloning and expression of a cellular high density lipoprotein-binding protein that is up-regulated by cholesterol loading of cells. J Biol Chem. 1992 Jun 15;267(17):12131–12141. [PubMed] [Google Scholar]
  21. Mehlin H., Daneholt B. The Balbiani ring particle: a model for the assembly and export of RNPs from the nucleus? Trends Cell Biol. 1993 Dec;3(12):443–447. doi: 10.1016/0962-8924(93)90034-x. [DOI] [PubMed] [Google Scholar]
  22. Moss B., Elroy-Stein O., Mizukami T., Alexander W. A., Fuerst T. R. Product review. New mammalian expression vectors. Nature. 1990 Nov 1;348(6296):91–92. doi: 10.1038/348091a0. [DOI] [PubMed] [Google Scholar]
  23. Motorin YuA, Wolfson A. D., Orlovsky A. F., Gladilin K. L. Mammalian valyl-tRNA synthetase forms a complex with the first elongation factor. FEBS Lett. 1988 Oct 10;238(2):262–264. doi: 10.1016/0014-5793(88)80492-7. [DOI] [PubMed] [Google Scholar]
  24. Musco G., Stier G., Joseph C., Castiglione Morelli M. A., Nilges M., Gibson T. J., Pastore A. Three-dimensional structure and stability of the KH domain: molecular insights into the fragile X syndrome. Cell. 1996 Apr 19;85(2):237–245. doi: 10.1016/s0092-8674(00)81100-9. [DOI] [PubMed] [Google Scholar]
  25. Negrutskii B. S., Deutscher M. P. Channeling of aminoacyl-tRNA for protein synthesis in vivo. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4991–4995. doi: 10.1073/pnas.88.11.4991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Piñol-Roma S., Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature. 1992 Feb 20;355(6362):730–732. doi: 10.1038/355730a0. [DOI] [PubMed] [Google Scholar]
  27. Plenz G., Gan Y., Raabe H. M., Müller P. K. Expression of vigilin in chicken cartilage and bone. Cell Tissue Res. 1993 Aug;273(2):381–389. doi: 10.1007/BF00312841. [DOI] [PubMed] [Google Scholar]
  28. Rumpel E., Kruse C., Müller P. K., Kühnel W. Expression of vigilin in the uterus of ovariectomized steroid-treated rats and during the estrous cycle. Ann Anat. 1996 Aug;178(4):337–344. doi: 10.1016/S0940-9602(96)80089-6. [DOI] [PubMed] [Google Scholar]
  29. Sanders J., Brandsma M., Janssen G. M., Dijk J., Möller W. Immunofluorescence studies of human fibroblasts demonstrate the presence of the complex of elongation factor-1 beta gamma delta in the endoplasmic reticulum. J Cell Sci. 1996 May;109(Pt 5):1113–1117. doi: 10.1242/jcs.109.5.1113. [DOI] [PubMed] [Google Scholar]
  30. Simos G., Tekotte H., Grosjean H., Segref A., Sharma K., Tollervey D., Hurt E. C. Nuclear pore proteins are involved in the biogenesis of functional tRNA. EMBO J. 1996 May 1;15(9):2270–2284. [PMC free article] [PubMed] [Google Scholar]
  31. Siomi H., Choi M., Siomi M. C., Nussbaum R. L., Dreyfuss G. Essential role for KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome. Cell. 1994 Apr 8;77(1):33–39. doi: 10.1016/0092-8674(94)90232-1. [DOI] [PubMed] [Google Scholar]
  32. Siomi H., Siomi M. C., Nussbaum R. L., Dreyfuss G. The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein. Cell. 1993 Jul 30;74(2):291–298. doi: 10.1016/0092-8674(93)90420-u. [DOI] [PubMed] [Google Scholar]
  33. Stapulionis R., Deutscher M. P. A channeled tRNA cycle during mammalian protein synthesis. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7158–7161. doi: 10.1073/pnas.92.16.7158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Urlaub H., Kruft V., Bischof O., Müller E. C., Wittmann-Liebold B. Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies. EMBO J. 1995 Sep 15;14(18):4578–4588. doi: 10.1002/j.1460-2075.1995.tb00137.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. von Ahsen U., Noller H. F. Identification of bases in 16S rRNA essential for tRNA binding at the 30S ribosomal P site. Science. 1995 Jan 13;267(5195):234–237. doi: 10.1126/science.7528943. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES