Abstract
Histone-poly(A) hybrid molecules were used for transport experiments with resealed nuclear envelopes and after attachment of a cleavable cross-linker (SASD) to identify nuclear proteins. In contrast to histones, the hybrid molecules cannot be accumulated in resealed nuclear envelopes, and in contrast to poly(A), the export of hybrids from preloaded nuclear envelopes is completely impaired. The experiments strongly confirm the existence of poly(A) as an export signal in mRNA which counteracts the nuclear location signals (NLS) in histones. The contradicting transport signals in the hybrid molecules impair translocation through the nuclear pore complex. The failure to accumulate hybrid molecules into resealed nuclear envelopes results from the covalent attachment of polyadenylic acid to histones in a strict 1∶1 molar ratio. This was demonstrated in control transport experiments where radiolabeled histones were simply mixed with nonlabeled poly(A) or radiolabeled poly(A) mixed with nonlabeled histones. In comparison, control uptake experiments with histones covalently linked to a single UMP-mononucleotide are strongly enhanced. Such controls exclude the conceivable possibility of a simple masking of the nuclear location signal in the histones by the covalent attached poly(A) moiety. Photoreactive histone-poly(A) hybrid analogs serve to identify nuclear envelope proteins-presumably in the nuclear pore-with molecular weights of 110, 80, and 71.4 kDa.
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References
Agutter, P. S. (1991).Between Nucleus and Cytoplasm, Chapman and Hall, London, pp. 95–116.
Agutter, P. S., and Ramsey, I. (1979). Further studies on the stimulation of the nuclear envelope NTPase by polyguanylic acid,Biochem. Soc. Trans. 7, 720–721.
Agutter, P. S., Harris, J. R., and Stevenson, I. (1977). Ribonucleic acid stimulation of mammalian liver nuclearenvelope nucleoside triphosphatase,Biochem J. 162, 671–679.
Benditt, J. O., Meyer, C., Fasold, H., Barnard, F. C., and Riedel, N. (1989). Interaction of a nuclear location signal with isolated nuclear envelopes and identification of signalbinding proteins by photoaffinity labeling,Proc. Natl. Acad. Sci. USA 86, 9327–9331.
Blobel, G., and Potter, V. R. (1966). Nuclei from rat liver: A isolation method that combines purity with high yield,Science 154, 1662–1665.
Bonner, W. M. (1975). Protein migration into nuclei I. Frog oocyte nucleiin vivo accumulate a class of microinjected oocyte nuclear proteins and exclude a class of microinjected cytoplamic proteins,J. Cell Biol. 64, 431–437.
Bradford, M. M. (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.
Cremer, G., Kalbas, M., Fasold, H., and Prochnow, D. (1992). Covalent attachment of ribonucleic acids to proteins,J. Protein Chem. 11, 553–560.
Dabauville, M. C., and Franke, W. W. (1982). Karyophilic proteins: Polypeptides synthesizedin vitro accumulate into the cytoplasm of amphibian oocytes,Proc. Natl. Acad. Sci. USA 79, 5302–5306.
Dingwall, C., Sharnick, S. V., and Laskey, R. A. (1982). A polypeptide domain that specifies migration of nucleoplasmin into the nucleus,Cell 30, 449–458.
Dworetzky, S. I., and Feldherr, C. M. (1988). Translocation of RNA-coated gold particles through the nuclear pore of oocytes,J. Cell. Biol. 106, 575–584.
Dworetzky, S. I., Landford, R. E., and Feldherr, C. M. (1988). The effects of variations into the number and sequence of targeting signals on nuclear uptake,J. Cell Biol. 107, 1279–1288.
Eckner, R., Ellmeier, W., and Birnstiel, M. L. (1991). Mature mRNA 3′ end formation stimulates RNA export from the nucleus,EMBO J. 10, 3513–3522.
Feldherr, C. M. (1962). The nuclear annuli as pathways for nucleocytoplasmic exchanges,J. Cell Biol. 14, 65–72.
Feldherr, C. M. (1972). InAdvances in Cell and Molecular Biology (Dupraw, E. J., ed.), Academic Press, New York, pp. 273–307.
Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963). The preparation of131I-labelled human growth hormone of high specific radioactivity,Biochem. J. 89, 114–123.
Hamm, J., and Mattaj, I. W. (1990). Monomethylated cap structures facilitate RNA export from the nucleus,Cell 63, 109–118.
Hill, C. S., and Thomas, J. O. (1990). Core histone-DNA interaction in sea urchin sperm chromatin,Eur. J. Biochem. 187, 145–153.
Jarmolowski, A., Boelens, W. C., Izaurralde, E., and Mattaj, I. W. (1994). Nuclear export of different classes of RNA is mediated by specific factors,J. Cell Biol. 124, 627–635.
Johns, E. W. (1964). Preparative methods for histone fractions from calf thymus,Biochem. J. 92, 55–59.
Kalderon, D., Richardson, W. D., Markham, A. F., and Smith, A. E. (1984). Sequence requirements for nuclear location of SV 40 large-T antigen,Nature 311, 33–38.
Lanford, R. E., Kanda, P., and Kennedy, R. C. (1986). Induction of ncuelar transport with a synthetic peptide homologous to the SV 40 T antigen transport signal,Cell 46, 575–582.
McDonald, J. R., and Agutter, P. S. (1980). The relationship between polyribonucleic binding and the phosphorylation and dephosphorylation of nuclear envelope proteins,FEBS Lett. 116, 145–148.
Moreland, R. B., Langevin, G. L., Singer, R. H., Garcea, R. L., and Hereford, L. A. (1987). Amino acid sequences that determine the nuclear localization of yeast histone 2B,Mol. Cell. Biol. 7, 4048–4057.
Paine, P. L., and Feldherr, C. M. (1972). Nucleoplasmic exchange of macromolecules,Exp. Cell Res. 74, 81–98.
Pandey, N. B., Sun, J. H., and Marzluff, W. (1991). Different complexes are formed on the 3′ end of histone mRNA with nuclear and polyribosomal proteins,Nucleic Acids Res. 19, 5653–5659.
Porschke, D., and Jung, M. (1985). The conformation of single stranded oligonucleotides and of oliogonucleotideoligopeptide complexes from their rotation relaxation in the nanosecond time range,J. Biomol. Struct. Dyn. 6, 1173–1183.
Prochnow, D., Riedel, N., Agutter, P. S., and Fasold, H. (1990). Poly(A) binding proteins located at the inner surface of resealed nuclear envelopes,J. Biol. Chem. 265, 6536–6539.
Prochnow, D., Thomson, M., Schröder, H. C., Müller, W. E. G., and Agutter, P. S. (1994). Efflux of RNA from resealed nuclear envelope ghosts,Arch. Biochem. Biophvs. 312, 579–587.
Richardson, C. C. (1965). Phosphorylation of nucelic acid by an enzyme from T4 bacteriophage-infectedEscherichia coli, Proc. Natl. Acad. Sci. USA 54, 158–165.
Riedel, N., and Fasold, H. (1987a). Preparation and characterization of nuclear-envelope vesicles from rat liver nuclei,Biochem. J. 241, 203–212.
Riedel, N., and Fasold, H. (1987b). Nuclear-envelope vesicles as a model system to study nucleocytoplasmic transport,Biochem. J. 241, 213–219.
Riedel, N., Bachmann, M., Prochnow, D., Richter, H.-P., and Fasold, H. (1987). Permeability measurements with closed vesicles from rat liver nuclear envelopes,Proc. Natl. Acad. Sci. USA 84, 3540–3544.
Sachs, A. B. (1993). Messenger RNA degradation in eukaryotes,cell 74, 413–421.
Schäfer, P., Aitken, S. J. M., Bachmann, M., Agutter, P. S., Müller, W. E. G., and Prochnow, D. (1993). Immunological evidence for the localization of a 110 kDa poly(A) binding protein from rat liver in nuclear envelopes and its phosphorylation by protein kinase C,Cell. Mol. Biol. 39, 703–714.
Schröder, H. C., Friese, U., Bachmann, M., Zaubitzer, T., and Müller, W. E. G. (1989). Energy requirements and kinetics of transport of poly(A)-free histone mRNA compared to poly(A)-rich mRNA from isolated L-cell nuclei,Eur. J. Biochem. 181, 149–158.
Silberklang, M., Gillum, A. M., and RajBhandary, U. L. (1979). Use ofin vitro 32P-labeling in the sequence analysis of nonradiactive tRNAs,Meth. Enzymol. 59, 58–109.
Stevens, B. J., and Swift, H. (1966). RNA transport from nucleus to cytoplasm inChironomus salivary glands,J. Cell. Biol. 31, 55–57.
Terns, M. P., Dahlberg, J. E., and Lund, E. (1993). Multiplecis-acting signals for export of pre-U1 snRNA from the nucleus,Genes Dev. 7, 1898–1908.
Tobian, J. A., Castano, J. G., and Zasloff, M. A. (1984). RNA nuclear transport inXenopus laevis oocytes: Studies with human initiator tRNA-met point mutations,J. Cell Biol. 99, 232a.
Tobian, J. A., Drinkard, L., and Zasloff, M. A. (1985). tRNA nuclear transport: Defining the critical regions of the human intiator tRNA-met point mutants,Cell 43, 415–422.
Wittop-Koning, T. H., and Schümperli, D. (1994). RNAs and ribonucleoproteins in recognition and catalysis,Eur. J. Biochem. 219, 25–42.
Yamasaki, L., Kanda, P., and Lanford, R. E. (1989). Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals,Mol. Cell Biol. 9, 3028–3036.
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Cremer, G., Wojtech, E., Kalbas, M. et al. Histone-poly(A) hybrid molecules as tools to block nuclear pores. J Protein Chem 14, 151–159 (1995). https://doi.org/10.1007/BF01980327
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DOI: https://doi.org/10.1007/BF01980327