Skip to main content

Overexpression of the nucleolar protein SURF-6 in mouse NIH/3T3 fibroblasts stabilizes pre-rRNA intragenic transcribed spacers

Abstract

The nucleolar protein SURF-6 is an evolutionarily conserved and vitally important eukaryotic protein; however, its function in mammals still remains unclear. In the present study, we have examined the effect of SURF-6 overexpression on the content of different pre-rRNA sequences in mouse NIH/3T3 fibroblasts. The overexpression of SURF-6 was induced by a Tet-On system of regulation of gene expression and two plasmid constructs. Plasmid pUHrT62-1 encoded the rtTA protein, a tetracycline-dependent factor of transcription activation, and plasmid pBI-SURF6 contained the coding sequences of the genes of the mouse SURF-6 protein and EGFP, whose expression was controlled by the rtTA-dependent promoter. An increase in the SURF-6 content was observed 24 and 48 h after the addition of the inductor doxycycline. The dot hybridization of total RNA from cells having an increased SURF-6 content with biotinylated oligonucleotide probes to different regions of mouse primary pre-rRNA transcripts revealed a sevenfold increase in the content of ITS2 and a twofold increase in the content of 5′ETS compared with their levels in cells transfected with the same plasmids but incubated without doxycycline. The content of fragments corresponding to 18S, 5.8S, and 28S rRNAs remained almost unchanged. These observations showed for the first time that the mammalian nucleolar protein SURF-6, similar to its homolog the yeast protein Rrp14, contributes to the stabilization (prevents the degradation) of pre-rRNA intragenic transcribed spacers. At present, no metazoan proteins that play a similar role in ribosome biogenesis have been described.

This is a preview of subscription content, access via your institution.

Abbreviations

DAPI:

4′,6′-diamidinophenylindole

5′ETS and 3′ETS:

5′- and 3′ external transcribed spacers of pre-rRNA, respectively

FISH:

fluorescent hybridization in situ

ITS1 and ITS2:

the first and the second transcribed spacers of pre-rRNA, respectively

pre-RNAs:

different forms of precursors of mature 18S, 5.8S, and 28S rRNAs

pUHrT62-1:

an auxiliary plasmid encoding the rtTA protein

pBI-EGFP:

the control plasmid encoding EGFP (green fluorescent protein)

pBI-SURF6:

a working plasmid encoding mouse SURF-6

PBS:

phosphate-buffered saline

PMSF:

phenylmethylsulfonyl fluoride

rDNA and rRNA:

ribosomal DNA and ribosomal RNA

rtTA:

a tetracycline-dependent factor of transcription activation

47S-45S:

a primary transcript of the common precursor of 18S, 5.8S, and 28S rRNAs

Tet-On system:

the Tet-On system of gene expression regulation

References

  1. Magoulas, C. and Fried, M., DNA Cell Biol., 1996, vol. 15, pp. 305–316.

    CAS  Article  PubMed  Google Scholar 

  2. Magoulas, C. and Fried, M., Gene, 2000, vol. 243, pp. 115–123.

    CAS  Article  PubMed  Google Scholar 

  3. Polzikov, M., Zatsepina, O., and Magoulas, C., Biochem. Biophys. Res. Commun., 2005, vol. 327, pp. 143–149.

    CAS  Article  PubMed  Google Scholar 

  4. Polzikov, M., Magoulas, Ch., and Zatsepina, O., Mol. Biol. Rep., 2007, vol. 34, pp. 155–160.

    CAS  Article  PubMed  Google Scholar 

  5. Oeffinger, M., Fatica, A., Rout, M.P., and Tollervey, D., Nucl. Acids Res., 2007, vol. 35, pp. 1354–1366.

    CAS  Article  PubMed  Google Scholar 

  6. Yamada, H., Horigome, C., Okada, N., Shirai, C., and Mizuta, K., RNA, 2007, vol. 13, pp. 1977–1987.

    CAS  Article  PubMed  Google Scholar 

  7. Magoulas, Ch., Zatsepina, O.V., Jordan, P.W., Jordan, E.G., and Fried, M., Eur. J. Cell Biol., 1998, vol. 75, pp. 174–183.

    CAS  Article  PubMed  Google Scholar 

  8. Gurchenkov, V.V., Polzikov, M.A., Magoulas, Sh., Romanova, L.G., and Zatsepina, O.V., Bioorg. Khim., 2005, vol. 31, pp. 578–585 [Russ. J. Bioorg. Chem. (Eng. Transl.), 2005, vol. 31, pp. 521–528].

    CAS  PubMed  Google Scholar 

  9. Puvion-Dutilleul, F., Mazan, S., Nicoloso, M., Pichard, E., Bachellerie, J.P., and Puvion, E., Eur. J. Cell Biol., 1992, vol. 58, pp. 149–162.

    CAS  PubMed  Google Scholar 

  10. Romanova, L.G., Anger, M., Zatsepina, O.V., and Schultz, R.M., Biol. Reprod., 2006, vol. 75, pp. 690–696.

    CAS  Article  PubMed  Google Scholar 

  11. Henras, A.K., Soudet, J., Gerus, M., Lebaron, S., Caizergues-Ferrer, M., Mougin, A., and Henry, Y., Cell. Mol. Life Sci., 2008, vol. 65, pp. 2334–2359.

    CAS  Article  PubMed  Google Scholar 

  12. Lazdins, I.B., Delannoy, M., and Sollner-Webb, B., Chromosoma, 1997, vol. 105, pp. 481–495.

    CAS  Article  PubMed  Google Scholar 

  13. Newton, K., Petfalski, E., Tollervey, D., and Caceres, J.F., Mol. Cell. Biol., 2003, vol. 23, pp. 8519–8527.

    CAS  Article  PubMed  Google Scholar 

  14. Kiss, T., Fayet, E., Jády, B.E., Richard, P., and Weber, M., Cold Spring Harb. Symp. Quant. Biol., 2006, vol. 71, pp. 407–417.

    CAS  Article  PubMed  Google Scholar 

  15. Ge, J., Rudnick, D.A., He, J., Crimmins, D.L., Ladenson, J.H., Bessler, M., and Mason, P.J., Mol. Cell. Biol., 2010, vol. 30, pp. 413–422.

    CAS  Article  PubMed  Google Scholar 

  16. Lebreton, A., Tomecki, R., Dziembowski, A., and Seraphin, B., Nature Lett., 2008, vol. 456, pp. 993–997.

    CAS  Article  Google Scholar 

  17. Lykke-Andersen, S., Brodersen, D.E., and Jensen, T.H., J. Cell Sci., 2009, vol. 122, pp. 1487–1494.

    CAS  Article  PubMed  Google Scholar 

  18. Savkur, R.S. and Olson, M.O., Nucleic Acid Res., 1998, vol. 26, pp. 4508–4515.

    CAS  Article  PubMed  Google Scholar 

  19. Okuwaki, M., J. Biochem., 2008, vol. 143, pp. 441–448.

    CAS  Article  PubMed  Google Scholar 

  20. Allmang, C., Petfalski, E., Podtelejnikov, A., Mann, M., Tollervey, D., and Mitchell, P., Genes Dev., 1999, vol. 13, pp. 2148–2158.

    CAS  Article  PubMed  Google Scholar 

  21. Kent, T., Lapik, Y.R., and Pestov, D.G., RNA, 2009, vol. 15, pp. 14–20.

    CAS  Article  PubMed  Google Scholar 

  22. Lopez, M.L., Rosenblad, M.A., and Samuelsson, T., RNA Biol., 2009, vol. 6, pp. 208–220.

    CAS  Article  Google Scholar 

  23. Fortin, K.R., Nicholson, R.H., and Nicholson, A.W., BMC Genomics, 2002, vol. 3, pp. 26–34.

    Article  PubMed  Google Scholar 

  24. Horsey, E.W., Jakovljevic, J., Miles, T.D., Harnpicharnchai, P., and Woolford, J.L., RNA, 2004, vol. 10, pp. 813–827.

    CAS  Article  PubMed  Google Scholar 

  25. Coute, Y., Kindbeiter, K., Belin, S., Dieckmann, R., Duret, L., Bezin, L., Sanchez, J.C., and Diaz, J.J., Mol. Cell. Proteomics, 2008, vol. 7, pp. 546–559.

    CAS  PubMed  Google Scholar 

  26. Turner, A.J., Knox, A.A., Prieto, J.L., McStay, B., and Watkins, N.J., Mol. Cell. Biol., 2009, vol. 29, pp. 3007–3017.

    CAS  Article  PubMed  Google Scholar 

  27. Nadano, D. and Sato, T.A.J., Biol. Chem., 2000, vol. 275, pp. 13967–13973.

    CAS  Article  Google Scholar 

  28. Mroczek, S. and Kufel, J., Nucleic Acid Res., 2008, vol. 36, pp. 2874–2888.

    CAS  Article  PubMed  Google Scholar 

  29. Urlinger, S., Baron, U., Thellmann, M., Hasan, M.T., Bujard, H., and Hillen, W., Proc. Natl. Acad. Sci. USA, 2000, vol. 97, pp. 7963–7968.

    CAS  Article  PubMed  Google Scholar 

  30. Stanek, D., Koberna, K., Pliss, A., Malínský, J., Masata, M., Vecerová, J., Risueño, M.C., and Raska, I., Chromosoma, 2001, vol. 110, pp. 460–470.

    CAS  Article  PubMed  Google Scholar 

  31. Bowman, L.H., Goldman, W.E., Goldberg, G.I., Hebert, M.B., and Schlessinger, D., Mol. Cell Biol., 1983, vol. 3, pp. 1501–1510.

    CAS  PubMed  Google Scholar 

  32. Veiko, N.N., Terekhov, S.V., Shubaeva, N.O., Smirnova, T.D., Ivanova, S.M., Egolina, N.A., Tsvetkova, T.G., Spitkovsky, D.M., and Lyapunova, N.A., Mol. Biol., 2005, vol. 39, pp. 264–275 [Mol. Biol. (Moscow) (Eng. Transl.), 2005, vol. 39, pp. 234–243].

    Article  Google Scholar 

  33. Grozdanov, P., Georgiev, O., and Karagyozov, L., Genomics, 2003, vol. 82, pp. 637–643.

    CAS  Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to O. V. Zatsepina.

Additional information

Original Russian Text © M.A. Polzikov, N.N. Veiko, O.O. Zharskaya, Ch. Magoulas, O.V. Zatsepina, 2010, published in Bioorganicheskaya Khimiya, 2010, Vol. 36, No. 5, pp. 661–671.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Polzikov, M.A., Veiko, N.N., Zharskaya, O.O. et al. Overexpression of the nucleolar protein SURF-6 in mouse NIH/3T3 fibroblasts stabilizes pre-rRNA intragenic transcribed spacers. Russ J Bioorg Chem 36, 610–619 (2010). https://doi.org/10.1134/S1068162010050092

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S1068162010050092

Key words

  • nucleolar protein SURF-6
  • ribosome biogenesis
  • NIH/3T3 cells