[en] BACKGROUND INFORMATION: Nucleolin is a major nucleolar protein which is highly expressed in rapidly dividing cells and cancer cell lines. This protein is claimed to be multifunctional and could play a role in rRNA (ribosomal RNA) synthesis, as well as in cell division or response to cellular stresses. Therefore, how nucleolin influences cell proliferation remained elusive so far. RESULTS: We have generated conditional nucleolin-knockout cells using the chicken B lymphocyte cell line DT40. Our results indicate that nucleolin is absolutely required for the proliferation and for the survival of these cells. Depletion of nucleolin drastically inhibits rDNA (ribosomal DNA) transcription while only slightly affecting pre-rRNA processing. This inhibition is accompanied by modifications of the shape and the structure of the nucleolus. The analysis of mutants of nucleolin, which lack two or three RNA-binding domains, shows that these domains harbour redundant functions and that nucleolin's roles in transcription, rRNA maturation and nucleolar shape can be partially uncoupled. CONCLUSIONS: The function of nucleolin in ribosomal synthesis could account for its effect on cell division and survival, but this vital role does not seem to be linked to sequence-specific RNA binding.
Attain, F.H., Bouvet, P., Dieckmann, T and Feigon, J. (2000) Molecular basis of sequence-specific recognition of pre-ribosomal RNA by nucleolin. EMBO J. 19, 6870-6881
Alvarez, M., Quezada, C., Navarro, C., Molina, A., Bouvet, P., Krauskopf, M. and Vera, M.I. (2003) An increased expression of nucleolin is associated with a physiological nucleolar segregation. Biochem. Biophys. Res. Commun. 301, 152-158
Angelov, D., Bondarenko, V.A., Almagro, S., Menoni, H., Mongelard, F., Hans, F., Mietton, F., Studitsky, V.M., Hamiche, A., Dimitrov, S. and Bouvet, R (2006) Nucleolin is a histone chaperone with FACT-like activity and assists remodeling of nucleosomes. EMBO J. 25, 1669-1679
Arabi, A., Wu, S., Ridderstrale, K., Bierhoff, H., Shiue, C., Fatyol, K., Fahlen, S., Hydbring, P., Soderberg, O., Grummt, I., Larsson, L.G. and Wright, A.P. (2005) c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription. Nat. Celt Biol. 7, 303-310
Arakawa, H., Lodygin, D. and Buerstedde, J.M. (2001) Mutant loxP vectors for selectable marker recycle and conditional knock-outs. BMC Biotechnol. 1, 7
Baba, T.W., Giroir, B.P. and Humphries, E.H. (1985) Cell lines derived from avian lymphomas exhibit two distinct phenotypes. Virology 144, 139-151
Bates, P., Mergny, J.L. and Yang, D. (2007) Quartets in G-major. The First International Meeting on Quadruplex DNA. EMBO Rep. 8, 1003-1010
Boisvert, F.M., van Koningsbruggen, S., Navascues, J. and Lamond, A.I. (2007) The multifunctional nucleolus. Nat. Rev. Mol. Cell. Biol. 8, 574-585
Borer, R.A., Lehner, C.F., Eppenberger, H.M. and Nigg, E.A. (1989) Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56, 379-390
Bouvet, P., Jain, C., Belasco, J.G., Amalric, F. and Erard, M. (1997) RNA recognition by the joint action of two nucleolin RNA-binding domains: genetic analysis and structural modeling. EMBO J. 16, 5235-5246
Buerstedde, J.M. and Takeda, S. (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 67, 179-188
Coller, H.A., Grandori, C., Temayo, P., Colbert, T., Lander, E.S., Eisenman, R.N. and Golub, T.R. (2000) Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc. Natl. Acad. Sci. U.S.A. 97, 3260-3265
De, A., Donahue, S.L., Tabah, A., Castro, N.E., Mraz, N., Cruise, J.L. and Campbell, C. (2006) A novel interaction of nucleolin with Rad51. Biochem. Biophys. Res. Commun. 344, 206-213
Derenzini, M., Passion, A. and Trere, D. (1990) Quantity of nucleolar silver-stained proteins is related to proliferating activity in cancer cells. Lab. Invest. 63, 137-140
Derenzini, M., Sirri, V., Passion, A., Trere, D., Roussel, P., Ochs, R.L. and Hernandez-Verdun, D. (1996) Quantitative changes of the two major AgNOR proteins, nucleolin and protein B23, related to stimulation of rDNA transcription. Exp. Cell Res. 219, 276-282
Derenzini, M., Trere, D., Pession, A., Govoni, M., Sirri, V. and Chieco, P. (2000) Nucleolar size indicates the rapidity of cell proliferation in cancer tissues. J. Pathol. 191, 181-186
Destouches, D., El Khoury, D., Hamma-Kourbali, Y., Krust, B., Albanese, P., Katsoris, P., Guichard, G., Briand, J.P., Courty, J. and Hovanessian, A.G. (2008) Suppression of tumor growth and angiogenesis by a specific antagonist of the cell-surface expressed nucleolin. PLoS ONE 3, e2518
Ghisolfi-Nieto, L., Joseph, G., Puvion-Dutilleul, F., Amalric, F. and Bouvet, P. (1996) Nucleolin is a sequence-specific RNA-binding protein: characterization of targets on pre-ribosomal RNA. J. Mol. Biol. 260, 34-53
Ginisty, H., Amalric, F. and Bouvet, P. (1998) Nucleolin functions in the first step of ribosomal RNA processing. EMBO J. 17, 1476-1486
Ginisty, H., Sarin, G., Ghisolfi-Nieto, L., Roger, B., Libante, V., Amalric, F. and Bouvet, P. (2000) Interaction of nucleolin with an evolutionarily conserved pre-ribosomal RNA sequence is required for the assembly of the primary processing complex. J. Biol. Chem. 275, 18845-18850
Ginisty, H., Amalric, F. and Bouvet, P. (2001) Two different combinations of RNA-binding domains determine the RNA binding specificity of nucleolin. J. Biol. Chem. 276, 14338-14343
Grandori, C., Gomez-Roman, N., Felton-Edkins, Z.A., Ngouenet, C., Galloway, D.A., Eisenman, R.N. and White, R.J. (2005) c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I. Nat. Cell Biol. 7, 311-318
Greasley, P.J., Bonnard, C. and Amati, B. (2000) Myc induces the nucleolin and BN51 genes: possible implications in ribosome biogenesis. Nucleic Acids Res. 28, 446-453
Grinstein, E., Du, Y., Santourlidis, S., Christ, J., Uhrberg, M. and Wernet, P. (2007) Nucleolin regulates gene expression in CD34-positive hematopoietic cells. J. Biol. Chem. 282, 12439-12449
Grisendi, S., Bernardi, R., Rossi, M., Cheng, K., Khandker, L., Manova, K. and Pandolfi, P.P. (2005) Role of nucleophosmin in embryonic development and tumorigenesis. Nature 437, 147-153
Hanakahi, L.A., Sun, H. and Maizels, N. (1999) High affinity interactions of nucleolin with G-G-paired rDNA. J. Biol. Chem. 274, 15908-15912
Hannan, K.M., Brandenburger, Y., Jenkins, A., Sharkey, K., Cavanaugh, A., Rothblum, L., Moss, T., Poortinga, G., McArthur, G.A., Pearson, R.B. and Hannan, R.D. (2003) mTOR-dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF. Mol. Cell. Biol. 23, 8862-8877
Hernandez-Verdun, D. (2006) The nucleolus: a model for the organization of nuclear functions. Histochem. Cell Biol. 126, 135-148
Khan, Q.A. and Dipple, A. (2000) Diverse chemical carcinogens fail to induce G1 arrest in MCF-7 cells. Carcinogenesis 21, 1611-1618
Khurts, S., Masutomi, K., Delgermaa, L., Arai, K., Oishi, N., Mizuno, H., Hayashi, N., Hahn, W.C. and Murakami, S. (2004) Nucleolin interacts with telom6rase. J. Biol. Chem. 279, 51508-51515
Kim, K., Dimitrova, D.D., Carta, K.M., Saxena, A., Daras, M. and Borowiec, J.A. (2006) Novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein a complex formation. Mol. Cell. Biol. 25, 2463-2474
Ma, N., Matsunaga, S., Takata, H., Ono-Maniwa, R., Uchiyama, S. and Fukui, K. (2007) Nucleolin functions in nucleolus formation and chromosome congression. J. Cell Sci. 120, 2091-2105
Maggi, Jr, L.B. and Weber, J.D. (2005) Nucleolar adaptation in human cancer. Cancer Invest. 23, 599-608
Mayer, C., Zhao, J., Yuan, X. and Grummt, I. (2004) mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability. Genes Dev. 18, 423-434
Menssen, A. and Hermeking, H. (2002) Characterization of the c-MYC-regulated transcriptome by SAGE: identification and analysis of c-MYC target genes. Proc. Nati. Acad. Sci. U.S.A. 99, 6274-6279
Neiman, P.E., Ruddell, A., Jasoni, C., Loring, G., Thomas, S.J., Brandvold, K.A., Lee, R., Burnside, J. and Delrow, J. (2001) Analysis of gene expression during myc oncogene-induced lymphomagenesis in the bursa of Fabricius. Proc. Natl. Acad. Sci. U.S.A. 98, 6378-6383
Nicoloso, M., Caizergues-Ferrer, M., Michot, B., Azum, M.C. and Bachellerie, J.P. (1994) U20, a novel small nucleolar RNA, is encoded in an intron of the nucleolin gene in mammals. Mol. Cell. Biol. 14, 5766-5776
Oskarsson, T. and Trumpp, A. (2005) The Myc trilogy: lord of RNA polymerases. Nat. Cell Biol. 7, 215-217
Pich, A., Chiusa, L. and Margaria, E. (2000) Prognostic relevance of AgNORs in tumor pathology. Micron 31, 133-141
Poortinga, G., Hannan, K.M., Snelling, H., Walkley, C.R., Jenkins, A., Sharkey, K., Wall, M., Brandenburger, Y., Palatsides, M., Pearson, R.B. et al. (2004) MAD1 and c-MYC regulate UBF and rDNA transcription during granulocyte differentiation. EMBO J. 23, 3325-3335
Raska, I., Shaw, P.J. and Cmarko, D. (2006) New insights into nucleolar architecture and activity. Int. Rev. Cytol. 255, 177-235
Rebane, A. and Metspalu, A. (1999) U82, a novel snoRNA identified from the fifth intron of human and mouse nucleolin gene. Biochim. Biophys. Acta 1446, 426-430
Rickards, B., Flint, S.J., Cole, M.D. and LeRoy, G. (2007) Nucleolin is required for RNA polymerase I transcription in vivo. Mol. Cell. Biol. 27, 937-948
Roger, B., Moisand, A., Amalric, F. and Bouvet, P. (2002) Repression of RNA polymerase I transcription by nucleolin is independent of the RNA sequence that is transcribed. J. Biol. Chem. 277, 10209-10219
Ruggero, D. and Pandolfi, P.P. (2003) Does the ribosome translate cancer? Nat. Rev. Cancer 3, 179-192
Sale, J.E. (2006) Stable non-targeted transfection of DT40. Subcell. Biochem. 40, 341-344
Sambrook, J. and Russel, D.W. (2001) Molecular Cloning: A Laboratory Manual, volume 1, pp. 7.31-37.40, Cold Spring Harbor Laboratory Press, Plainview, NY
Saribasak, H. and Arakawa, H. (2006) Targeted transfection of DT40 cells. Subcell. Biochem. 40, 419-421
Serin, G., Joseph, G., Faucher, C., Ghisolfi, L., Bouche, G., Amalric, F. and Bouvet, P. (1996) Localization of nucleolin binding sites on human and mouse pre-ribosomal RNA. Biochimie 78, 530-538
Serin, G., Joseph, G., Ghisolfi, L., Bauzan, M., Erard, M., Amalric, F. and Bouvet, P. (1997) Two RNA-binding domains determine the RNA-binding specificity of nucleolin. J. Biol. Chem. 272, 13109-13116
Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E. and Tsien, R.Y. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567-1572
Shibata, Y., Muramatsu, T., Hirai, M., Inui, T., Kimura, T., Saito, H., McCormick, L.M., Bu, G. and Kadomatsu, K. (2002) Nuclear targeting by the growth factor midkine. Mol. Cell. Biol. 22, 6788-6796
Stefanovsky, V.Y., Pelletier, G., Hannan, R., Gagnon-Kugler, T., Rothblum, L.I. and Moss, T. (2001) An immediate response of ribosomal transcription to growth factor stimulation in mammals is mediated by ERK phosphorylation of UBF. Mol. Cell 8, 1063-1073
Storck, S., Shukla, M., Dimitrov, S. and Bouvet, P. (2007) Functions of the histone chaperone nucleolin in diseases. Subcell. Biochem. 41, 125-144
Takagi, M., Absalon, M.J., McLure, K.G. and Kastan, M.B. (2005) Regulation of p53 translation and induction after DNA damage by ribosomal protein L26 and nucleolin. Cell 123, 49-63
Takao, N., Kato, H., Mori, R., Morrison, C., Sonada, E., Sun, X., Shimizu, H., Yoshioka, K., Takeda, S. and Yamamoto, K. (1999) Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation. Oncogene 18, 7002-7009
Thiry, M. and Goessens, G. (1996) The Nucleolus During the Cell Cycle, pp. 1-144, R.G. Landes, New York
Thiry, M., Lepoint, A. and Goessens, G. (1985) Re-evaluation of the site of transcription in Ehrlich tumour cell nucleoli. Biol. Cell 54, 57-64
Tsai, R.Y. and McKay, R.D. (2002) A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. Genes Dev. 16, 2991-3003
Ugrinova, I., Monier, K., Ivaldi, C., Thiry, M., Storck, S., Mongelard, F. and Bouvet, P. (2007) Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication. BMC Mol. Biol. 8, 66
Verrou, C., Zhang, Y, Zurn, C., Schamel, W.W. and Reth, M. (1999) Comparison of the tamoxifen regulated chimeric Cre recombinases MerCreMer and CreMer. Biol. Chem. 380, 1435-1438
Yuan, X., Zhou, Y., Casanova, E., Chai, M., Kiss, E., Grone, H.J., Schutz, G. and Grummt, I. (2005) Genetic inactivation of the transcription factor TIF-IA leads to nucleolar disruption, cell cycle arrest, and p53-mediated apoptosis. Mol. Cell 19, 77-87
Zhao, J., Yuan, X., Frodin, M. and Grummt, I. (2003) ERK-dependent phosphorylation of the transcription initiation factor TIF-IA is required for RNA polymerase I transcription and cell growth. Mol. Cell 11, 405-413
