Nucleolar proteome dynamics


The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses1,2,3,4. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling5, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.

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Figure 1: The nucleolar proteome.
Figure 2: Determination of nucleolar protein dynamics.
Figure 3: Comparison of different methods to measure nucleolar protein dynamics.
Figure 4: Dynamic profiles of nucleolar proteins.


  1. 1

    Visintin, R. & Amon, A. The nucleolus: the magician's hat for cell cycle tricks. Curr. Opin. Cell Biol. 12, 752 (2000)

    CAS  Article  Google Scholar 

  2. 2

    Guarente, L. Link between aging and the nucleolus. Genes Dev. 11, 2449–2455 (1997)

    CAS  Article  Google Scholar 

  3. 3

    Sherr, C. J. & Weber, J. D. The ARF/p53 pathway. Curr. Opin. Genet. Dev. 10, 94–99 (2000)

    CAS  Article  Google Scholar 

  4. 4

    Olson, M. O. Sensing cellular stress: another new function for the nucleolus? Sci. STKE [online] pe10 (2004) (doi:10.1126/stke.2242004pe10)

  5. 5

    Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Mauramatsu, M., Smetana, K. & Busch, H. Quantitative aspects of isolation of nucleoli of the Walker carcinosarcoma and liver of the rat. Cancer Res. 25, 693–697 (1963)

    Google Scholar 

  7. 7

    Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Andersen, J. S. et al. Directed proteomic analysis of the human nucleolus. Curr. Biol. 12, 1–11 (2002)

    Article  Google Scholar 

  9. 9

    Trinkle-Mulcahy, L., Sleeman, J. E. & Lamond, A. I. Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells. J. Cell Sci. 114, 4219–4228 (2001)

    CAS  PubMed  Google Scholar 

  10. 10

    Li, D., Meier, U. T., Dobrowolska, G. & Krebs, E. G. Specific interaction between casein kinase 2 and the nucleolar protein Nopp140. J. Biol. Chem. 272, 3773–3779 (1997)

    CAS  Article  Google Scholar 

  11. 11

    Yamamoto, R. T., Nogi, Y., Dodd, J. A. & Nomura, M. RRN3 gene of Saccharomyces cerevisiae encodes an essential RNA polymerase I transcription factor which interacts with the polymerase independently of DNA template. EMBO J. 15, 3964–3973 (1996)

    CAS  Article  Google Scholar 

  12. 12

    Charroux, B. et al. Gemin4. A novel component of the SMN complex that is found in both gems and nucleoli. J. Cell Biol. 148, 1177–1186 (2000)

    CAS  Article  Google Scholar 

  13. 13

    Lamond, A. I. & Earnshaw, W. C. Structure and function in the nucleus. Science 280, 547–553 (1998)

    CAS  Article  Google Scholar 

  14. 14

    Misteli, T. Protein dynamics: implications for nuclear architecture and gene expression. Science 291, 843–847 (2001)

    ADS  CAS  Article  Google Scholar 

  15. 15

    Lamond, A. I. & Sleeman, J. E. Nuclear substructure and dynamics. Curr. Biol. 13, R825–R828 (2003)

    CAS  Article  Google Scholar 

  16. 16

    Leung, A. K. & Lamond, A. I. In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles. J. Cell Biol. 157, 615–629 (2002)

    CAS  Article  Google Scholar 

  17. 17

    Ong, S. E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002)

    CAS  Article  Google Scholar 

  18. 18

    Blagoev, B., Ong, S. E., Kratchmarova, I. & Mann, M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nature Biotechnol. 22, 1139–1145 (2004)

    CAS  Article  Google Scholar 

  19. 19

    Perry, R. P. & Kelley, D. E. Inhibition of RNA synthesis by actinomycin D: characteristic dose-response of different RNA species. J. Cell. Physiol. 76, 127–139 (1970)

    CAS  Article  Google Scholar 

  20. 20

    Raghavan, A. et al. Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes. Nucleic Acids Res. 30, 5529–5538 (2002)

    CAS  Article  Google Scholar 

  21. 21

    Tschochner, H. & Hurt, E. Pre-ribosomes on the road from the nucleolus to the cytoplasm. Trends Cell Biol. 13, 255–263 (2003)

    CAS  Article  Google Scholar 

  22. 22

    Dragon, F. et al. A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature 417, 967–970 (2002)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Eisen, M. B., Spellman, P. T., Brown, P. O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA 95, 14863–14868 (1998)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Tamm, I., Hand, R. & Caliguiri, L. A. Action of dichlorobenzimidazole riboside on RNA synthesis in L-929 and HeLa cells. J. Cell Biol. 69, 229–240 (1976)

    CAS  Article  Google Scholar 

  25. 25

    Mattsson, K., Pokrovskaja, K., Kiss, C., Klein, G. & Szekely, L. Proteins associated with the promyelocytic leukemia gene product (PML)-containing nuclear body move to the nucleolus upon inhibition of proteasome-dependent protein degradation. Proc. Natl Acad. Sci. USA 98, 1012–1017 (2001)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Olsen, J. V., Ong, S. E. & Mann, M. Trypsin cleaves exclusively C-terminal to Arginine and lysine residues. Mol. Cell. Proteomics 6, 608–614 (2004)

    Article  Google Scholar 

  27. 27

    Leung, A. K. et al. Quantitative kinetic analysis of nucleolar breakdown and reassembly during mitosis in live human cells. J. Cell Biol. 166, 787–800 (2004)

    CAS  Article  Google Scholar 

  28. 28

    Masson, C. et al. Conditions favoring RNA polymerase I transcription in permeabilized cells. Exp. Cell Res. 226, 114–125 (1996)

    CAS  Article  Google Scholar 

  29. 29

    Boisvert, F. M., Hendzel, M. J. & Bazett-Jones, D. P. Promyelocytic leukemia (PML) nuclear bodies are protein structures that do not accumulate RNA. J. Cell Biol. 148, 283–292 (2000)

    CAS  Article  Google Scholar 

  30. 30

    Scherl, A. et al. Functional proteomic analysis of human nucleolus. Mol. Biol. Cell 13, 4100–4109 (2002)

    CAS  Article  Google Scholar 

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We thank A. Fox for providing the HeLaYFP–p68 cell line and other members of the Lamond and the Mann laboratories for help and discussions. Work in the Center for Experimental Bioinformatics (CEBI) is supported by a grant from the Danish National Research Foundation. A.I.L. is a Wellcome Trust Principal Research Fellow and is funded by a Wellcome Trust Programme grant; A.K.L.L. was funded by a Croucher studentship; Y.W.L. was funded by The Human Frontier Science Program, which is also acknowledged for a network grant entitled ‘Functional organization of the cell nucleus investigated through proteomics and molecular dynamics’.

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Correspondence to Angus I. Lamond or Matthias Mann.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Legends

This document contains the legends for Supplementary Figures S1 and S2, and legends for Supplementary Tables 1 and 2. (DOC 30 kb)

Supplementary Figure S1

Electron micrograph details of isolated HeLa nucleoli that are structurally intact and transcriptionally active. (PDF 2216 kb)

Supplementary Figure S2

Hierarchical clustering of 302 nucleolar proteins arranged by similarity in their response to Actinomycin-D treatment. (PDF 331 kb)

Supplementary Table 1

The nucleolar proteome. (XLS 3108 kb)

Supplementary Table 2

Nucleolar proteome dynamics after actinomycin D treatment. (XLS 3590 kb)

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Andersen, J., Lam, Y., Leung, A. et al. Nucleolar proteome dynamics. Nature 433, 77–83 (2005).

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