Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit

Abstract

The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments1,2,3,4. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed5,6,7,8,9,10,11,12. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the ‘beak’. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Ltv1, Enp1 and Rps3 form an extractable pre-ribosomal subcomplex.
Figure 2: Structural comparison of pre-40S and mature 40S subunits.
Figure 3: Phosphorylation state induces 40S subunit maturation.
Figure 4: Hrr25 kinase regulates 40S subunit maturation.

References

  1. 1

    Warner, J. R. Nascent ribosomes. Cell 107, 133–136 (2001)

    CAS  Article  Google Scholar 

  2. 2

    Fatica, A. & Tollervey, D. Making ribosomes. Curr. Opin. Cell Biol. 14, 313–318 (2002)

    CAS  Article  Google Scholar 

  3. 3

    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 

  4. 4

    Granneman, S. & Baserga, S. J. Crosstalk in gene expression: coupling and co-regulation of rDNA transcription, pre-ribosome assembly and pre-rRNA processing. Curr. Opin. Cell Biol. 17, 281–286 (2005)

    CAS  Article  Google Scholar 

  5. 5

    Bassler, J. et al. Identification of a 60S preribosomal particle that is closely linked to nuclear export. Mol. Cell 8, 517–529 (2001)

    CAS  Article  Google Scholar 

  6. 6

    Harnpicharnchai, P. et al. Composition and functional characterization of yeast 66S ribosome assembly intermediates. Mol. Cell 8, 505–515 (2001)

    CAS  Article  Google Scholar 

  7. 7

    Saveanu, C. et al. Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps. EMBO J. 20, 6475–6484 (2001)

    CAS  Article  Google Scholar 

  8. 8

    Fatica, A., Cronshaw, A. D., Dlakic, M. & Tollervey, D. Ssf1p prevents premature processing of an early pre-60S ribosomal particle. Mol. Cell 9, 341–351 (2002)

    CAS  Article  Google Scholar 

  9. 9

    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 

  10. 10

    Grandi, P. et al. 90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors. Mol. Cell 10, 105–115 (2002)

    CAS  Article  Google Scholar 

  11. 11

    Nissan, T. A., Bassler, J., Petfalski, E., Tollervey, D. & Hurt, E. 60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm. EMBO J. 21, 5539–5547 (2002)

    CAS  Article  Google Scholar 

  12. 12

    Schäfer, T., Strauss, D., Petfalski, E., Tollervey, D. & Hurt, E. The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes. EMBO J. 22, 1370–1380 (2003)

    Article  Google Scholar 

  13. 13

    Loar, J. W. et al. Genetic and biochemical interactions among Yar1, Ltv1 and Rps3 define novel links between environmental stress and ribosome biogenesis in Saccharomyces cerevisiae. Genetics 168, 1877–1889 (2004)

    CAS  Article  Google Scholar 

  14. 14

    Frank, J., Verschoor, A. & Boublik, M. Multivariate statistical analysis of ribosome electron micrographs. L and R lateral views of the 40 S subunit from HeLa cells. J. Mol. Biol. 161, 107–133 (1982)

    CAS  Article  Google Scholar 

  15. 15

    Frank, J., Verschoor, A. & Boublik, M. Computer averaging of electron micrographs of 40S ribosomal subunits. Science 214, 1353–1355 (1981)

    ADS  CAS  Article  Google Scholar 

  16. 16

    Spahn, C. M. et al. Structure of the 80S ribosome from Saccharomyces cerevisiae–tRNA–ribosome and subunit–subunit interactions. Cell 107, 373–386 (2001)

    CAS  Article  Google Scholar 

  17. 17

    Wimberly, B. T. et al. Structure of the 30S ribosomal subunit. Nature 407, 327–339 (2000)

    ADS  CAS  Article  Google Scholar 

  18. 18

    Ferreira-Cerca, S., Poll, G., Gleizes, P. E., Tschochner, H. & Milkereit, P. Roles of eukaryotic ribosomal proteins in maturation and transport of pre-18S rRNA and ribosome function. Mol. Cell 20, 263–275 (2005)

    CAS  Article  Google Scholar 

  19. 19

    Geerlings, T. H., Faber, A. W., Bister, M. D., Vos, J. C. & Raue, H. A. Rio2p, an evolutionarily conserved, low abundant protein kinase essential for processing of 20S pre-rRNA in Saccharomyces cerevisiae. J. Biol. Chem. 278, 22537–22545 (2003)

    CAS  Article  Google Scholar 

  20. 20

    Ho, Y., Mason, S., Kobayashi, R., Hoekstra, M. & Andrews, B. Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 94, 581–586 (1997)

    ADS  CAS  Article  Google Scholar 

  21. 21

    Leger-Silvestre, I. et al. The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast. EMBO J. 23, 2336–2347 (2004)

    CAS  Article  Google Scholar 

  22. 22

    Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnol. 17, 1030–1032 (1999)

    CAS  Article  Google Scholar 

  23. 23

    Siniossoglou, S. et al. A novel complex of nucleoporins, which includes Sec13p and a Sec13p homolog, is essential for normal nuclear pores. Cell 84, 265–275 (1996)

    CAS  Article  Google Scholar 

  24. 24

    Beltrame, M. & Tollervey, D. Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA. EMBO J. 11, 1531–1542 (1992)

    CAS  Article  Google Scholar 

  25. 25

    Gadal, O. et al. Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p. Mol. Cell. Biol. 21, 3405–3415 (2001)

    CAS  Article  Google Scholar 

  26. 26

    Milkereit, P. et al. A Noc complex specifically involved in the formation and nuclear export of ribosomal 40 S subunits. J. Biol. Chem. 278, 4072–4081 (2003)

    CAS  Article  Google Scholar 

  27. 27

    Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 10, 953–961 (1998)

    Article  Google Scholar 

Download references

Acknowledgements

We thank S. Merker, P. Ihrig, J. Reichert, J. Pfannstiel and J. Lechner for performing mass spectrometry, and M. Seedorf and G. Dieci for the gift of antibodies. B.B. acknowledges support by a grant from EU-NOE (3D-Repertoire). E.H. is recipient of grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie. E.P and D.T. were supported by the Wellcome Trust. Author Contributions Experiments were designed and data were analysed and interpreted by T.S. and E.H. Strain constructions, DNA recombinant work, fluorescence microscopy and biochemical analyses (affinity purification, gel filtration, sucrose gradient centrifugation and in vitro assays) were performed by T.S. Negative-staining electron microscopy was conducted by B.M. and U.A., and cryoelectron microscopy and three-dimensional reconstruction by B.B. E.P. and D.T. performed rRNA processing analyses. The manuscript was written by T.S. and E.H. All authors discussed the results and commented on the manuscript.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Ed Hurt.

Ethics declarations

Competing interests

Three-dimensional reconstructions of mature 40S and pre-40S ribosomal subunits have been deposited in the EMBL-EBI Molecular Structure Database (http://www.ebi.ac.uk/msd/) and can be retrieved under accession numbers EMD-1211 and EMD-1212. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This document comprises a Supplementary Methods section, additional references and Supplementary Figure Legends. (DOC 55 kb)

Supplementary Movie 1

Animated rotation around the longitudinal axis of a mature 40S ribosomal subunit. 3D reconstruction as derived from cryo electron micrographs. (AVI 2817 kb)

Supplementary Movie 2

Animated rotation around the longitudinal axis of an affinity-purified pre-40S particle. 3D reconstruction as derived from cryo electron micrographs. (AVI 2853 kb)

Supplementary Figure 1

Ribosomal protein Rps3 interacts with mature 40S subunits in a stable salt-resistant manner. (PDF 1804 kb)

Supplementary Figure 2

Structural classification of 40S and pre-40S ribosomal subunits by negative staining and cryo electron microscopy. (PDF 3941 kb)

Supplementary Figure 3

Depletion of ribosomal protein Rps3 induces pre-rRNA processing defects and inhibits formation and nuclear export of 40S subunits. (PDF 2833 kb)

Supplementary Figure 4

Ribosome biogensis factors Ltv1 and Enp1 are phosphorylated in vivo and in vitro. (PDF 1487 kb)

Supplementary Figure 5

Neither phosphorylation nor dephosphorylation alone induce in vitro maturation of affinity-purified pre-40S particles. (PDF 1648 kb)

Supplementary Figure 6

Repression of HRR25 induces pre-rRNA processing defects and inhibits formation and nuclear export of ribosomal 40S subunits. (PDF 1638 kb)

Supplementary Figure 7

Repression of RIO2 does not affect ATP-induced phosphorylation of Ltv1, Enp1 and Rps3 in affinity-purified pre-40S particles. (PDF 3574 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Schäfer, T., Maco, B., Petfalski, E. et al. Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit. Nature 441, 651–655 (2006). https://doi.org/10.1038/nature04840

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing