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.
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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.
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.
This document comprises a Supplementary Methods section, additional references and Supplementary Figure Legends. (DOC 55 kb)
Animated rotation around the longitudinal axis of a mature 40S ribosomal subunit. 3D reconstruction as derived from cryo electron micrographs. (AVI 2817 kb)
Animated rotation around the longitudinal axis of an affinity-purified pre-40S particle. 3D reconstruction as derived from cryo electron micrographs. (AVI 2853 kb)
Ribosomal protein Rps3 interacts with mature 40S subunits in a stable salt-resistant manner. (PDF 1804 kb)
Structural classification of 40S and pre-40S ribosomal subunits by negative staining and cryo electron microscopy. (PDF 3941 kb)
Depletion of ribosomal protein Rps3 induces pre-rRNA processing defects and inhibits formation and nuclear export of 40S subunits. (PDF 2833 kb)
Ribosome biogensis factors Ltv1 and Enp1 are phosphorylated in vivo and in vitro. (PDF 1487 kb)
Neither phosphorylation nor dephosphorylation alone induce in vitro maturation of affinity-purified pre-40S particles. (PDF 1648 kb)
Repression of HRR25 induces pre-rRNA processing defects and inhibits formation and nuclear export of ribosomal 40S subunits. (PDF 1638 kb)
Repression of RIO2 does not affect ATP-induced phosphorylation of Ltv1, Enp1 and Rps3 in affinity-purified pre-40S particles. (PDF 3574 kb)
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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
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