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.
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Nature Communications (2016)