1. Biochemie-Zentrum der Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
2. M. E. Müller Institute for Structural Biology, Biozentrum, Universität Basel, CH-4056 Basel, Switzerland
3. Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK
4. EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany

Correspondence to: Ed Hurt¹ Correspondence and requests for materials should be addressed to E.H. (Email: cg5@ix.urz.uni-heidelberg.de). 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.

Abstract

The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments^{1, 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 formed^{5, 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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