The EMBO Journal (2010) 29, 2026–2036 | & 2010 European Molecular Biology Organization | Some Rights Reserved 0261-4189/10 www.embojournal.org Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA–protein cross-linking THE EMBO JOURNAL EMBO open This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without speciﬁc permission. Sander Granneman, Elisabeth Petfalski, Agata Swiatkowska and David Tollervey* Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, Scotland, UK Understanding of eukaryotic ribosome synthesis has been slowed by a lack of structural data for the pre-ribosomal particles. We report rRNA-binding sites for six late-acting 40S ribosome synthesis factors, three of which cluster around the 30 end of the 18S rRNA in model 3D structures. Enp1 and Ltv1 were previously implicated in ‘beak’ struc- ture formation during 40S maturation—and their binding sites indicate direct functions. The kinase Rio2, putative GTPase Tsr1 and dimethylase Dim1 bind sequences involved in tRNA interactions and mRNA decoding, indi- cating that their presence is incompatible with translation. The Dim1- and Tsr1-binding sites overlap with those of homologous Escherichia coli proteins, revealing conserva- tion in assembly pathways. The primary binding sites for the 18S 30-endonuclease Nob1 are distinct from its cleavage site and were unaltered by mutation of the catalytic PIN domain. Structure probing indicated that at steady state the cleavage site is likely unbound by Nob1 and ﬂexible in the pre-rRNA. Nob1 binds before pre-rRNA cleavage, and we conclude that structural reorganization is needed to bring together the catalytic PIN domain and its target. The EMBO Journal (2010) 29, 2026–2036. doi:10.1038/ emboj.2010.86; Published online 7 May 2010 Subject Categories: RNA; structural biology Keywords: CRAC; pre-ribosome; ribosome synthesis; RNP structure; yeast Introduction Biogenesis of the mature 40S and 60S ribosomal subunits is an exceptionally complex process in eukaryotes, which requires the activities of roughly 200 synthesis factors for rRNA maturation and subunit assembly (reviewed in Henras et al, 2008). In the nucleolus, RNA polymerase I generates a polycistronic precursor rRNA (35S pre-RNA) that contains the sequences for the mature 18S, 5.8S and 25S rRNA, ﬂanked by the external transcribed spacers (50-ETS and *Corresponding author. Wellcome Trust Centre for Cell Biology and Centre for Systems Biology at Edinburgh, University of Edinburgh, Michael Swann Building, King’s Buildings, Mayﬁeld Road, Edinburgh EH9 3JR, Scotland, UK. Tel.: þ 44 131 650 7092; Fax: þ 44 131 650 7040; E-mail: d.tollervey@ed.ac.uk Received: 30 November 2009; accepted: 14 April 2010; published online: 7 May 2010 2026 The EMBO Journal VOL 29 | NO 12 | 2010 30-ETS) and separated by the internal transcribed spacers (ITS1 and ITS2; Supplementary Figure 1). The 35S pre- rRNA is cleaved at processing sites A0, A1 and A2, within a large, B90S complex, to yield a pre-40S particle that contains the 20S pre-rRNA and a pre-60S particle containing the 27SA2 pre-rRNA. The pre-60S particles undergo a complex matura- tion pathway in the nucleolus, nucleoplasm and cytoplasm (reviewed in Henras et al, 2008). In contrast, the pre-40S particles are rapidly exported to the cytoplasm. Here, the 20S pre-rRNA is modiﬁed close to the 30 end of the 18S rRNA sequence by the dimethylase Dim1 (Lafontaine et al, 1994), before cleavage by the PIN-domain endonuclease Nob1 to generate the mature 18S rRNA (Pertschy et al, 2009) (Supplementary Figure 1). Notably, both Dim1 and Nob1 bind the nuclear 90S pre-ribosomes and are exported to the cytoplasm together with the pre-40S particles (Schafer et al, 2003). This implies a mechanism that inhibits the premature activity of these enzymes on the substrates with which they are associated, possibly involving structural reorganization of the pre-40S particles. Dim1 is essential for viability, but its essential function is not 18S rRNA methylation, but pre-rRNA cleavage at sites A1 and A2 (Lafontaine et al, 1995). The basis of this requirement was, however, unclear. Structural reorga- nization of the pre-40S particle does occur, most notably in formation of the beak structure; a prominent feature of the mature subunit that is absent from pre-40S particles (Schafer et al, 2006). This reorganization involves phosphorylation of Enp1 and Ltv1 by the kinase Hrr25, and subsequent dephos- phorylation of Rps3 but the roles of Enp1 and Ltv1 in this process were unclear. Late pre-40S particles contain a differ- ent protein kinase, Rio2, the targets for which are unknown, and a putative GTPase, Tsr1. Both are needed for 20S–18S processing but, again, their actual roles are unclear. Afﬁnity puriﬁcation and mass spectrometry allowed the composition of many pre-ribosomal complexes to be deter- mined, whereas genetic analyses showed that loss of ribo- some synthesis factors impeded pre-rRNA processing at speciﬁc stages (see Fromont-Racine et al, 2003; Henras et al, 2008). However, in few cases it was clear how these factors actually participated in pre-rRNA processing. Numerous sub-complexes and other protein–protein interac- tions between ribosome synthesis factors have been identi- ﬁed (reviewed in Henras et al, 2008; Tarassov et al, 2008) but little is known about the architecture and structure of these RNP complexes. EM structural analyses of pre-ribosomes have given important insights (Nissan et al, 2004; Schafer et al, 2006; Ulbrich et al, 2009) but these studies are techni- cally challenging, due in part to instability and heterogeneity of puriﬁed particles. The most informative single piece of data that could be provided (short of actual crystal structures of pre-ribosomes) would be the locations of the binding sites for the proteins on & 2010 European Molecular Biology Organization