the pre-rRNA. The H. marismortui homologue of Rpl26 (HmRpl24) is an initiator protein for 50S assembly (Nowotny and Nierhaus, 1982) and appears critical for the tertiary structure of 23S Domain I (Klein et al, 2004). HmRpl24 binds in the centre of 5.8S rRNA and to the region corresponding to H24 near the 50-end of 5.8S (Klein et al, 2004). Notably, the binding sites for Rpl26 include at least four rRNA regions that are dispersed in the primary and secondary structures, including contacts at H19, in proximity to the Erb1 binding site, and the 50-end of 5.8S (Figures 3D and 8). We propose that interactions between the A3-cluster proteins bring these regions together to promote Rpl26 binding. In turn, Rpl26 may be required for correct 5.8S rRNA folding, correct positioning of the 50-region of 5.8S and binding of further r-proteins, including Rpl17, Rpl35 and Rpl37. Erb1 appears to dissociate from pre-ribosomes after C2 is cleaved (Figure 5), suggesting that this bridging interaction is & 2011 European Molecular Biology Organization Pre-60S ribosome synthesis S Granneman et al Figure 8 Speculative model for the reorganization of the ITS1- 5.8S–ITS2-25S region during Rat1-dependent removal of ITS1. (A) Schematic representation of the secondary structure of the nascent pre-rRNA. Domain I of the 25S rRNA sequence is indicated in green, domain II is indicated in light blue, domain III is indicated in orange and 5.8S rRNA is indicated in dark blue. ITS2 is drawn in the ‘ring’ conformation predicted for the nascent transcript (Joseph et al, 1999). Rat1 binds before A3 cleavage and is depicted on 5.8S H3/4, whereas Cic1 and Nop15 are depicted in grey bound to the ITS2 ‘ring’ conformation. (B) Binding sites for the A3-cluster proteins are indicated in grey. Nop4 binding sites are indicated in green. Interactions between these factors (dashed black lines) are predicted to bring all of these positions into close proximity. Coloured segments represent binding sites for r-proteins Rpl25, Rpl26, Rpl37 and Rpl17. These proteins each contact multiple rRNA sequences that are dispersed in the 11 and 21 structures but associate in the ribosomal particles. Stable pre-rRNA binding by these r-proteins requires the A3-cluster proteins (Sahasranaman et al, submitted), which may bring together the dispersed compo- nents of the r-protein binding sites. Major Rat1 binding sites are located in the 50-ends of 5.8S and 25S, which are independent of the A3-cluster protein Nop15 and Nop4. In the absence of the A3 cluster, we speculate that the 50-end of ITS2 rapidly adopts a highly base-paired conformation preventing access to A3 and C2 for Rat1, bound to 5.8S H3/4 and/or 25S H11, which are in close proximity. Interactions between Rat1 and sequences 50 to A3 and C2 likely precede cleavage, but have been omitted for simplicity. (C) Degradation of ITS1 by Rat1 makes the 50-end of the 5.8S rRNA (blue) accessible for base pairing with the 25S rRNA se- quences (green), resulting in the formation of 5.8S–25S interactions H3 and H4. We speculate that formation of these stem structures triggers stable binding of r-protein Rpl17, Rpl26 and Rpl37. After removal of ITS1, ITS2 is cleaved at C2. The ITS2 30-region is very rapidly processed back to the 25S 50-end, and we predict that this involves Rat1 bound at 25S H11. Degradation of ITS2 is likely accompanied by removal of Nop15 and Cic1. (D) The remaining A3-cluster proteins dissociate from the mature 25S rRNA and 6S pre- rRNA. Long-range interactions that were initially established by the A3-cluster proteins are now maintained by the r-proteins via their multipartite binding elements (indicated by coloured lines). required only at early stages of 60S assembly. This is consistent with bridging by the A3-cluster proteins establish- ing long-range interactions that are subsequently maintained by the r-proteins. Future structural analyses and assays for r-protein association in strains depleted of individual ribo- some synthesis factors will be required to test these models. Conclusions Previous analyses indicated that recruitment of the 18S rRNA 30-endonuclease Nob1 to the pre-ribosomes is distinct from its activation for pre-rRNA cleavage (Lamanna and Karbstein, 2009; Pertschy et al, 2009; Granneman et al, 2010). This also appears to be the case for Rat1, suggesting that this might be a more general property of eukaryotic pre-rRNA processing enzymes. Pre-rRNA cleavage or digestion is an irreversible step and separation of binding and activation of the proces- sing enzymes presumably permits better regulation and quality control during pre-ribosome assembly. Rat1 can terminate Pol I transcription by degradation of the nascent transcript (El Hage et al, 2008). Rat1 also degrades the excised A2-A3 fragment (Petfalski et al, 1998), and is therefore capable of initiating degradation at site A2. This makes it important that exonuclease degradation from A2 and A3 be delayed until the transcript is released from the polymerase by cleavage at site B0, almost 4 kb away (Figure 1A). We propose that the structural reorganization of ITS2, induced by A3-cluster binding, is important for The EMBO Journal VOL 30 | NO 19 | 2011 4017