proximity to the binding sites for Rpl25 and Rpl35 (Figures 3D, 4A and B). Nop7 is genetically linked to Rpl25 (Oefﬁnger et al, 2002) and cells lacking Rpl35A accumulate 27SA3 pre- rRNA and are defective in ITS2 processing (Babiano and de la Cruz, 2010). In the yeast 60S structure, the N-terminal do- main of Rpl25 makes extensive contacts with 5.8S, whereas the C-terminal region mainly contacts Domain III of 25S (Figure 3D). Both CRAC and genetic data, therefore, support direct interactions between Nop7, Rpl25 and Rpl35. Notably, Rpl35, together with Rpl17 and Rpl26, binds in the vicinity of the peptide exit tunnel in mature 60S subunits (Figure 4E and F). Cic1 and Nop15 crosslinked preferentially to the 50-end of the ITS2 spacer region (Figure 3A and D). Although many of the RNAs crosslinked to Nop15 and Cic1 originated from the same region, analysis of shorter fragments and speciﬁc mutations in the RNAs allowed us to distinguish the respec- tive RNA binding sites (Supplementary Figure S3). Cic1 also consistently crosslinked to H10 in 25S (Figure 3A), albeit less frequently. High-throughput data suggested that Cic1 and Nop15 interact (Tarassov et al, 2008), and the locations of their RNA binding sites strongly support their direct associa- tion in pre-60S ribosomes. The major Nop12 binding sites were located at H8 (Figure 3A), near the 30-end of the 5.8S rRNA, and in H10, which is formed by base pairing between the 30-end of 5.8S and the 50-end of the 25S rRNA (Figure 3D, ITS2 proximal stem). Thus, Nop12 could have a role in formation or stabilization of the interaction between the 5.8S and 25S rRNAs. The proximity of Nop12 binding sites to A3-cluster protein crosslinking sites prompted us to re-analyze the role of Nop12 in 5.8S processing. Because cells lacking Nop12 are cold-sensitive (Wu et al, 2001) pre-rRNA processing was analysed in nop12D cells grown at 18 1C for 24 h. Similar to A3-cluster-depleted cells, nop12D cells accumulated 35S and 27SA3 pre-rRNAs (Supplementary Figure S4B and C, lanes 11 and 12), albeit modestly. We conclude that Nop12 is a novel A3-cluster protein. Nop4 crosslinking sites were dispersed in the primary rRNA sequence and mapped to 25S rRNA domains II and III (Figure 3C and D). In the yeast 60S crystal structure, these sites are B30–80 A˚ apart and cluster near the 50-end of 5.8S (Figure 4C and D). Nop4 is an B80-kDa protein and could conceivably contact many of these sites simultaneously, bringing domains II and III in proximity to each other. The Nop4 binding sites in H26, H32, H33 and H47 are close to 5.8S rRNA and to binding sites for Rpl17, Rpl35 and Rpl37. We predict Nop4 directly contacts these ribosomal proteins. The binding site in H60 is adjacent to Rpl19 binding sites (Figures 3D, 4C and D). Consistent with previous data (Berge`s et al, 1994), Nop4 depletion leads to delayed cleavage Pre-60S ribosome synthesis S Granneman et al at site A2, with more severe inhibition at A3, B1L and concomitant decrease in 27SB levels (Supplementary Figure S4B and C, lanes 7 and 8). The 27SB1L/27SB1S ratio was not signiﬁcantly altered in these cells (Supplementary Figure S4D, compare lane 2 with lane 1), suggesting Rat1 can process residual 27SA3 generated in the absence of Nop4. We conclude that cleavage at A3 and B1L requires Nop4 binding near the 50-end of 5.8S, whereas Rat1-dependent processing from A3 requires A3-cluster proteins binding near the 30-end of 5.8S. Validation of CRAC data To validate the CRAC data, we performed immunoprecipita- tion experiments with HTP-tagged proteins and the non- tagged parental strain and analysed co-precipitated RNAs by northern blot analysis, primer extension analysis and EtBr staining (Figure 5). The data were quantiﬁed by calcu- lating fold enrichment over background levels observed in experiments with untagged strains (Supplementary Figure S5). Consistent with previous reports, tagged Nop7, Cic1 and Nop15 co-precipitated 27SA/B and 7S pre-rRNAs (Nissan et al, 2002; Fatica et al, 2003; Oefﬁnger & Tollervey, 2003; Figure 5B and C). HTP-tagged Nop7 also associated with 6S pre-rRNA, a 5.8S rRNA precursor that is 30-extended by only 8–9 nt (Figure 5B, lane 12). These observations are consistent with the CRAC data and indicate that ITS2 may be a major determinant for pre-60S binding by Cic1 and Nop15, but not by Nop7. The association of Nop4, Nop12 and Erb1 with pre-rRNA species has not previously been analysed in detail. Both Nop4 and Nop12 co-precipitated 27SA/B and 7S pre-rRNAs, albeit in modest amounts compared with other proteins tested (Figure 5B and C, lanes 11 and 13). Nop12 and Nop4 also reproducibly showed modest enrichment of mature 5.8S and 25S (Figure 5A and B, lanes 11 and 13; Supplementary Figure S5A), suggesting that these proteins remain associated with pre-ribosomes after removal of ITS2. HTP-tagged Erb1 efﬁciently precipitated 27S pre-rRNA, but precipitated low amounts of 7S and 50-extended 25S pre-rRNAs (25S’ and 26S) compared with other proteins tested (Figure 5B and C, lane 10; Supplementary Figure S5B and C), implying that Erb1 dissociates from pre-ribosomes following cleavage of 27SB pre-rRNA at site C2. In the 25S rRNA secondary structure, the Erb1 and Nop7 binding sites appear distant from the 30-end of 5.8S and ITS2 (Figure 3D). However, in the yeast 60S structure (Ben-Shem et al, 2010), these sites are in the vicinity of the Nop12 binding sites and B30–50 A˚ (equivalent to 10–15 nt) from the 30-end of 5.8S rRNA (Figure 4A and B). Nop7 and Erb1 directly interact in pre-ribosomes (Tang et al, 2008), suggest- Figure 3 Overview of CRAC results and locations of protein–RNA interaction sites in the 25S and 5.8S rRNA secondary structures. (A) Results from 2 to 5 independent CRAC experiments. (B) Results from untagged strain. (C) Illumina-Solexa results from Nop4 (red line) and negative control (untagged strain; blue line). Sequences were aligned to the rDNA reference sequence using blast and plotted using gnuplot. Locations of mature rRNA sequences, spacers and cleavage site are indicated below the x axis. The y axis displays the total number of times each nucleotide within an RNA fragment was mapped to the reference sequence. The location of the peaks in the secondary structure of the rRNA is indicated with helix (H) numbers (Klein et al, 2004). The asterisks indicate frequent contaminants. (D) Locations of minimal binding sites for the ribosome synthesis factors are displayed on the 5.8S/25S rRNA secondary structures (http://www.rna.ccbb.utexas.edu/) and the ‘ring model’ for yeast ITS2 structure (Joseph et al, 1999; Cote et al, 2002). Large 25S rRNA domains are indicated with dashed boxes. The 5.8S rRNA sequence is coloured red. Locations of r-protein binding sites are boxed, based on their locations in the yeast 60S crystal structure (Ben-Shem et al, 2010) and previous genetic studies (van Beekvelt et al, 2000). Two Rat1 binding sites in helices 3/5 and 11 are shown in light blue. Crosslinking sites in the spacer regions are shown in Supplementary Figure S9. & 2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 19 | 2011 4011