Pre-60S ribosome synthesis S Granneman et al Figure 6 Rat1 can bind helices 3, 4 and 11 in pre-rRNA independently of Nop4 and Nop15. (A) Depletion of Nop4 and A3-cluster protein Nop15 reduces binding to the 30 5.8S–50 ITS2 region but does not affect crosslinking to the 50-ETS and helices 3, 4 and 11. Average hits for each nucleotide in the rDNA were calculated using 2000 rDNA reads from two independent experiments. Hit densities are the sum of hits for each nucleotide in a nucleotide sequence and were plotted as percentages relative to the results of the Rat1-HTP CRAC experiments. (B, C) Distribution of reads mapped to the 50-ETS (B) and the 5.8S-ITS2 regions (C, D). Average hits per nucleotide from 2000 rDNA-mapped reads from two independent experiments are shown. Reads mapped to the 50 ETS are shown in (C), whereas reads mapped to ITS1-5.8S-ITS2 region are shown in (D). The shaded area in (D) covers the region from which hit densities were calculated. Cic1 and Nop15 are required to establish the conformation of ITS2 Based on phylogenetics and structure probing, two distinct structures were proposed for ITS2, termed the ‘ring’ (Figure 7C) and ‘hairpin’ (Figure 7D) structures (Yeh and Lee, 1990; Joseph et al, 1999). These share the same distal stems but differ in the 25S and 5.8S proximal regions. Notably, the 50-end of 25S and 30-end of 5.8S are base paired in the mature ribosome, and are brought into proximity by the hairpin but not by the ring structure. Subsequent genetic data indicated that ITS2 undergoes a conformational change from the ring to hairpin structure during maturation (Cote et al, 2002). Structurally conserved regions of ITS2 are important for pre-rRNA processing (shaded in blue in Figure 7C and D; van der Sande et al, 1992; Van Nues et al, 1995; Peculis and Greer, 1998; Joseph et al, 1999; Cote et al, 2002), including domains II, III.A and III.B. These were proposed to be binding sites for ribosome synthesis factors (Van Nues et al, 1995; Cote et al, 2002) and they are indeed the crosslinking sites for Cic1 and Nop15 (see Figure 7C and D). Deletions and mutations in these regions abrogated 25S rRNA synthesis and caused 27SA accumulation, with a strong block in conversion of 27SA to 27SB (Van Nues et al, 1995; Cote et al, 2002); processing defects similar to those observed A3- cluster protein-depleted cells (Supplementary Figure S4B and C; Van Nues et al, 1995). These ﬁndings strongly implicated Cic1 and Nop15 in the structural rearrangement of ITS2. To assess the RNA secondary structure, we performed in vivo and in vitro chemical modiﬁcation experiments using dimethyl-sulphate (DMS) (Figure 7), which preferen- tially methylates adenines and cytosines in single stranded or ﬂexible RNA not bound by proteins. In vitro chemical probing reactions were performed on refolded total RNA, which helps to identify RNA regions potentially protected by protein binding in vivo. We analysed DMS modiﬁcations in condi- 4014 The EMBO Journal VOL 30 | NO 19 | 2011 tional (PGAL) Nop15 and Cic1 strains, grown for 12 h in glucose medium to deplete the proteins. As controls, we analysed the parental strain (BY4741) and the strain depleted of Nop4, which did not signiﬁcantly crosslink to ITS2 (Figure 3C). DMS modiﬁcations were mapped by primer extension analysis and autoradiography and quantiﬁed by normalizing signal intensities for each band to remove differ- ences between lanes (Figure 7E). Note that primer extension was performed on total RNA, revealing the ‘average’ second- ary structure of all 27S pre-rRNA species. The 30 most region of ITS2 could not be analysed but the distal region (stems VI and V) adopts virtually the same secondary structure in vitro or in vivo and in the depleted and parental strains (Figure 7B). In contrast, ITS2 stem II has a more accessible structure in vivo than in refolded RNA, as judged by the intensity of the primer extension stops at nucleotides C2 and C5 (indicated by open circles in Figure 7A, compare lane 9 with lane 1, and see Supplementary Figure S8). This is consistent with C2 and C5 base pairing with G22 and G25 in vitro (dotted arrows in Figure 7), forming a longer stem II very similar to that proposed in the hairpin model (Figure 7D). In Cic1- and Nop15-depleted cells, C2 and C5 in stem II were almost completely protected from DMS modiﬁcation in vivo (Figure 7A, compare lanes 3, 4 with lane 1). We conclude that binding of Cic1 (and perhaps also Nop15) to this region blocks formation of these base-pairing interactions, maintaining the shorter stem II structure of the ring structure. Notably, these nucleotides are located in a highly conserved region required for A3-B1S processing (Van Nues et al, 1995). Stem III (stem III.A in the ring model) was more protected from DMS modiﬁcation in vivo compared with the refolded RNA (Figure 7A, compare lane 9 with lane 1), but this protection was lost in Nop15-depleted cells (Figure 7A, compare lane 4 with lane 1). The CRAC data indicate that & 2011 European Molecular Biology Organization