Pre-60S ribosome synthesis S Granneman et al Figure 7 Cic1 and Nop15 are required to maintain a ﬂexible RNA conformation in the 50-end of ITS2. (A, B) The 50-end of ITS2 adopts a different conformation in Nop15- and Cic1-depleted cells. Parental strain (BY4741), and Nop4, Nop15 and Cic1 GAL depletion strains were grown in YPG/R to exponential phase and subsequently grown for 12 h in glucose. Dimethyl-sulphate (DMS) probing was performed in vivo in cell culture (lanes 1–4) or on in vitro refolded total RNA extracted from the same cultures (lanes 9–12). To control for natural primer extension stops, primer extension was performed on unmodiﬁed total RNA (lanes 5–8). DMS modiﬁcations were mapped by primer extension and detected by autoradiography following gel electrophoresis. The positions of the modiﬁed nucleotides are indicated on the right side of each panel. Results for the ITS1 and 5.8S are shown in Supplementary Figure S7. Oligonucleotides used for primer extensions (Supplementary Table S1) are indicated below each panel. Position of helices and RNA domains is indicated on the left of each panel. Coloured circles indicate changes in DMS reactivity in Nop15 (green circles) and Cic1-depleted cells (purple circles). Small circles depict weak DMS modiﬁcations; closed circles indicate an increased DMS reactivity; open circles indicate a decreased DMS reactivity. (C, D) Overview of the chemical probing results on the ‘ring’ (C) and ‘hairpin’ secondary structure models for ITS2. Positions of processing sites (C1, C2 and E) are indicated by arrows. Changes in DMS modiﬁcations are highlighted as described above. Red circles indicate nucleotides that were methylated in vitro, whereas black circles indicate in vivo modiﬁed nucleotides. (E) Quantiﬁcation of primer extension results shown in (A). The data were normalized by removing differences in signal intensity between lanes. Y axis indicates the signal intensities calculated by the AIDA software. X axis indicates pixel positions in the lanes. Positions of 5.8S sequences and ITS2 domains II, III and IV are indicated below the x axis. Nop15 binds to this region, which would largely explain the observed differences in DMS modiﬁcation in stem III. Depletion of Nop4 did not dramatically affect the in vivo DMS modiﬁcation pattern in ITS2 compared with the other strains tested (Figure 7A and B). Notably, we did observe a reduction in DMS reactivity in depleted cells at G319 in ITS1, between the A3 and B1L cleavage sites (Supplementary Figure S7, indicated with arrows); however, primer extension stops & 2011 European Molecular Biology Organization at this position were also found in unmodiﬁed RNA, indicat- ing that it represents a natural primer extension stop (Supplementary Figure S7, lanes 5–8). The signiﬁcance of this observation remains unclear. We conclude that Cic1 and Nop15 binding is necessary to maintain or establish a more ﬂexible and open structure in the 50-end of ITS2, potentially preventing premature forma- tion of the hairpin structure. The EMBO Journal VOL 30 | NO 19 | 2011 4015