Pre-60S ribosome synthesis S Granneman et al Figure 4 Location of Nop4, Nop7, Erb1 and Nop12 and RNA binding sites in 60S crystal structure. (A, B) Images, rotated by 901, showing the protein neighbourhood around the 30-end of the yeast 5.8S rRNA. The 5.8S rRNA is shown in wheat color, with binding sites for Nop12 (orange and blue), Nop7 (red) and Erb1 (purple) indicated as coloured nucleotide strands. Images were generated using pymol. Ribosomal proteins (Rpl15, Rpl17, Rpl25, Rpl26, Rpl35 and Rpl37) binding to this region are indicated as surface representations. Helix numbers are indicated with ‘H’. The double arrows indicate the distance (in A˚) between the 30-end region of 5.8S and the Nop7 and Erb1 binding sites. (C, D) Images showing the location of the Nop4 (blue) binding sites that surround the 50-end of 5.8S. Ribosomal proteins Rpl15, Rpl17 and Rpl37 that bind in this region are indicated as surface representations. Helix numbers are indicated with ‘H’. (E, F) Images showing the location of the Nop7 (red) binding site in the structure of the 25S rRNA (wheat color) and 5.8S 30-end (light blue). Superimposed are structures for r-proteins Rpl35 (orange), Rpl25 (dark purple) and Rpl15 (green), Rpl26 (yellow) and Rpl17 (light purple). The polypeptide exit tunnel is indicated. The double arrow indicates the distance (in A˚) between the 30-end of 5.8S and the base of the helix containing the Nop7 binding site. ing their participation in long-range interactions bringing domains III and I of 25S close to the 30-end of 5.8S (Figure 4B). Collectively, UV crosslinking, protein–protein interaction and pre-rRNA processing data strongly indicate that A3- cluster proteins physically and functionally interact with the pre-60S region surrounding the 30-end of 5.8S and with ITS2. Altered Rat1 binding in strains depleted of Nop4 or Nop15 Sites of Rat1 crosslinking may reﬂect targets undergoing active degradation, pre-rRNA docking sites directed by pro- tein association or both. To distinguish between these possi- bilities, Rat1 crosslinking was analysed in strains depleted of Nop4 or the A3 cluster protein Nop15. 4012 The EMBO Journal VOL 30 | NO 19 | 2011 & 2011 European Molecular Biology Organization