Pre-60S ribosome synthesis S Granneman et al trols, four independent CRAC experiments were performed with the non-tagged parental strain (Figure 3B). Sanger sequencing of Nop4 CRAC cDNA libraries had indicated multiple pre-rRNA binding sites and therefore high-through- put Solexa sequencing was performed together with a negative control to obtain sufﬁcient sequence depth for each binding site (Figure 3C). Recovered sequences that overlapped with control peaks in H66, H79 and H99 at the 30-end of the 25S were considered background (asterisks in Figure 3). Crosslinking sites were precisely identiﬁed by the presence of multiple point deletions or substitutions at a speciﬁc position in sequence reads (see Granneman et al, 2009), or a minimal RNA binding site was determined from overlapping sequences. In CRAC analyses, Erb1 was crosslinked to the 50-region of the 25S rRNA over H21 and H22 (Figure 3A and D). In the yeast 60S structure, this region forms a compact structure, probably associated with Rpl42, Rpl8, Rpl28 and Rpl15 (Figure 4A and B; Ben-Shem et al, 2010). The average length of RNAs crosslinked to Erb1 was relatively long (59 nt), possibly because this structure confers protection during the RNase digestion used to generate the cloned fragments. Nop7 predominately crosslinked to H54 in 25S rRNA (Figure 3A and D). This site is within Domain III, in close Figure 2 Rat1 crosslinking sites over the pre-rRNAs. (A) Rat1 crosslinks primarily to spacer regions in the pre-rRNA. A histogram that displays the distribution of rDNA-mapped reads along the entire rDNA sequence is shown. The red line indicates 100 000 averaged hits from two independent Rat1 CRAC experiments. The blue line indicates the distribution of 10 000 rDNA hits from the negative control experiment. The asterisks indicate frequent contaminants. The rDNA is schematically represented below the x axis, with processing sites included. The y axis displays the total number of times a nucleotide within an RNA fragment was mapped to the rDNA sequence. (B) The dashed lines point to expanded views of hits over the 50-ETS and ITS1-25S region with schematics showing Rat1 substrates. Positions of potential crosslinking sites in spacer regions are shown in Supplementary Figure S9. Figure 1 Pre-rRNA processing and protein interactions. (A) Schematic representation of pre-rRNA processing in yeast. The locations of processing sites on the 35S pre-rRNA are indicated. The positions of oligonucleotides used for northern hybridization (020) and primer extension (007) are shown. In the nucleolus, the 35S pre-rRNA, part of a 90S-sized complex called the SSU processome or 90S pre-ribosome, is processed at sites A0, A1 and A2, leading to the formation of 43S and 66S pre-ribosomes. Proteins studied in the CRAC analysis and their association with pre-ribosomal complexes are indicated. The 43S pre-ribosome is exported to the cytoplasm where Nob1 cleaves at site D, yielding the mature 18S rRNA and 40S subunit. 66S pre-ribosomes containing 27SA2 pre-rRNA are either processed at A3 or at B1L, which requires the presence of Nop4. Pre-ribosomes containing 27SA3 pre-rRNA are exonucleolytically trimmed by the Rat1, Rrp17 and Xrn1 50–30 exonucleases, yielding the 27SBS pre-rRNA. This maturation step requires the presence of Nop12, Nop7, Nop15 and Erb1 (depicted as coloured circles). After this step, the 27SB is cleaved at C2 by an unknown endonuclease, followed by exonucleolytic and endonucleolytic trimming of 7S by Rrp6 and Rrp44, two components of the exosome complex. Pre-ribosomes containing the 6S pre-rRNA are exported to the cytoplasm and matured by Ngl2 after which Nop12 and Nop4 dissociate. (B) Overview of known and predicted protein–protein interactions in 66S pre- ribosomes. The interaction map depicts interactions between the various assembly factors and r-proteins in 66S pre-ribosomes. Black lines; physical interactions among proteins shown to be part of subcomplexes or interacting as recombinant proteins (Krogan et al, 2004; Miles et al, 2005). Dashed red lines; yeast two-hybrid interactions (Ito et al, 2001; Miles et al, 2005). Red lines; interactions from protein-fragment complementation assays (PCAs), which detect proteins located within B80 A˚ (Tarassov et al, 2008). Dashed black lines; protein–protein interactions predicted from our CRAC data. Note that these may be mediated by RNA. & 2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 19 | 2011 4009