Rat1-HTP strains carrying PGAL::3xHA-NOP4 or PGAL:: 3xHA-NOP15 were compared with the parental Rat1-HTP strain. Cells were harvested at an OD600 of B0.5 after 12 h of depletion in glucose-containing minimal medium. At this depletion time point, only modest defects in growth and pre-rRNA processing were observed (Supplementary Figure S6A and B). Crosslinked RNAs were cloned using barcoded 50 Pre-60S ribosome synthesis S Granneman et al linkers (Supplementary Table S1) and pooled cDNA libraries were Illumina deep sequenced. Depletion of Nop4 or Nop15 caused a B1.6-fold reduction in the total number of reads that mapped to the rDNA (Figure 6A). To normalize the data sets, 2000 reads mapping to the rDNA from two independent experiments were randomly selected and the averaged num- ber of hits for each nucleotide in the rDNA was plotted. Only binding sites present in all four Rat1-HTP CRAC experiments were considered. The results for the 50 ETS and a region comprising ITS1, 5.8S and ITS2 are presented in Figure 6C and D. Average hit densities (sum of the hits for nucleotides in a region) for the 50 ETS and 5.8S-ITS2 boundary region (nts 2970–3170, shaded region in Figure 6D) from the normalized data sets are plotted in Figure 6B. Numerous changes were observed, but these were strikingly similar following depletion of either Nop4 or Nop15. Three classes of changes were apparent. Loss of Nop4 or Nop15 did not dramatically alter the distribution of Rat1 crosslinking to the 50-ETS region including A0-A1 (Figure 6C), whereas crosslinking was increased 2–3-fold over H3/4 at the 50-end of 5.8S and H11 near the 50-end of 25S, and also over the 50-region of ITS1. Binding of Rat1 at all of these sites, therefore, appears to be independent of Nop4 and the A3 cluster. In marked contrast, crosslinking was dramatically (B70%) reduced, across the A2-A3 and 5.8S 30 to C2 (Figure 6B and D). Notably, association of 50 with A3 was equally reduced in strains depleted of Nop4 (which inhibits proces- sing before A3 cleavage) and Nop15 (which inhibits proces- sing following A3 cleavage), indicating that loss of A2-A3 fragment synthesis is not solely responsible for the effect. Rat1 association over the 5.8S 30- to ITS2 50-region and the number of reads that terminated at C2 (the 30-end of 7S pre-rRNA) and the level of 7S were similarly reduced (Supplementary Figure S6C). We could not detectably co-precipitate 7S with HTP-tagged Rat1 (unpublished obser- vation), but binding to ITS2 may be transient in vivo and unstable during the extended incubations needed for precipitation. We propose that Rat1 binding 50 to A3 at least in part represents binding to 27SA2 and precedes cleavage at A3, while binding 50 to C2 represents binding to 27SA2 and/or 27SB and precedes cleavage at C2. Pre-binding of Rat1 at the A3 and C2 cleavage sites may explain the strikingly low steady-state abundance of the 30 products of these cleavages (27SA3 and 26S pre-rRNA, respectively) in wild-type cells. Rat1 association at these sites is apparently dependent on both Nop4 and the A3 cluster. Figure 5 Nop4, Nop7, Nop12, Erb1 and Nop15 associate with 66S pre-rRNAs. Immunoprecipitations were performed with HTP-tagged proteins and the non-tagged parental strain ( lanes). (A) Schematic representation of pre-rRNA species that are detected by oligonu- cleotides 020 and 007. Red lines indicate the positions of these oligonucleotides on the rDNA. RNAs were resolved on 1.2% agarose and 8% polyacrylamide/7 M urea gels and detected by northern hybridization (B) or primer extension (C) using oligonu- cleotides 020 and 007, respectively (Supplementary Table S1). Input and supernatant indicate 0.1% of RNA extracted from cell lysates and supernatants after immunoprecipitation. Note that in the north- ern blots shown in (B) longer exposures are shown for inputs and supernatants. Rat1-1/xrn1D primer extension products were loaded (C, lane 15) as markers for 26S and 50-extended (50-ext) 25S. & 2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 19 | 2011 4013