Pestov et al, 2001; Gadal et al, 2002; Oefﬁnger et al, 2002; Fatica et al, 2003; Oefﬁnger and Tollervey, 2003; Miles et al, 2005). Loss of these factors (including Rlp7, Cic1/Nsa3, Erb1, Nop7 and Nop15) leads to accumulation of the 27SA3 pre- rRNA and reduced synthesis of 27SBS and mature 5.8SS without concomitant loss of 27SBL or 5.8SL. A further factor, Nop12, is non-essential but required for efﬁcient 25S rRNA synthesis (Wu et al, 2001) and, together with Brx1 and Ebp2 is predicted to be physically close to Rlp7, Cic1/Nsa3, Erb1, Nop7 and Nop15 within the pre-60S particle (Tarassov et al, 2008; Figure 1B). The role of Nop12 in 5.8S rRNA maturation was not assessed (Wu et al, 2001). A further protein, Nop4 (Nop77) was predicted to bind in this region (Tarassov et al, 2008), but its depletion confers a different phenotype, with speciﬁc inhibition of cleavage at both A3 and B1L (Berge`s et al, 1994; Sun and Woolford, 1994, 1997). To better understand the roles of the non-enzymatic ribo- some synthesis factors implicated in 5.8S 50-maturation, we identiﬁed their sites of interaction with the pre-rRNA using the crosslinking and analyses of cDNA (CRAC) crosslinking technique (Supplementary Figure S1; Granneman et al, 2009). Unexpectedly, binding sites for ﬁve factors required for exonucleolytic removal of the A3-B1S fragment clustered near the 30-end of the 5.8S rRNA (E-site), not the 50-end. In contrast, Nop4, required for endonucleolytic cleavages in ITS1, crosslinked near the 50-end of 5.8S. This suggests that a ribonucleoprotein complex, hereafter referred to as ‘A3 clus- ter’, assembled near the 30-end of 5.8S rRNA is required for the activity of the 50-exonucleases acting at the 50-end of 5.8S, whereas endonucleolytic cleavage in ITS1 requires Nop4 binding to the 50-end of 5.8S. High-throughput CRAC on the Rat1 50-exonuclease indicated signiﬁcant crosslinking at the 30- end of 5.8S and in ITS2 and we furthermore demonstrate that Rat1 can bind the 50-end of 25S independently of Nop4 and A3- cluster protein Nop15. We propose that A3-cluster proteins act to coordinate events at the 3 0 - and 50-ends of 5.8S rRNA, ensuring that 50-end maturation of 5.8S precedes ITS2 cleavage. Results Rat1 is associated with multiple sites in the pre-rRNA Rat1 has several distinct targets in the pre-rRNA; it accurately processes the 50-ends of the mature 5.8S (A3-B1S) and 25S rRNAs (C2-C1) and completely degrades the excised A0-A1 and A2-A3 pre-rRNA fragments (Figures 1 and 2). In addition, Rat1 functions in the 50-maturation of intronic snoRNAs, surveil- lance of mRNAs with defects in splicing and 50-capping, and transcription termination on both Pol I and Pol II genes. Given this large number of substrates, we anticipated that Rat1 binding would be very transient. To maximize the recovery of RNA binding sites, we therefore developed a novel UV crosslinking system that allows UV irradiation of large vo- lumes of cells actively growing in culture medium. This cross- linking device (the ‘Megatron’) consists of a 1.2-m metal tube with a central 205 W, 254 nm UV lamp, and can irradiate 2.5 l of culture in B1 min (Supplementary Figure S1A). Cells expressing HTP-tagged Rat1 and a non-tagged negative control strain (BY4741) were UV irradiated in the Megatron. A3-B1S processing also requires the 50-exonuclease Rrp17 and the Rat1 cofactor Rai1 (Fang et al, 2005; Oefﬁnger et al, 2009). Both were tested as HTP-tagged constructs but neither gave usable crosslinking efﬁciencies. Illumina-Solexa deep sequencing of & 2011 European Molecular Biology Organization Pre-60S ribosome synthesis S Granneman et al two independent Rat1 CRAC cDNA libraries revealed that around a quarter of the reads mapped to rDNA repeats. A multiple sequence alignment of rDNA-mapped reads is shown in Supplementary Table S2. As expected, many other putative Rat1 substrates were identiﬁed, which will be discussed else- where (manuscript in preparation). Prominent peaks of Rat1 crosslinking were observed over the 50-region of the 50-ETS (Figure 2B), which is degraded by pathways including endonuclease cleavage (Lebreton et al, 2008; Schaeffer et al, 2009; Schneider et al, 2009). These data indicate that, like its human counterpart Xrn2 (Wang and Pestov, 2010), Rat1 is involved in degradation of the cleaved 50-ETS fragments. A very prominent peak was present at 30-end of the 50-ETS, directly upstream of the A1 cleavage site, in agreement with the reported role of Rat1 in degrada- tion of the excised A0-A1 fragments (Petfalski et al, 1998). Inspection of the regions surrounding the known Rat1 pre- rRNA processing substrates, A3-B1 in ITS1 and C2-C1 in ITS2 (Figure 2) revealed apparent similarities. High levels of cross- linking were seen immediately 50 to the A3 and C2 cleavage sites; over the A2-A3 region in ITS1 and over the 30-region of 5.8S and between the 30-end of 5.8S (site E) and cleavage site C2 in ITS2. Reads including the 30-region of 5.8S predomi- nately extended through site E, at least 2 nt into ITS2 (Supplementary Figure S2A), indicating that binding oc- curred on the pre-rRNA, rather than on mature 5.8S rRNA. Substantial Rat1 crosslinking was also observed 30 to the Rat1 targets, over the 50-regions of the mature 5.8S (helices H3 and H4) and 25S rRNAs (H11) (Figure 2). In contrast, crosslinking was much lower over the Rat1 processing substrates A3-B1 and C2-C1. The intermediates in A3-B1 and C2-C1 processing are almost undetectable in wild-type yeast indicating high processivity during Rat1 processing. Finally, hits located at H66, H79 and H99 were also frequently found in negative control experiments (marked with asterisks in Figures 2A, B and 3C) and were therefore considered background. Rat1 also participates in degradation of the excised A2-A3 spacer fragment (Petfalski et al, 1998). This may contribute to Rat1 crosslinking over this region, but mutational analyses (below) indicate that this is not the major source. The location of site C2 was originally inferred from ﬁnger- printing of in vivo labelled RNA and predicted to lie within the G133–G136 region (Veldman et al, 1980). However, Rat1-associated sequences frequently extended B8 nt further 30 to terminate at U140 and A141 in ITS2 (Supplementary Figure S2A and C). This end was previously detected by primer extension in a rat1 mutant (Geerlings et al, 2000) and the same end is observed in the wild-type (see below). 7S pre- rRNA was immunoprecipitated using HTP-tagged Nop7 and 30-ends were mapped by RNA cloning (Supplementary Figure S2B). The results conﬁrmed that the 30-end of 7S predominately falls at U140 and A141. We conclude that the major endonuclease cleavage site lies between A141 and G142, as proposed previously (Geerlings et al, 2000), which we refer to herein as site C2. We conclude that Rat1 is associated with sites ﬂanking its target regions for pre-rRNA processing. Binding sites for Nop7, Nop12, Erb1, Cic1 and Nop15 cluster around the 30-end of the 5.8S rRNA Based on published data (Miles et al, 2005; Tarassov et al, 2008), a group of synthesis factors were predicted to physi- The EMBO Journal VOL 30 | NO 19 | 2011 4007