Pre-60S ribosome synthesis S Granneman et al Discussion The eukaryotic pre-rRNA processing and ribosome assembly pathways are strikingly complex making coordination and timing of key importance. Following cleavage of site A3 by the endonuclease RNase MRP, the 50-exonuclease Rat1 digests the pre-rRNA back to the major 50-end of the 5.8S rRNA at site B1S (Schmitt and Clayton, 1993; Henry et al, 1994; Lygerou et al, 1996; Figure 1A). We initially assumed that A3 cleavage to generate a free 50-end would be necessary and sufﬁcient for recruitment of Rat1, which shows robust, processive activity in vitro on non-speciﬁc substrates (Kenna et al, 1993). However, subsequent data revealed that A3 processing requires a group of eight proteins (Nop12, Nop7, Erb1, Ytm1, Rlp7 Nop15, Nsa3/Cic1 and Rrp1), termed the A3 cluster (Tang et al, 2008) (Sahasranaman et al, submitted) (this work). Similarly, Nop4 and other factors are required in vivo for A3 cleavage (Berge`s et al, 1994; Sun and Woolford, 1994), even though this site can be cleaved by RNase MRP alone in vitro (Lygerou et al, 1996). Rat1 showed strong pre-rRNA binding over several differ- ent regions, reﬂecting its diverse targets for processing and degradation. Strong signals were seen over the A0-A1 region of the 50-ETS, a known Rat1 target, and at sites further 50, presumably reﬂecting a role in spacer degradation following endonuclease cleavage by the PIN domain of the Rrp44 component of the exosome complex (Lebreton et al, 2008; Schaeffer et al, 2009; Schneider et al, 2009; Wang and Pestov, 2010). In contrast, fewer hits were recovered over the A3-B1S and C2-C1 regions that are targets for Rat1 processing during pre-rRNA maturation. The steady-state levels of A3-cleaved and C2-cleaved pre-rRNAs (27SA3 and 26S, respectively, in Figure 1A) are very low, indicating that exonuclease digestion very rapidly follows endonuclease cleavage, and processing intermediates are essentially undetectable. These observa- tions indicate that Rat1 is actively recruited to, and highly processive on, the 27SA3 and 26S pre-rRNAs. Rat1 was strongly crosslinked to regions immediately 50 to the A3 and C2 cleavage sites, suggesting that it is bound to these locations before cleavage. Rat1 binding 50 to A3 pre- sumably also partly reﬂects its role is A2-A3 fragment degra- dation (Petfalski et al, 1998). However, the similar effects of depletion of Nop4 and Nop15, which differ in their effects on A3 processing, strongly indicates that loss of A2-A3 is not solely responsible for the reduction. In contrast, crosslinking of Rat1 to the 50-region of 5.8S (H3/4) and H11 in the 50-region of 25S rRNA was independent of the A3-cluster protein Nop15 and of Nop4. This suggests that these helices provide distinct access points for Rat1 in pre-ribosomes, potentially representing initial binding sites. Model for links between pre-ribosome structure and processing from site A3 CRAC analyses placed Nop7, Erb1, Cic1, Nop12 and Nop15 on the surface of the pre-60S ribosomes in the vicinity of the 5.8S 30–25S 50 stem. Using the CRAC data, we can extend the interaction map for the A3-cluster proteins (see Figure 1B). Some of these interactions are superimposed onto a schematic of the pre-rRNA in Figure 8, allowing potential mechanisms to be proposed. In the mature 60S structure, both the 50- and 30-regions of 5.8S rRNA are base paired to the 25S rRNA. However, based 4016 The EMBO Journal VOL 30 | NO 19 | 2011 on in vitro chemical modiﬁcation experiments, it was pro- posed that in the 35S pre-rRNA the 5.8S 50-region is initially base paired to ITS1 (Yeh et al, 1990), forming a stable stem- loop structure that will be strongly favoured over the long- range interaction with 25S during folding of the nascent transcript. Our in vivo chemical probing experiments are largely consistent with this model (Supplementary Figure S7), as are analyses of the intermediates detected in rat1 mutants in vivo (Henry et al, 1994) and phylogenetic analyses (Van Nues et al, 1994). Rat1 processing of ITS1 will free the 50-end of 5.8S, which might be a pre- requisite for the establishment of base pairing with 25S (Figure 8). The 30-end of 5.8S and 50-end of 25S are separated by ITS2, which was previously reported to be refolded during pre-rRNA maturation from a more open ‘ring’ conformation into a closed ‘hairpin’ structure (Yeh and Lee, 1990; Peculis and Greer, 1998; Joseph et al, 1999). In the hairpin structure, a proximal stem brings the 5.8S and 25S rRNAs into close proximity, presumably promoting base pairing between them. The ring-to-hairpin transition was shown to be an active process involving unidentiﬁed ribosome synthesis factors (Peculis and Greer, 1998). The locations of the binding sites for Nop15 and Cic1 are in strikingly good agreement with the binding sites predicted for these factors. Moreover, the phenotypes of ITS2 mutations expected to block these interactions confer processing defects similar to those seen in A3-cluster mutants. In vivo structure probing indicated that depletion of Nop15 and Cic1 indeed shifted the conformation of ITS2 from the ring to the hairpin structure. The hairpin structure is predicted to be more thermodynamically stable and is very likely to fold spontaneously. Binding of Nop15 and Cic1 blocks folding of the hairpin, potentially establish- ing the timing of base pairing between 5.8S 30- and 25S 50-regions (Figure 8A). A3-cluster proteins show interdependent pre-rRNA binding (i.e., depletion of a single protein causes loss of many or all A3-cluster proteins; Tang et al, 2008) (Sahasranaman et al, submitted). This implies that they interact—so their binding sites, which are dispersed in the pre-rRNA sequence, must all be brought into proximity. Notably, several 60S r-proteins make contact with multiple sites in the rRNA, which must also be brought together during subunit assembly. We spec- ulate that this is a key function of the ribosome synthesis factors. Nop7 directly binds Erb1, forming an inter-domain bridge in the 27S pre-rRNA. Cells lacking these A3-cluster proteins fail to assemble r-proteins Rpl35, Rpl37, Rpl26 and Rpl17 in pre-60S complexes (Sahasranaman et al, submitted). All of these r-proteins have binding sites in close proximity to Nop12, Erb1 and Nop7 crosslinking sites (and presumably also Cic1 and Nop15; Figure 3D) and contact both the 25S rRNA and the 5.8S rRNA (Ben-Shem et al, 2010). Nop4 crosslinking sites were located near the binding sites for Rpl17, Rpl35 and Rpl37 in each of two different domains. In addition, Nop7 was genetically linked to Rpl25 and cross- linked adjacent to an Rpl25 binding site in 25S domain III. The Nop4, Nop7 and Erb1 interactions with pre-rRNA bring together sites that are distant in the primary sequence. We speculate that this establishes an assembly platform for Rpl17, Rpl25, Rpl26, Rpl35 and Rpl37, allowing these proteins to simultaneously interact with multiple regions of & 2011 European Molecular Biology Organization