The EMBO Journal (2011) 30, 4006–4019 | & 2011 European Molecular Biology Organization | Some Rights Reserved 0261-4189/11 www.embojournal.org A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease THE EMBO JOURNAL EMBO open Sander Granneman*, Elisabeth Petfalski and David Tollervey* Wellcome Trust Centre for Cell Biology and Centre for Systems Biology at Edinburgh, University of Edinburgh, Edinburgh, Scotland The 50-exonuclease Rat1 degrades pre-rRNA spacer frag- ments and processes the 50-ends of the 5.8S and 25S rRNAs. UV crosslinking revealed multiple Rat1-binding sites across the pre-rRNA, consistent with its known functions. The major 5.8S 50-end is generated by Rat1 digestion of the internal transcribed spacer 1 (ITS1) spacer from cleavage site A3. Processing from A3 requires the ‘A3-cluster’ proteins, including Cic1, Erb1, Nop7, Nop12 and Nop15, which show interdependent pre-rRNA bind- ing. Surprisingly, A3-cluster factors were not crosslinked close to site A3, but bound sites around the 5.8S 30- and 25S 50-regions, which are base paired in mature ribosomes, and in the ITS2 spacer that separates these rRNAs. In contrast, Nop4, a protein required for endonucleolytic cleavage in ITS1, binds the pre-rRNA near the 50-end of 5.8S. ITS2 was reported to undergo structural remodelling. In vivo chemical probing indicates that A3-cluster binding is required for this reorganization, potentially regulating the timing of processing. We predict that Nop4 and the A3 cluster establish long-range interactions between the 5.8S and 25S rRNAs, which are subsequently maintained by ribosomal protein binding. The EMBO Journal (2011) 30, 4006–4019. doi:10.1038/ emboj.2011.256; Published online 2 August 2011 Subject Categories: RNA; proteins Keywords: CRAC; ribosome synthesis; rRNA processing; RNP structure; yeast Introduction During ribosome subunit biogenesis in eukaryotes, the nas- cent pre-rRNA can either undergo cotranscriptional cleavage by the small subunit processome or be transcribed into the 35S pre-rRNA, which is cleaved within the 90S pre-ribosome complex. In either pathway, the early cleavages separate the *Corresponding authors. S Granneman, Centre for Systems Biology at Edinburgh (CSBE), University of Edinburgh, CH Waddington Building, Mayﬁeld Road, Kings Buildings, Edinburgh EH9 3JD, Scotland. Tel.: þ 44 131 651 9082; Fax: þ 44 131 651 9068; E-mail: sgrannem@staffmail.ed.ac.uk or D Tollervey, Wellcome Trust Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Mayﬁeld Road, Kings Buildings, Edinburgh EH9 3JR, Scotland. Tel.: þ 44 131 650 7092; Fax: þ 44 131 650 7040; E-mail: d.tollervey@ed.ac.uk Received: 23 December 2010; accepted: 29 June 2011; published online: 2 August 2011 4006 The EMBO Journal VOL 30 | NO 19 | 2011 pre-40S and pre-60S complexes, which are then matured independently (Figure 1A). During 60S maturation, internal transcribed spacers 1 and 2 (ITS1 and ITS2) are removed from the pre-rRNA by endonuclease cleavage followed by exonu- clease digestion to yield the mature 5.8S and 25S rRNA. The 60S ribosome maturation pathway involves several intermediate particles and a many different proteins. Several late steps in 60S maturation have recently been characterized (see Lo et al, 2010 and Panse and Johnson, 2010), but much less is known about early pre-60S particles or the actual function of most of the protein components. Understanding of yeast ribosome assembly will be greatly facilitated by the recent determination of the crystal structure of the mature 80S ribosomes (Ben-Shem et al, 2010). Early in the eukaryotic lineage, the 5.8S rRNA was derived from the 50-end of an ancestral 23S rRNA-like molecule by insertion of the ITS2 region. Reﬂecting this, the 5.8S rRNA associates with the 25S rRNA via two regions of extended base pairing between the 50-region of 5.8S (nts 4–13) and 25S rRNA (nts 404–413), and between the 30-region of 5.8S (nts 139–155) and 25S rRNA (nts 4–19). 5.8S–25S base pairing is established before or at C2 cleavage, since the 7S pre-rRNA remains associated with 25S rRNA following de- proteinization (DT, unpublished). However, secondary struc- ture models of the ITS1 region of the 27SA pre-rRNAs strongly predict that the 50-region of 5.8S is base paired to the 30-end of ITS1 in a stem-loop that is incompatible with base pairing to 25S rRNA, implying the presence of a con- formational switch (Yeh et al, 1990). It is currently unclear whether the transfer of the 50-region of 5.8S from ITS1 base pairing to 25S base pairing is provoked by exonuclease digestion of ITS1 from A3 to B1S, or is required for this processing to take place. The 50-end of the yeast 5.8S rRNA is heterogeneous due to the use of two alternative processing pathways (Henry et al, 1994). The major, short form of the rRNA (5.8SS) has a 50-end at site B1S (Figure 1A). This is generated by exonuclease digestion from an upstream cleavage site (A3), which is the target for cleavage by RNase MRP. Processing from A3 to B1S involves two exonucleases: the Rat1–Rai1 heterodimer (Schmitt and Clayton, 1993; Henry et al, 1994; Lygerou et al, 1996; Xue et al, 2000) and Rrp17 (Oefﬁnger et al, 2009). In addition, the cytoplasmic exonuclease Xrn1 can degrade the A3 -B1S region when Rat1 is inactive (Henry et al, 1994), but probably does not contribute to normal processing (El Hage et al, 2008). Formation of the 50-end of the less abundant 5.8SL rRNA does not require RNase MRP or the 50-exonucleases and is believed to involve an unidentiﬁed endonuclease (Faber et al, 2006). Similarly, 50 heterogeneity is observed for 5.8S rRNA in metazoans, plants and other eukaryotes (Henry et al, 1994), suggesting the use of conserved processing pathways. In addition to the exonucleases, A3 processing requires components of the pre-60S particles (Dunbar et al, 2000; & 2011 European Molecular Biology Organization