Pre-60S ribosome synthesis S Granneman et al regulating the timing of 50-processing of the 5.8S rRNA. ITS2 was introduced into an ancestral 23S-like rRNA early in eukaryotic evolution and this mechanism offers a potential rationale for the advantage of having, and retaining, an ITS2 region. Materials and methods Yeast strains and media Saccharomyces cerevisiae strain BY4741 (MATa; his3D1; leu2D0; met15D0; ura3D0) was used as the parental strain (Brachmann et al, 1998). The HTP carboxyl-tagged strains were generated by PCR as described (Rigaut et al, 1999). Strains were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose), YPG/R (1% yeast extract, 2% peptone, 2% galactose, 2% rafﬁnose) or yeast minimal medium (Formedium) lacking uracil and tryptophan, at 30 1C. CRAC and sequence analyses In vivo CRAC, western and northern blot analyses were performed essentially as described previously (Granneman et al, 2009) with the following modiﬁcations: For the Rat1 CRAC experiments shown in Figure 6, cells were pre-grown in ﬁlter sterilized synthetic minimal medium containing galactose and rafﬁnose, but lacking uracil and tryptophan (SG/R-URA-TRP), to an OD600 of 1.0, subsequently shifted to glucose containing medium (SD-URA-TRP) for 12 h to an OD600 of B0.5. Cultures were UV irradiated in the Megatron (Supplementary Figure S1A) at room temperature for 2 100 s (equivalent to an average dose of B1.6 mJ/cm ) and cells were harvested by centrifugation, washed with phosphate-buffered saline (PBS), and stored at 80 1C. Megatron parts were purchased from UVO3 (http://www.uvo3.co.uk; contact Peter Wadsworth http://Peter@uvo3.co.uk). Sanger sequencing of cDNAs was performed as described (Granneman et al, 2009). Negative control cDNAs were generated from RNA extracted from excised membrane fragments from mock experiments run in parallel. For the high- throughput sequencing analysis, crosslinked RNAs were sequen- tially ligated to L3 and barcoded L5 adaptors and ampliﬁed by RT/ PCR (see Supplementary Table S1 for oligonucleotide sequences). Oligonucleotides were purchased from Integrated DNA technologies (IDT). Illumina sequencing (single end 50-bp reads) was performed according to the manufacturer’s procedure and reads were aligned to the yeast genome using novoalign (http://www.novocraft.com). Data analyses were performed using pyCRAC, a set of Python tools for high throughput sequence analysis (Webb, Tollervey and Granneman, manuscript in preparation). References Babiano R, de la Cruz J (2010) Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae. 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Proc Natl Acad Sci USA 97: 13027–13032 El Hage A, Koper M, Kufel J, Tollervey D (2008) Efﬁcient termination of transcription by RNA polymerase I requires the 50 exonuclease Rat1 in yeast. Genes Dev 22: 1069–1081 4018 The EMBO Journal VOL 30 | NO 19 | 2011 Chemical footprinting experiments Parental (BY4741) and GAL depletion strains (GAL::3HA-nop4, GAL::3HA-cic1 and GAL::3HA-nop15) were grown in YPG/R to logarithmic phase, shifted to YPD and grown for 12 h to an OD600 of B0.5. For the in vivo modiﬁcation experiment, 200 ml of 33% DMS solution (diluted in ethanol) was added to 10 ml of cells and shaken for 3 min at room temperature. DMS was quenched using 10 ml of 0.7 M b-mercaptoethanol and 5 ml of water-saturated isopropanol. Cell pellets were subsequently washed once with 10 ml 0.7 M b-mercaptoethanol and once with ice-cold phosphate saline (PBS). RNA extractions were performed as previously described (Tollervey, 1987). For the in vitro DMS experiments, RNA extracted from 5 ODs of cells was dissolved in 400 ml of refolding buffer (50 mM HEPES- KOH pH 7.5, 150 mM NaAc, 1.5 mM MgCl2). RNA was refolded by slowly cooling heat denatured RNA (65 1C 10 min) to room temperature. Refolded RNA was incubated with 20 ml 33% DMS solution (diluted in ethanol) for 3 min at room temperature and reactions were quenched by adding 160 ml of 0.7 M b-mercaptoetha- nol. One microgram of total RNA was used for primer extension reactions. Primer extension products were resolved on 6% polyacrylamide/7 M urea gels and visualized by autoradiography. Chemical probing data were quantiﬁed as described in the main text using a Fuji FLA5000 phosphoimager and the Aida software package. Supplementary data and methods Supplementary data and Materials and Methods are available at The EMBO Journal Online (http://www.embojournal.org). Acknowledgements We thank Ernst Granneman, Peter Davis and the Darwin workshop for help with designing and modifying the Megatron; John Woolford and Aarti Sahasranaman for communicating unpublished results; Adam Ben-Shem, Lasse Jenner, Gulnara Yusupova and Marat Yusupov for making the yeast 80S ribosome crystal structure available; Tollervey laboratory members for helpful suggestions; the Edinburgh Gene Pool Sanger and Solexa Sequencing Facilities; and the Swann Building kitchen staff. This work was supported by the Welcome Trust, the BBSRC (BB/D019621/1), EMBO long-term fellowship (SG) and a Marie Curie EIF fellowship (SG). Author contributions: SG and DT designed the research. SG, EP performed the research and analysed the data. SG and DT wrote the paper. Conﬂict of interest The authors declare that they have no conﬂict of interest. Faber AW, Vos HR, Vos JC, Raue HA (2006) 50-end formation of yeast 5.8SL rRNA is an endonucleolytic event. 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