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. Author manuscript; available in PMC: 2013 May 15.
Published in final edited form as: Nat Struct Mol Biol. 2012 Jul 1;19(8):744–753. doi: 10.1038/nsmb.2308

Figure 6.

Figure 6

Fun12 binding to 25S rRNA is required for efficient 20S processing.

(a) 25S region involved in subunit joining. Fun12 binding sites are indicated by circled nucleotides. The snR75 interaction that directs methylation at Gm2288 is indicated in red. The accessory guide interaction is indicated in blue, along with the new target for snR75Mut2 at Gm2307. (b) Constructs expressing wild-type or mutant snR75. Red box; methylation guide. Blue box; accessory guide. In snR75Mut1, the accessory guide is non-functional. In snR75Mut2, the accessory guide is extended and confers ectopic methylation at Gm2307. The snoRNAs are expressed from the intron of the ACT1 gene flanked by PGAL and TADH, in a strain deleted for the SNR78-72 cluster. (c,d) Effects of expression of snR75Mut2 on pre-rRNA processing in vivo. (c) FISH analysis as in Figure 4d. (d) Northern analysis of pre-rRNAs using the probes indicated on the left. Mature rRNAs were visualized by EthBr staining. (e) Cleavage assay as in Figure 2 in lysates from cells expressing snR75 or snR75Mut2. Lanes 1 and 4, cleavage without added GTP. Lanes 2 and 5, with 1mM GTP. Lanes 3 and 6, with 1mM GTP plus 100pmol of purified ribosomal subunits (ribs). (f) Quantification of cleavage efficiency.