The last nucleotide of the guide region can influence the site to be 2΄-O-methylated in the target RNA. (A) A schematic representation of the conservation of the active guide and D΄/D box sequences of the S. cerevisiae snoRNAs (24). The diagram was prepared using the WebLogo software (29). (B) Comparison of the 3΄ nucleotide of the active and inactive (no guide) guide regions of the available yeast box C/D snoRNAs (24). (C) Secondary structure of the hU24 box C΄/D΄ motif, in the context of the artificial snoRNA targeting sites 1316 in the 18S rRNA. The C΄ and D΄ boxes are shown in white on a black background. The arrow indicates the site to be modified. The mutations to the D΄ box are shown to the left. (D) Constructs expressing artificial snoRNAs containing the wild-type and mutant hU24 C΄/D΄ motifs (as indicated above each lane) were transformed into yeast cells. RNA was extracted from the cells and analysed by primer extension to detect rRNA methylation. The position of the stop corresponding to methylation of the target nucleotides, S1315 and S1316 in the 18S rRNA, are indicated. The levels of the various snoRNAs were determined by Northern blotting (Supplementary Figure S2D).