An unusual D΄ box and the extra base-pairing sequence are required for multi-site 2΄-O-methylation by the snR13 snoRNA. (A) A schematic representation of the conservation of the active guide and D΄/D box sequences of the snR13 snoRNA (24).The consensus for the C΄ and D΄ sequences is shown above the image. The diagram was prepared using the WebLogo software (29). (B) Secondary structure models of the snR13 box C΄/D΄ motif, guide region and accessory guide with the natural rRNA target site (shown in red) in the 25S 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 and the mutation to the accessory guide is shown on the right. Arrows indicate the site in the rRNA to be modified. Note, in the alternative structure a longer, five nucleotide box D΄ is used. (C) Constructs expressing wild-type and mutant snR13 snoRNAs (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, L2280 and L2281 in the 25S rRNA, are indicated. The levels of the various snoRNAs were determined by Northern blotting (Supplementary Figure S2E). (E) The data presented in (D) was analysed using imagequant software and the relative levels of the bands corresponding to the L2280 and L2281, relative to the signal seen for the modification at L2288, were calculated and plotted. The data were adjusted to the WT signal at L2280. Error bars indicate standard deviation from three separate experiments.