The U24 and snR13 C΄/D΄ motifs both directed methylation six nucleotides from the originally predicted D΄ box (Figure 1E) but neither element, in the context of the artificial snoRNA system, targeted methylation of two adjacent nucleotides. We therefore next investigated whether other elements in the snoRNAs contribute to multi-site modification of the target region. To our surprise, many of the mutations to guide and/or C΄/D΄ regions of yeast U24 frequently affected snoRNA levels to the extent that we could not draw any conclusions from our analysis of this snoRNA (data not shown). We therefore turned our attention to the snR13 snoRNA which directs 2΄-O-methylation of nucleotides L2280 and L2281 in the 25S rRNA. snR13 contains an extra base-pairing region or accessory guide (Figure 5A and B; (24)). The snR13 D΄ box is evolutionarily well conserved and again possibly has two D΄ boxes, one four nucleotides (Figure 5B; UCGA) and one five nucleotides in length (CUCGA). Based on the work presented above, the CU present at the 5΄ end of the D΄ box likely contributes to the targeting of the nucleotide six nucleotides into the guide. Given that multi-site modification was not achieved with the artificial snoRNA system, we analysed the role of the extra base-pairing region and the 5΄ end of the D' box in the level and specificity of 2΄-O-methylation by the natural snR13 snoRNA. An empty vector or plasmids expressing wild-type snR13 or mutated snR13 (Figure 5B and C), were introduced into a yeast strain where the endogenous snR13 gene had been deleted. RNA was extracted from these strains and 2΄-O-methylation was analysed by primer extension.