To investigate this further, we generated a series of mutants in the U18 C΄/D΄ region in the artificial construct to test the importance of the stem II sequence in multi-site 2΄-O-methylation (Figure 2B). For these experiments we also mutated the extra base-pairing element in the U18 C΄/D΄ such that it base-paired upstream of the 18S rRNA target site to fully replicate the base-pairing interactions seen with the wild-type U18 snoRNA and the 25S rRNA (U18mutX; Figure 2B and Supplementary Figure S1C). It is important to note that we found that the extra base-pairing played no role in multi-site modification or methylation efficiency by the U18C΄/D΄ region (data not shown). The constructs were transformed into yeast cells, the expression of the snoRNAs was monitored by Northern blotting (Supplementary Figure S2B) and the ability to direct 2΄-O-methylation was analysed by primer extension.