From the analysis of U18 and snR48, our data indicate that the presence of a C at the 3΄ end of the guide region, particularly where the D΄ box lacks a 5΄ C, can influence the site methylated in the rRNA and extend the D΄ box to five nucleotides. From this we predict that C would not frequently be found at the 3΄ end of the guide region. We therefore compared the sequence of the last three nucleotides of guides, before the D or D΄ box, known to direct 2΄-O-methylation in the S. cerevisiae snoRNAs. There was no sequence bias in the nucleotides three and two positions before the D/D΄ box (Figure 4A). In contrast, in 60% of the RNAs a U was present in the last position of the guide, A was represented normally, G slightly under-represented and C only present in two cases (Figure 4A). In one case, snR13, the C present at the 3΄ end of the guide is probably part of a longer D΄ box (see later). The data therefore indicate that in S. cerevisiae, U is the preferred nucleotide for the last position in the guide region and that C is significantly under-represented. We next compared the sequence of the last nucleotide of the guide region in 2033 active and 556 inactive (i.e. the guide regions that are not conserved and have no obvious target) guides adjacent to the D΄ box for all available yeast snoRNAs that were characterised in our previous bioinformatics analysis (24). While there was a slight bias towards C, and against G, in the inactive guides, this revealed no real significant sequence bias (Figure 4B). However, in the active guides, the sequence was again biased towards U, with C found at this position in only 9% of the snoRNAs. Interestingly, G was also under-represented at this position. These data therefore support our hypothesis that a C at the 3΄ nucleotide of the guide region may interfere with 2΄-O-methylation specificity.