We were next interested in analysing the sequence and structural features of the C΄/D΄ motif of the snR48 snoRNA from S. cerevisiae. To this end, we generated a series of mutations in the C΄ and D΄ boxes (Figure 3C) and analysed their effect on 2΄-O-methylation (Figure 3D). Mutation C΄9,10 AA-CC, which altered nucleotides just downstream of the C΄ box, had no noticeable effect on methylation at either site while the D΄6,7 AG-CC and C΄3,4 GA-CC mutations severely reduced and completely abolished modification at both sites, respectively. The D΄6,7 AG-CC mutation altered the last nucleotide of the predicted D΄ box and suggests that the 3΄ end of the D΄ motif is required for both modification events. A slight reduction in methylation at site S1314 was observed with the D΄5,6 UU-CC and D΄8,9 GA-CC mutations. The D΄1,2 AU-CC and C΄4,5 AA-CC mutations severely reduced 2΄-O-methylation at site S1314, while not significantly affecting modification of S1316. Mutations C΄-1,1 AA-CC and C΄4,5 AA-UU reduced methylation at both sites. Interestingly, the C΄4,5 AA-CC and C΄4,5 AA-UU mutations change the same 2 nt but have different outcomes. Deletion of the first 2 nt of box D΄ (Figure 3C; D΄1,2 ΔAU) replaced the AU of the D΄ box with the UU of the guide region and moved the predicted target sites (now S1316 and S1318). This mutant resulted in methylation of S1316 but not S1318 (Figure 3D). Our data indicate that the whole of the D΄ box is essential for the multi-site modification of the target region guided by snR48. However, only the 3΄ A of the D΄ box was absolutely essential for rRNA modification. As with U18, the insertion of C's in the 5΄ end of the D΄ region (mutants D΄1,2 AU-CC) significantly influenced the nucleotide modified. The C΄ box of snR48 appears to be comparable to C΄ boxes found in other yeast snoRNAs. Our data are consistent with snR48 containing two different structures for the C΄/D΄ motif using two overlapping D΄ boxes. However, the interaction between the consensus C΄ box and the D΄ box contains one to two Watson–Crick base-pairs and three non-Watson–Crick base-pairs making it difficult to predict exactly how these boxes and, especially very divergent sequences, interact with each other (see Discussion). This point is exemplified by the fact that the D΄1,2 AU-CC mutation would be predicted to block methylation at S1316, in a similar manner to the D΄1,3 UG-AA mutation, but actually affects 2΄-O-methylation at site S1314 and suggests not only that the complete D΄ region is required for both modifications but that a C at the 5΄ end of the D' box determines which nucleotide is modified.