Mutation of the first GA di-nucleotide in the C΄ box (C΄3,4 GA-CC) abolished methylation at both sites (Figure 2C) while mutation of the last A in the D΄ box (D΄4 A-C) abolished methylation at S1316 and severely reduced methylation at S1315. Interestingly, mutation of the second GA di-nucleotide in box C΄ (C΄7,8 GA-CC) abolished methylation at S1316 but not at S1315. From this we concluded that the GA base-pairs are needed for total activity and that changes to stem II affect the site targeted for methylation. Further mutation of the stem II sequence revealed that deletion of the G insertion (C΄5 ΔG) had little effect on the methylation of S1316 and S1315. However, mutation of this G to a U or A (C΄5 G-U; C΄5 G-A) biased the modification to site S1315 over S1316. Mutation D΄1 A-C, which generates the consensus D΄ box resulted in a reduction in 2΄-O-methylation at nucleotide S1316 but no change to methylation at S1315. Conversely, mutation D΄-1 U-C, which places a C at the 3΄ end of the guide region, resulted in methylation of S1316 and a reduction in the signal at S1315 to background levels (Figure 2C; compare D΄-1 U-C to hU24). These data indicate that the sequence of stem II in U18, in particular the 5΄ nt of the D΄ box and the 3΄ nucleotide of the guide, dictate which nucleotides are modified in the target sequence. Importantly, neither of these nucleotides is a C, the consensus nucleotide for the 5΄ end of box D΄, and mutation of either nucleotide to a C dramatically alters the methylation pattern. This therefore implies that the sequence/structure of stem II and, in particular, suboptimal sequences at the 5΄ end of box D΄ can result in the modification of two different nts in the target sequence.