No 2΄-O-methylation of sites L2280 and L2281 was seen in the strain lacking snR13 and methylation was restored at these two sites when the wild-type snR13 was expressed from a plasmid (Figure 5D; compare lanes 1 and 2). Mutation of the nucleotide just before the D΄ box to an A (D΄-1 C-A) resulted in a reproducible increase in modification at site L2280 with a corresponding decrease to modification at site L2281 (Figure 5C (lane 4) and D). Interestingly, this change was not seen when this nucleotide was changed to a U (D΄-1 C-U; lane 6). The C to U change would still allow base-pairing with the second G in the C΄ box (RUGAUGA) while a C to A change would likely abolish this interaction. Mutation of the extra-base-pairing region (Xmut) resulted in a significant reduction of 2΄-O-methylation by snR13 at L2280 while modification of nucleotide L2281 was not detectable (lane 3). Interestingly, combining Xmut with the D΄-1 C-A mutation (Xmut-D΄-1 C-A; lane 5) resulted in a slight reduction at modification at site L2280 but a dramatic reduction in 2΄-O-methylation at L2281. In contrast, combining Xmut with the D΄-1 C-U mutation gave the same result as the Xmut alone (Xmut-D΄-1 C-U; lane 7). Extra base-pairing can affect the level of methylation (24) but we were surprised to find that this base-pairing interaction can also affect target specificity. Taken together, our data show that the combination of an unusual D΄ box and extra base-pairing lead to the efficient 2΄-O-methylation of two sites in the target region by snR13.