The use of the S. kluyveri D' box resulted in the preferential 2΄-O-methyation of site S1314 and all but abolished modification at S1316 (Figure 3D; D΄2,3 UG-AA). This is consistent with the fact that this mutation would interrupt base-pairing with the lower D΄ box and therefore methylation at S1316. Interestingly, reverse transcriptase stops, above background levels, were also seen 1 nt above and below S1314 indicating that the methylation machinery bound to this motif is somewhat promiscuous. The L. elongisporus C΄/D΄ motif (Figure 3D; L.elo) also directed modification of S1314 and, to a lesser extent, S1313 but not S1316. This indicates that snR48 C΄/D΄ motifs generally direct the 2΄-O-methylation of multiple sites in the target region. However, the pattern of modification in different yeast species can vary from that seen in S. cerevisiae depending on the sequence of the C΄/D΄ motif.