While archaeal sRNAs are compact with highly conserved sequence motifs, the eukaryotic snoRNAs are significantly more varied (18). In archaea the C/D and C΄/D΄ motifs are highly conserved and very similar. In eukaryotes, while the C/D and C΄/D΄ motifs share the same consensus, the C/D motif is considerably more conserved than the C΄/D΄ motif (24). The eukaryotic C΄ box can contain insertions although it is unclear how this would be accommodated with respect to protein-binding (24). Furthermore, several C΄/D΄ motifs that are clearly active in directing 2΄-O-methylation, vary considerably from the consensus (24). In eukaryotes, an extra, accessory rRNA base-pairing element, that stimulates snoRNP-catalysed methylation by interacting with the rRNA adjacent to the target region of the snoRNA has been described in more than half of the box C/D snoRNAs (24). In yeast, the U18, U24, snR13 and snR48 snoRNAs have been suggested to modify more than one nucleotide in the target region using a single guide and C΄/D΄ region (Figure 1C). Recent deep-sequencing approaches indicate that, for each snoRNA, both sites are almost completely modified (15,16). In each case, deletion of the snoRNA abolishes both modifications (25,26) and, in the absence of alternative methytransferases, it is likely that the C΄/D΄ motifs of these snoRNAs direct multiple modifications. It is, however, unclear how the specificity of box C/D snoRNP catalysed methylation can be altered to enable the 2΄-O-methylation of multiple sites in one target region. We therefore set out to investigate the ability of the U18, U24, snR13 and snR48 snoRNAs to direct multiple modifications in a single target region.