Having shown that the unusual C΄/D΄ motif, in particular the D΄ box, in U18 results in the modification of two sites in the target region, we were next interested to determine the elements needed for the unusual activity of the snR48 C΄/D΄ motif. Yeast snR48 guides the 2΄-O-methylation of two nucleotides (G2791 and G2793) in the snR48 target region of the 25S rRNA (helix 88 in domain V). Interestingly, by comparing the available yeast RNA sequences we found that the unusual D΄ box region of snR48 is a highly conserved 6 nt region (AUGUUA) that does not contain the CUGA consensus (Figure 3A). Conversely, the C΄ motif, in particular the 5΄ half, fits to the consensus sequence and is as conserved as most yeast C΄ boxes (24). As with U18, we found two possible base-pairing alternatives for the C΄/D΄ motif that may explain the ability of this motif to direct two 2΄-O-methylation events in the same target (Figure 3B). This secondary structure is also conserved, with only a couple of exceptions, across a diverse range of fungi. The UG in the D΄ box of S. cerevisiae snR48 (from 5΄ positions 2 and 3) is an AA in S. kluyveri snR48. This means that only one of the two base-pairing options seen in the S. cerevisiae snR48 is possible in the S. kluyveri RNA (Figure 3B). In addition, the C΄/D΄ motif in the snR48 snoRNAs from Lodderomyces elongisporus, Candida parapsilosis, Verticillium dahliae, Phaeosphaeria nodorum, Aspergillus flavus and Glomerella graminicola (Figure 3B and data not shown) also allow for only one of the two base-pairing interactions. To test the methylation activity of the different snR48 C΄/D΄ motifs, we mutated the S. cerevisiae sequence in the artificial construct to mimic the sequence of the S. kluyveri snR48 D΄ motif (Figure 3C; mutant D΄2,3 UG-AA). In addition, we cloned the C΄/D΄ region from the L. elongisporus snR48 snoRNA into the artificial snoRNA construct (Supplementary Figure S1). The ability of these artificial snoRNAs to direct methylation was then analysed as described above.