The D΄ box interacts with the C΄ box to form a protein-binding site that functions to position multiple proteins, including the catalytic subunit fibrillarin/Nop1, onto the snoRNA. Based on the structure of the Archaeal box C/D sRNPs (22), both the C/D and C΄/D΄ motifs are bi-partite structures. The GA base-pairs and the first U in the C/C΄ box, form the L7Ae (Snu13 in yeast) binding site (Figure 6A and B). Stem II is bound by Nop5 (Nop58 or Nop56 in yeast). Importantly, the only sequence specific contacts between Nop5 and stem II are between Q296 and R339 in the Nop domain of Nop5 the C in the D/D΄ box and the last GA in the C/C΄ box (Figure 6A and B). Nop5 is homologous to both Nop56 and Nop58 and the Q296 and R339 are evolutionarily highly conserved. We therefore assume that Nop56 would interact with stem II of the eukaryotic C΄/D΄ motif in a similar manner. The catalytic subunit, fibrillarin (Nop1 in yeast) does not contact the C/D or C΄/D΄ motifs and is held in position by its interaction with Nop5. Assuming that this is also the case in eukaryotes, the docking site of Nop56 on the C΄/D΄ motif would therefore dictate where Nop1 is positioned on the substrate RNA (Figure 6A). Based on our data, the C at the 5΄ end of D box appears a major determinant for Nop56 binding and moving this C one nucleotide upstream in the snoRNA causes a similar move in the position of Nop1 on the target rRNA and therefore a one nucleotide shift in the target nucleotide. Figure 6C shows the predicted association of Snu13, Nop56 and Nop1 (fibrillarin) on the artificial snoRNA containing the hU24 C΄/D΄ motif to demonstrate this. Interestingly, only one hydrogen bond is predicted to form between the D΄ box C nucleotide and Nop56, while four hydrogen bonds are expected between Nop56 and the G and A in box C΄. From this we predict that the importance of the C in the D΄ box lies, in part, in positioning the G and A in the C΄ box and therefore creating the Nop56-binding site. Our data indicate that the use of alternative D΄ motifs, which differ in the use of a ‘normal’ and extended D΄ box, accounts for the modification of two nucleotides in the target region by snR13, snR48 and U18. We believe that, for each snoRNA, the cell contains two distinct snoRNPs, which differ in the folding of the C΄/D΄ motif, based on the D΄ box used, which in turn, alters the binding site of Nop56 to the snoRNA. Figure 6D shows the predicted association of Snu13, Nop56 and Nop1 (fibrillarin) with the two alternative structures of the U18 C΄/D΄ motif to illustrate this point. As Nop56 positions Nop1 in the snoRNP, the alternative binding of Nop56 leads to different positioning of Nop1 in the two snoRNPs, relative the rRNA, and therefore results in the modification of different nucleotides in the target region. Furthermore, our data fit nicely with the archaeal structure and support the idea that RNA–protein contacts in the eukaryotic snoRNP will be very similar to those seen in the archaeal box C/D sRNP.