Site‐specific cross‐linking analyses reveal an asymmetric protein distribution for a box C/D snoRNP

Methylation of the ribose 2′‐hydroxyl, the most widespread modification of ribosomal and splicesomal RNAs, is guided by the box C/D class of small nucleolar RNAs (snoRNAs). Box C/D small nucleolar ribonucleoproteins (snoRNPs) contain four core proteins: fibrillarin, Nop56, Nop58 and 15.5 kDa. We constructed U25 snoRNAs containing a single photoactivatable 4‐thiouridine at each U position within the conserved box C/D and C′/D′ motifs. Proteins assembled on the snoRNA after injection into Xenopus oocyte nuclei were identified by cross‐linking, and reconstituted particles characterized by functional rescue and mutational analyses. Our data argue that box C/D snoRNPs are asymmetric, with the C′ box contacting Nop56 and fibrillarin, the C box interacting with Nop58, and the D and D′ boxes contacting fibrillarin. No cross‐link to 15.5 kDa was detected; its binding is disrupted by 4‐thiouridine substitution in position 1 of the C box. Repositioning the guide sequence of U25 upstream of box D instead of D′ revealed that both C/D motifs have the potential to function as guide centers, but, surprisingly, there was no alteration in protein cross‐linking.