The 15.5-kD protein and its yeast homolog Snu13p bind U4 snRNA, U3 snoRNA, and the C/D box snoRNAs. In U4 snRNA, they associate with a helix-bulge-helix (K-turn) structure. U3 snoRNA contains two conserved pairs of boxes, C′/D and B/C, which were both expected to bind the 15.5-kD/Snu13 protein. Only binding to the B/C motif was experimentally demonstrated. Here, by chemical probing of in vitro reconstituted RNA/protein complexes, we demonstrate the independent binding of the 15.5-kD/Snu13 protein to each of the two motifs. Due to a highly reduced stem I (1 bp), the K-turn structure is not formed in the naked B/C motif. However, gel-shift experiments revealed a higher affinity of Snu13p for the B/C motif, compared to the C′/D motif. A phylogenetic analysis of U3 snoRNA, coupled with an analysis of Snu13p affinity for variant yeast C′/D and B/C motifs, and a study of the functionality of a truncated yeast U3 snoRNA carrying base substitutions in the C′/D and B/C motifs, revealed that conservation of the identities of residues 2 and 3 in the B/C K-turn is more important for Snu13p binding and U3 snoRNA function, than conservation of the identities of corresponding residues in the C′/D K-turn. This suggests that binding of Snu13p to K-turns with a very short helix I imposes sequence constraints in the bulge. Altogether, the data demonstrate the strong importance of the binding of the 15.5-kD/Snu13 protein to the C′/D and B/C motifs for both U3 snoRNP assembly and activity.

The eukaryal Snu13p/15.5K protein binds K-turn motifs in U4 snRNA and snoRNAs. Two Snu13p/15.5K molecules bind the nucleolar U3 snoRNA required for the early steps of preribosomal processing. Binding of one molecule on the C'/D motif allows association of proteins Nop1p, Nop56p, and Nop58p, whereas binding of the second molecule on the B/C motif allows Rrp9p recruitment. To understand how the Snu13p-Rrp9p pair recognizes the B/C motif, we first improved the identification of RNA determinants required for Snu13p binding by experiments using the systematic evolution of ligands by exponential enrichment. This demonstrated the importance of a U.U pair stacked on the sheared pairs and revealed a direct link between Snu13p affinity and the stability of helices I and II. Sequence and structure requirements for efficient association of Rrp9p on the B/C motif were studied in yeast cells by expression of variant U3 snoRNAs and immunoselection assays. A G-C pair in stem II, a G residue at position 1 in the bulge, and a short stem I were found to be required. The data identify the in vivo function of most of the conserved residues of the U3 snoRNA B/C motif. They bring important information to understand how different K-turn motifs can recruit different sets of proteins after Snu13p association.