A structural, phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA

• Published in: RNA (Cambridge) 2003-07, Vol.9 (7), p.821-838
• Main Authors: Marmier-Gourrier, Nathalie, Cléry, Antoine, Senty-Ségault, Veronique, Charpentier, Bruno, Schlotter, Florence, Leclerc, Fabrice, Fournier, Régis, Branlant, Christaine
• Format: Article
• Language: English
• Subjects: Base Sequence; Binding Sites; Chemical Sciences; Genetic Variation; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Organic chemistry; Phylogeny; Protein Binding; Ribonucleoproteins, Small Nuclear - genetics; RNA, Fungal - chemistry; RNA, Fungal - genetics; RNA, Fungal - metabolism; RNA, Small Nuclear - chemistry; RNA, Small Nuclear - genetics; RNA, Small Nuclear - metabolism; RNA, Small Nucleolar - genetics; Saccharomyces cerevisiae Proteins - genetics; Templates, Genetic; Transcription, Genetic
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.2130503

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.