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Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 UHP-CNRS, Université Henri Poincaré Nancy 1, 54506 Vandoeuvre-Lès-Nancy cedex, France
Reprint requests to: Christiane Branlant, Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 UHP-CNRS, Université Henri Poincaré Nancy 1, Boulevard des Aiguillettes, 54506 Vandoeuvre-Lès-Nancy cedex, France; e-mail: christiane.branlant{at}maem.uhp-nancy.fr; fax: 33 3 83 68 43 07.
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.
Keywords: U3 snoRNA; 15.5-kD/Snu13 protein; snoRNP, RNA–protein interaction; K-turn structure; RNA evolution
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