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

  1. NATHALIE MARMIER-GOURRIER1,
  2. ANTOINE CLÉRY1,
  3. VÉRONIQUE SENTY-SÉGAULT,
  4. BRUNO CHARPENTIER,
  5. FLORENCE SCHLOTTER,
  6. FABRICE LECLERC,
  7. RÉGIS FOURNIER, and
  8. CHRISTIANE BRANLANT
  9. Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 UHP-CNRS, Université Henri Poincaré Nancy 1, 54506 Vandoeuvre-Lès-Nancy cedex, France

Abstract

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.

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