A structural, phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA.
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.
Marmier-Gourrier
Nathalie
N
Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 UHP-CNRS, Université Henri Poincaré Nancy 1, 54506 Vandoeuvre-Lès-Nancy cedex, France.
Cléry
Antoine
A
Senty-Ségault
Veronique
V
Charpentier
Bruno
B
Schlotter
Florence
F
Leclerc
Fabrice
F
Fournier
Régis
R
Branlant
Christaine
C
D001483:Base Sequence; D001665:Binding Sites; D014644:Genetic Variation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010802:Phylogeny; D011485:Protein Binding; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D020537:RNA, Small Nucleolar; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C440569:Snu13 protein, S cerevisiae