Analysis of sequence and structural features that identify the B/C motif of U3 small nucleolar RNA as the recognition site for the Snu13p-Rrp9p protein pair.
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.
Cléry
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Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567, Université Henri Poincaré, Nancy I, BP 239, 54506 Vandoeuvre-lès-Nancy, France. christiane.branlant@maem.uhp-nancy.fr
Senty-Ségault
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Leclerc
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Raué
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HA
Branlant
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C
D020029:Base Pairing; D001483:Base Sequence; D017124:Conserved Sequence; D006147:Guanine; D008969:Molecular Sequence Data; D011485:Protein Binding; D020871:RNA Stability; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000737:chemistry* / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism*; D052156:SELEX Aptamer Technique; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017384:Sequence Deletion
D012331:RNA, Fungal; D020537:RNA, Small Nucleolar; C117378:RNA, U3 small nucleolar; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D029701:Saccharomyces cerevisiae Proteins; C440569:Snu13 protein, S cerevisiae; C117377:ribonucleoprotein, U3 small nucleolar; D006147:Guanine