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Molecular and Cellular Biology, February 2007, p. 1191-1206, Vol. 27, No. 4
0270-7306/07/$08.00+0     doi:10.1128/MCB.01287-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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{triangledown} ,{dagger}

A. Cléry,1 V. Senty-Ségault,1 F. Leclerc,1 H. A. Raué,2 and C. Branlant1*

Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567, Université Henri Poincaré, Nancy I, BP 239, 54506 Vandoeuvre-lès-Nancy, France,1 Section Biochemistry and Molecular Biology, Department of Chemistry, Faculty of Sciences and Institute Molecular Biology Science, BioCenter Amsterdam, Vrije Universiteit, Amsterdam, The Netherlands2

Received 14 July 2006/ Returned for modification 4 August 2006/ Accepted 20 November 2006

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.

* Corresponding author. Mailing address: Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567, Université Henri Poincaré, Nancy I, BP 239, 54506 Vandoeuvre-lès-Nancy, France. Phone: 33 383684303. Fax: 33 383684307. E-mail: christiane.branlant{at}maem.uhp-nancy.fr.

{triangledown} Published ahead of print on 4 December 2006.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, February 2007, p. 1191-1206, Vol. 27, No. 4
0270-7306/07/$08.00+0     doi:10.1128/MCB.01287-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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