Two RNA-binding Domains Determine the RNA-binding Specificity of Nucleolin*
- Guillaume Serin,
- Gérard Joseph,
- Laurence Ghisolfi,
- Marielle Bauzan,
- Monique Erard,
- François Amalric and
- Philippe Bouvet‡
- From the Laboratoire de Biologie Moléculaire Eucaryote, Institut de Biologie Cellulaire et de Génétique du CNRS, UPR 9006, 118 route de Narbonne, 31062 Toulouse Cedex, France
Abstract
Nucleolin is an abundant nucleolar RNA-binding protein that seems to be involved in many aspects of ribosome biogenesis. Nucleolin contains four copies of a consensus RNA-binding domain (CS-RBD) found in several other proteins. In vitroRNA-binding studies previously determined that nucleolin interacts specifically with a short RNA stem-loop structure. Taken individually, none of the four CS-RBDs interacts significantly with the RNA target, but a peptide that contains the first two adjacent CS-RBDs (R12) is sufficient to account for nucleolin RNA-binding specificity and affinity. The full integrity of these two domains is required, since N- or C-terminal deletion abolishes the specific interaction with the RNA. Mutation of conserved amino acids within the RNP-1 sequence of CS-RBD 1 or 2 drastically reduces the interaction with the RNA, whereas mutation of the analogous residues in CS-RBDs 3 and 4 has no effect in the context of the R1234G protein (which corresponds to the C-terminal end of nucleolin). Our results demonstrate that nucleolin RNA-binding specificity is the result of a cooperation between two CS-RBDs (RBDs 1 and 2) and also suggests a direct or indirect involvement of the RNP-1 consensus sequence of both CS-RBDs in the recognition of the RNA target.
Footnotes
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↵* This work was supported by grants from the Association pour la Recherche sur le Cancer and the Région midi-Pyrénées.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed. Tel.: 33-5-61-33-59-52; Fax: 33-5-61-33-58-86; E-mail:bouvet{at}ibcg.biotoul.fr.
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↵1 The abbreviations used are: CS-RBD, consensus RNA-binding domain; NRE, nucleolin recognition element; CD, circular dichroism; hnRNP, heteronuclear ribonucleoprotein; snRNP, small nuclear ribonucleoprotein; PABP, poly(A)-binding protein; PCR, polymerase chain reaction; NS, nonspecific; SELEX, systematic evolution of ligands by exponential enrichment.
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- Received December 31, 1996.
- Revision received February 19, 1997.
