Two RNA-binding domains determine the RNA-binding specificity of nucleolin.
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 vitro RNA-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.
Serin
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Laboratoire de Biologie Moléculaire Eucaryote, Institut de Biologie Cellulaire et de Génétique du CNRS, UPR 9006, 118 route de Narbonne, 31062 Toulouse Cedex, France.
Joseph
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Ghisolfi
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L
Bauzan
M
M
Erard
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M
Amalric
F
F
Bouvet
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P
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D000097796:Nucleolin
D009687:Nuclear Proteins; D009841:Oligonucleotides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin