No 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 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.

Nucleolin is an abundant nucleolar protein which is involved in the early stages of ribosome assembly. The central 40-kDa domain of nucleolin comprises four RNA recognition motifs (RRM) which are presumed to be involved in specific interactions with pre-rRNA. In order to examine in detail the role of this central domain and the contribution of the N-terminal and C-terminal domains of nucleolin to RNA binding, we have used an Escherichiu coli expression system to synthezise polypeptides corresponding to various combinations of the three domains and their subdomains. By means of an in-vitro binding assay and a synthetic RNA corresponding to a specific recognition site in pre-rRNA we have been able to demonstrate conclusively that the central 40-kDa domain is indeed responsible for the specificity of RNA recognition and that the N-terminal domain can be removed without affecting RNA binding. Most interestingly, it appears that the C-terminal 10-kDa domain, which is rich in glycine and arginine residues, is essential for efficient binding of nucleolin to RNA, but does not itself contribute to the specificity of the interaction. Circular dichroic spectroscopic probing of the RNA component shows that the C-terminal domain significantly modifies the RNAbinding properties of the central RRM core. Finally, infrared spectroscopic studies reveal that the central 40-kDa domain is structured in a helices and p sheets and that the interaction with the specific pre-rRNA site induces subtle changes in the p sheet conformation.Just as newly synthesized hnRNA associates in the nucleus with a discrete set of proteins, pre-rRNA is organised within the nucleolus into specific ribonucleoproteic complexes. One of the most abundant proteic factors to have been identified in the dense fibrillar component of the nucleolus is a 100-kDa phosphoprotein, called nucleolin [l, 21. Based on this finding and the observed association between nucleolin and prerRNA [3, 41, it was supposed that this nucleolar protein was implicated in the early stages of ribosoinal assembly. Recently, direct evidence of a molecular interaction between nucleolin and pre-rRNA has been obtained. Several sites of interaction have been mapped within the 18s and 28s ribosomal RNA sequences as well as in the 5' external transcribed spacer (5'-ETS), and it has been shown that this last site can be specifically recognised by nucleolin in an in-vitro system IS].Interestingly, in addition to its role in preribosome biogenesis, nucleolin is associated with both nucleolar chromatin in interphase [6, 71 and nucleolar organiser regions in mitosis [8, 91. Indeed, we have shown that nucleolin can modulate chromatin condensation [7,10, 111 and that this function correlates well with the observed homology between the 280-amino-acid N-terminal domain of nucleolin and high-mobility-group proteins. The remaining 430 amino acids of the protein sequence comprise two other domains: a central do- main, made up of four closely related 80 -90-amino-acid repeats and a C-terminal domain, 85 re...