Gérard Joseph

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 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.

Nucleolin — pre-rRNA interactions and preribosome assembly

Nucleolln (713 residues in hamster) is an ubiquitous protein in most eukaryotic cells that is localized in the nucleolus (i). The primary structure of the protein reveals a highly polar NH2-termlnal domain, four RNA binding domains (2) and a PheGly-Arg C-termlnal domain (3). In vitro, the protein presents affinity for RNA and for chromatln. Similarly, in vivo, nucleolln is found associated with nascent pre-rENA in prerlbosomes (4) and with nucleolar chromatin (4).

Identification of the ‘NORE’ (N-Oct-3 responsive element), a novel structural motif and composite element

N-Oct-3 is a neuronal transcription factor widely expressed in the developing mammalian central nervous system, and necessary to maintain neural cell differentiation. The key role of N-Oct-3 in the transcriptional regulation of a multiplicity of genes is primarily due to the structural plasticity of its so-called ‘POU’ (acronym of Pit, Oct, Unc) DNA-binding domain. We have recently reported about the unusual dual neuro-specific transcriptional regulation displayed by N-Oct-3 [Blaud,M., Vossen,C., Joseph,G., Alazard,R., Erard,M. and Nieto,L. (2004) J. Mol. Biol., 339, 1049–1058]. To elucidate the underlying molecular mechanisms, we have now made use of molecular modeling, DNA footprinting and electrophoretic mobility shift assay techniques. This combined approach has allowed us to uncover a novel mode of homodimerization adopted by the N-Oct-3 POU domain bound to the neuronal aromatic amino acids de-carboxylase and corticotropin-releasing hormone gene promoters and to demonstrate that this pattern is induced by a structural motif that we have termed ‘NORE’ (N-Oct-3 responsive element), comprising the 14 bp sequence element TNNRTAAATAATRN. In addition, we have been able to explain how the same structural motif can also induce the formation of a heterodimer in association with hepatocyte nuclear factor 3β(/Forkhead box a2). Finally, we discuss the possible role of the NORE motif in relation to neuroendocrine lung tumor formation, and in particular the development of small cell lung cancer.