Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains |
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Authors: | Armas Pablo Agüero Tristán H Borgognone Mariana Aybar Manuel J Calcaterra Nora B |
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Institution: | 1 División Biología del Desarrollo, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas—Área Biología General, Dpto. de Ciencias Biológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina 2 Dpto. Biología del Desarrollo, INSIBIO (Consejo Nacional de Investigaciones Científicas y Técnicas—Universidad Nacional de Tucumán), Chacabuco 461, T4000ILI San Miguel de Tucumán, Tucumán, Argentina |
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Abstract: | Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development. |
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Keywords: | CNBP cellular nucleic-acid-binding protein ssDNA single-stranded DNA NC nucleocapsid HIV-1 human immunodeficiency virus type 1 EMSA electrophoretic mobility shift assay UTR untranslated region GST glutathione-S-transferase EDTA ethylenediaminetetraacetic acid FDx fluorescein dextran HRP horseradish peroxidase TBE Tris-Borate-EDTA buffer |
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