Dissecting CNBP, a Zinc-Finger Protein Required for Neural Crest Development, in Its Structural and Functional Domains

ARMAS, P; AGÜERO, T.; BORGOGNONE, M; AYBAR, M. J.; CALCATERRA, N.B.

JOURNAL OF MOLECULAR BIOLOGY

Elsevier

Año: 2008 vol. 384 p. 1043 - 1056

0022-2836

Cellular nucleic-acid-binding protein (CNBP) plays an essential role inforebrain and craniofacial development by controlling cell proliferation andsurvival to mediate neural crest expansion. CNBP binds to single-strandednucleic acids and displays nucleic acid chaperone activity in vitro. The CNBPfamily shows a conserved modular organization of seven Zn knuckles andan arginine-glycine-glycine (RGG) box between the first and second Znknuckles. The participation of these structural motifs in CNBP biochemicalactivities has still not been addressed. Here, we describe the generation ofCNBP mutants that dissect the protein into regions with structurally andfunctionally distinct properties. Mutagenesis approaches were followed togenerate: (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 theRGGbox alone; and (iii) a C-terminal deletion that eliminated the threelast Zn knuckles. Mutant proteins were overexpressed in Escherichia coli,purified, and used to analyze their biochemical features in vitro, oroverexpressed in Xenopus laevis embryos to study their function in vivoduring neural crest cell development. We found that the Zn knuckles arerequired, but not individually essential, for CNBP biochemical activities,whereas the RGG box is essential for RNA–protein binding and nucleic acidchaperone activity. Removal of the RGG box allowed CNBP to preserve aweak single-stranded-DNA-binding capability. A mutant mimicking thenatural N-terminal proteolytic CNBP form behaved as the RGG-deletedmutant. By gain-of-function and loss-of-function experiments in Xenopusembryos, we confirmed the participation of CNBP in neural crestdevelopment, and we demonstrated that the CNBP mutants lacking theN-terminal region or the RGG box alone may act as dominant negatives invivo. Based on these data, we speculate about the existence of a specificproteolytic mechanism for the regulation of CNBP biochemical activitiesduring neural crest development.