Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains

ARMAS, PABLO; AGÜERO, TRISTAN; BORGOGNONE, MARIANA; AYBAR, MANUEL; CALCATERRA, NORA

Buenos Aires, Argentina

Congreso; IV International Meeting of the Latinamerican Society of Developmental Biology; 2008

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 G-rich single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. Through these biochemical activities CNBP may be involved in gene expression control of several putative targets at the transcriptional and translational levels. 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’s biochemical activities and biological functions has still not been addressed. Here, we describe the generation of site-directed CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. These mutagenesis approaches generated: (i) amino acid replacements 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. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. CNBP function during the early steps of neural crest development was analyzed in vivo by gain-of-function and loss-of-function experiments. Xenopus laevis embryos were microinjected with mRNAs coding for wild-type and different CNBP mutant forms and subsequently analyzed by whole-mount in situ hybridization of foxD3 and c-myc genes. The expanded expression territories of these neural crest marker genes after the overexpression of CNBP confirm its participation in the early steps of neural crest development. The reduced expression areas of these neural crest marker genes after the overexpression of CNBP mutants lacking the N-terminal region or the RGG box alone demonstrate that they may act as dominant negatives in vivo. The co-injection of the mRNAs coding for wild-type and CNBP mutants rescued the of the loss-of-function effect caused by the overexpression of CNBP mutants alone, confirming the specificity of the previous results. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP functionality during neural crest development.