CNBP: a single-stranded nucleic acid binding protein that may be involved in Neural Crest Development

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

Los Cocos, Córdoba, Argentina

Workshop; 1st International Workshop “Latest concepts in Developmental Biology; 2006

Society of Developmental Biology

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein widespread and strikingly conserved among vertebrates, which biological function is not completely known. It consists of seven CCHC zinc knuckles and an RGG box between the first two zinc knuckles. Main activities assigned to CNBP range from transcriptional regulation to translational control during vertebrates development. Recently, it was reported that homozygous mutant mice CNBP-/- are lethal and unable to develop normal forebrain structures, while heterozygotes present various craniofacial defects, may be due to miss-regulation of c-myc proto-oncogene expression. We used Bufo arenarum CNBP (bCNBP) to achieve information about its developmental nucleic acid binding capabilities and biochemical and molecular activities. Embryonic bCNBP bound in vitro to ssRNA and ssDNA probes that represent developmentally regulated 5’UTR sequences, including Xenopus c-myc 5’UTR, showing a differential pattern depending on the developmental stage, specially around the mid-blastula transition (MBT). CNBP also showed nucleic acid chaperone activity in assays that analyze the annealing promotion of complementary oligonucleotides in vitro. Furthermore, by means of site directed mutagenesis, we identified the major motif involved in these biochemical activities as the RGG box. Taking advantage of the high level of phylogenetic conservation of CNBP among vertebrates, and considering the fact that bCNBP is 99.4% identical to its Xenopus laevis homologous xCNBP, we decided to test CNBP in vivo function by overexpression of mRNA coding for wild type and mutant bCNBPs. Microinjected embryos were analyzed by in situ hibridization using probes for c-myc proto-oncogene and FoxD3 gene, both involved in neural crest development. Microinjection with wild type bCNBP mRNA caused a marked increase of c-myc and FoxD3 expression, while microinjection with deletion mutant bCNBP mRNAs, including those lacking RGG box, caused a clear decrease of the expression of these markers. Although these results do not completely explain CNBP molecular mechanisms of action, nor its hierarchy, they suggest that CNBP may be exerting some regulative activity on genes involved in neural crest inductive cascade, acting perhaps as a nucleic acid chaperone by binding, remodelling and stabilizing nucleic acids secondary structures.