Nora B Calcaterra | PubMed Health

PubMed2006

In vitro embryonic developmental phosphorylation of the cellular nucleic acid binding protein by cAMP-dependent protein kinase, and its relevance for biochemical activities.

The zinc-finger cellular nucleic acid binding protein (CNBP) is a strikingly conserved single-stranded nucleic acid binding protein essential for normal forebrain formation during mouse and chick embryogenesis. CNBP cDNAs from a number of vertebrates have been cloned and analysed. CNBP is mainly conformed by seven retroviral Cys-Cys-His-Cys zinc-knuckles and a glycine/arginine rich region box. CNBP amino acid sequences show a putative Pro-Glu-Ser-Thr site of proteolysis and several putative phosphorylation sites. In this study, we analysed CNBP phosphorylation by embryonic kinases and its consequences on CNBP biochemical activities. We report that CNBP is differentially phosphorylated by Danio rerio embryonic extracts. In vitro CNBP phosphorylation is basal and constant at early embryonic developmental stages, it begins to increase after mid-blastula transition stage reaching the highest level at 48 hours postfertilization stage, and decreases thereafter to basal levels at 5 days postfertilization. The cAMP-dependent protein kinase (PKA) was identified as responsible for phosphorylation on the unique CNBP conserved putative phosphorylation site. Site-directed mutagenesis replacing the PKA phospho-acceptor amino acid residue impairs CNBP phosphorylation, suggesting that phosphorylation may not only exist in D. rerio but also in other vertebrates. CNBP phosphorylation does not change single-stranded nucleic acid binding capability. Instead, it promotes in vitro the annealing of complementary oligonucleotides representing the CT element (CCCTCCCC) from the human cellular myelocytomatosis oncogene (c-myc) promoter, an element responsible for c-myc enhancer transcription. Our results suggest that phosphorylation might be a conserved post-translational modification that allows CNBP to perform a fine tune expression regulation of a group of target genes, including c-myc, during vertebrate embryogenesis.

Amino Acid Sequence↗

PubMed2004

Zebrafish cellular nucleic acid-binding protein: gene structure and developmental behaviour.

Here we analyse the structural organisation and expression of the zebrafish cellular nucleic acid-binding protein (zCNBP) gene and protein. The gene is organised in five exons and four introns. A noteworthy feature of the gene is the absence of a predicted promoter region. The coding region encodes a 163-amino acid polypeptide with the highly conserved general structural organisation of seven CCHC Zn knuckle domains and an RGG box between the first and the second Zn knuckles. Although theoretical alternative splicing is possible, only one form of zCNBP is actually detected. This form is able to bind to single-stranded DNA and RNA probes in vitro. The analysis of zCNBP developmental expression shows a high amount of CNBP-mRNA in ovary and during the first developmental stages. CNBP-mRNA levels decrease while early development progresses until the midblastula transition (MBT) stage and increases again thereafter. The protein is localised in the cytoplasm of blastomeres whereas it is mainly nuclear in developmental stages after the MBT. These findings suggest that CNBP is a strikingly conserved single-stranded nucleic acid-binding protein which might interact with maternal mRNA during its storage in the embryo cell cytoplasm. It becomes nuclear once MBT takes place possibly in order to modulate zygotic transcription and/or to associate with newly synthesised transcripts.

Alternative Splicing↗