Cellular nucleic acid binding protein: analysis of its biochemical activity and behaviour during the early development of Bufo arenarum and Danio rerio

ARMAS, P.; LOMBARDO, V. A.; WEINER, A. M. J.; BOLOGNA, N.; CALCATERRA, N. B.

Guaruja, San Pablo

Congreso; 2nd International Meeting of the Latin American Society of Developmental Biology (LASDB); 2005

Sociedad Latinoamericana de Biología del Desarrollo (LASDB)

Cellular nucleic acid binding protein (CNBP) is widespread vertebrates. This protein shows a striking sequence conservation and general structural organisation. It consists of seven CCHC zinc knuckles and an RGG box between the first two zinc knuckles. We used the Bufo arenarum CNBP (bCNBP) to achieve information about its nucleic acid binding capabilities, possible cellular targets, and biochemical and molecular activities. We found that embryonic bCNBP is able to bind in vitro to RNA and ssDNA probes that represent developmentally regulated 5´UTR sequences. The binding showed a differential pattern according to the nature of the probe and the developmental stage. CNBP binds to RNA as dimer whereas monomer and dimer bind to ssDNA and the embryonic binding capability decreased after the mid-blastula transition (MBT). We also showed that CNBP acts as a nucleic acid chaperone by promoting the annealing of complementary oligonucleotides in vitro. Furthermore, we identified the major motif involved in these biochemical properties as the RGG box. Zebrafish CNBP (zCNBP) was also able to bind to RNA and ssDNA probes, showing a biochemical behaviour similar to that observed for bCNBP. zCNBP contains four putative phosphorylation sites in Ser4, Tyr41, Tyr85 and Tyr145. We determined that CNBP was in vivo and in vitro phosphorylated by zebrafish embryo extracts. The in vitro phosphorylation was differential during early development. Phosphorylation levels were low and uniform during the first stages, reached a maximum between 24 and 48 hours post-fertilisation and decreased afterwards. The phosphorylation of a natural proteolytic CNBP product, which contained the Ser4, allowed us to propose that this amino acid residue is the main embryonic kinase substrate. The phosphorylation increased the protein capability of binding to ssDNA probes in vitro and improved its ability to promote strand annealing of complementary oligonucleotides in vitro. Cellular functions assigned to CNBP range from transcriptional regulation to translational control. The CNBP biochemical activities in addition to their post-translational regulation by phosphorylation may be the basis to understand the diverse ways in which CNBP controls genes and mRNAs expression.