Cellular Nucleic Acid Binding Protein (cnbp) Functional Studies

CNBP encodes a 19 kDa protein containing seven tandem zinc finger repeats of 14 amino acid residues (Cys-X₂-Cys- X₄-His-X₄-Cys), which is also named as zinc finger 9, ZNF9. Human CNBP has two transcripts,αandβ, which encodes 177 and 170 amino acids respectively. The amino acid sequence of CNBP is highly conserved during evolution, especially in vertebrates. This striking conservation, coupled with the fact that homologous genes have been found in various organisms, suggests that CNBP may play an essential biological role across different species. CNBP can interact with DNA and RNA, and exert dual functions in transcriptional and translational regulation. CNBP is widely expressed in different organs, and plays an important role during embryonic development. The biological functions and mechanisms of CNBP have not been clearly elucidated till now.We had cloned the full length cDNA of human CNBP from HeLa cDNA library and found 3 novel transcripts, including CNBPβtranscript variant 1 (coding 171 amino acids), CNBPβtranscript variant 2(coding 172 amino acids), and CNBPαtranscript variant 1 (coding 178 amino acids).Recombinant CNBPβprotein was expressed in E.coli and purified by affinity column. Polyclonal antibody against CNBP was obtained by immuning rabbits.The transcriptional regulation activity of CNBP was examined by Dual-Luciferase reporter assay system. We found that both CNBPαand CNBPβhad remarkable transactivity , and the C-terminus of CNBPβincluding a potential phosphorylation site at Thr165 was important in mediating transactivation. Stable HeLa cell line with suppressed level of CNBP expression by RNAi showed negative trend in cell cycle progression, cell proliferation and colony formation. The results were opposite in cell line with elevated level of CNBP expression. These data indicated that CNBP had a positive role in cell proliferation.We searched CNBPβ-interacting proteins by yeast two-hybrid screening, but no protein was found.We searched CNBP downstream genes by gene-chip from HeLa cells with suppressed level of CNBP expression or not, and 583 differential expressed genes were found. These differential expressed genes were mostly involved in development and translational regulation. We selected 15 candidate genes as downstream genes of CNBP. We found CNBP could positively regulate four candidate genes including CPS1, BMP2, ALPI and SARS, and proved that there might be a cis-acting element in the promoter of ALPI, which was sensitive to the change of CNBP in expression level.In conclusion, we identified 3 novel transcripts of CNBP and firstly charactered it as a transactivation factor, whose transactivity was depended on C-terminal domain of CNBP. We also discovered that CNBP had a positive role in cell proliferation. Candidates of downstream genes provided possible directions for further studies.