Zebrafish P53 Protein And Its Mrna Interaction Studies

The tumor suppressor p53 gene, which is an important factor involved in the occurrence and development of cancer, plays a key role in regulating the cell cycle, cell growth, and development. More than half of human malignancies are due to a mutation or functional disorder of the p53 gene. Under normal conditions, the expression of p53 protein is maintained at low levels through the ubiquitin degradation pathway. In response to various cellular stresses, such as DNA damage, hypoxia, or nutrient deprivation, the tumor suppressor gene produces p53 protein as a sequence-specific transcription factor to regulate expression of a series of target genes involved in governing cell cycle arrest, apoptosis, and DNA repair. The expression of p53 protein is regulated at different levels, including transcriptional regulation, translational regulation, and post-translational regulation.Translational regulation of p53 is an important mechanism involved in the response to various stresses. Studies have shown that p53 protein can bind with the 5'UTR or 3'UTR region localized at its cognate mRNA and repress its translation. The UTRs of both human and murine p53 are predicted to form stable stem-loop structures that are capable of inhibiting translation. Moreover, some protein factors may interact with the 5'UTR or 3'UTR domain of p53 mRNA, resulting in enhancement or decreasing in p53 translation. HuR and RPL26 protein have been shown to bind with the 3'UTR of p53 mRNA and regulate it by enhancing translation or increasing its stability. This type of regulation can protect cells or organisms against damage or abnormality.p53 protein is an important regulation factor that can bind to p53 mRNA to regulate its translation in human and murine. To determine if a similar interaction exists in zebrafish and if the interaction affects zebrafish development, we cloned and expressed p53 protein from zebrafish in Escherichia coli. The binding activity of p53 protein and its mRNA was determined by UVcross-linking and immunoprecipitation: RT-PCR analysis. And then the p53mRNA was cloned into the psiCHECK-2 vector, between the XhoI and Not I sites, immediatedly 3'downstream from the Renilla luciferase gene. The constructed luciferase reportor and eukaryotic expression vector for p53 were cotransfected into cells or were microinjected into zebrafish embryos. The luciferase activities were measured with Dual luciferase reporter system assay. Our results demonstrated that p53 protein could enhance luciferase translation by binding to its mRNA. Further experiments were carried out, immunoprecipitation: RT-PCR, microinjection of zebrafish showed that 53 protein could specifically bind to its own mRNA in zebrafish embryos. This interation play important role in zebrafish development.Our result demonstrated that p53 protein could specifically bind to its own mRNA in zebrafish embryos. p53 protein could enhance mRNA translation in cells and in zerafish embryos under stresses. This interatction may be an important protective mechanism for zebrafish development facing stresses. Our result also provided evidences that zebrafish may be developed as useful model for studying the anti-tumor agents targeting p53 signal pathway.