| p53is an important transcription factor and tumor suppressor, whose expression level needs to be strictly monitored and regulated. The low level of p53under normal circumstances in vivo is mainly maintained by the ubiquitination-26S proteasome degradation pathway. Current reports have shown that the regulation of p53by nucleolar factors such as RPL5and RPL11is also through Mdm2-mediated ubiquitination pathway. However, so far little is known about the non-proteasome degradation pathways regulating p53protein levels. Our study shows that a nucleolar factor, Def, can mediate p53degradation through a specific cysteine proteinase CAPN3, rather than through the ubiqutination-26S proteasome pathway. In addition, we also found that the lack of Def will induce p53accumulation in the nucleoli. As being reported, CAPN3is the only cysteine protease which has been found in the nucleoli. So, our study likely defines a new nucleolar turn over pathway for p53. In zebrafish, capn3has two homologous genes, capn3a and capn3b. By using TALEN technique, we have generated capn3a and capn3b mutants, respectively, and these mutant lines are valuable materials for us to study the biological functions of CAPN3in the future.Loss of function of Def not only up-regulates the expression of p53protein and its isoforms, but also affects the development of the digestive organs. But still nothing is known about the influence of Def overexpression on the liver. We generated several independent zebrafish Tg(fabp10a:def) transgenic lines which persistently over-expressed Def specially in the liver. Microarray results showed the remarkable change of gene expression patterns between the adult livers of Tg(fabp10a:def) and wide type fishes. We also found that over-expression of Def will affect18S pre-rRNA processing as well as will disrupt the structural integrity of liver cells and even will induce liver tumors. Therefore, Def expression levels in vivo must be controlled within a specific range. In addition, we also found Def performs its functions through cell-autonomous manner. Finally, we cloned a novel p53isoform in zebrafish,â–³113bp53, which is a splice variant ofâ–³113p53(â–³113p53is an isoform of p53as reported previously).â–³113bp53andâ–³113p53share the same promoter as well as the same transcription start site. Both of them can be induced by the loss of function of Def or Camptothecine treatment or other stresses. Both of them function to anti-apoptosis induced by p53. Furthermore, we foundâ–³113bp53is created due to a deletion of CATT four bases naturally occurred in the wide type zebrafish.In conclusion, in this study we used zebrafish and human cell lines as the research models, and combined various technologies used in molecular biology, genetics, cell biology, developmental biology to study the regulation of p53by the nucleolar protein Def. We defined a novel nucleolar protein degradation pathway through Def-CAPN3, and found that the pathway plays as a key role in maintaining the nucleolar p53homeostasis. This study has also identified a new p53isoform, namelyâ–³113bp53in zebrafish. These findings not only deepen our understanding of the biological functions of the nucleoli and extend the study of the p53pathway, but also provide new clues and theories for understanding the ribosome related human diseases and help us to develop new therapeutic approaches for these diseases. |
