Def Defines A Novel Nucleolar Pathway Leading P53and Δ113p53/Δ133p53to Ubiquitination-independent Degradation

Tumor suppressor gene p53is subject to exquisitely fine regulation and acts as a transcription factor to control or regulate the expression of thousands of genes. Regulation of p53turnover is essential to keep p53activity under control in a normal cell. Multiple E3ligases have been identified to mediate p53degradation through the ubiquitination-26S proteasome pathway. Besides, the free20S proteasome is found to mediate p53degradation by default in an ubiquitination-independent manner, and this process can be inhibited by NQO-1in the presence of high concentration of NADH. However, so far very little is known regarding p53turnover through any proteasome-independent pathway(s).△113p53/A133p53are N-terminal truncated p53isoforms whose transcriptional expression is initiated by an alternative p53promoter which is totally p53-dependent. Overexpression of A113p53/△133p53functions to antagonize p53-induced apoptosis. Also, nothing is known about the regulation of the turnover of△113p53/△133p53. Liver development is coordinated by dozens of spatiotemporal factors, including the signaling molecules from mesoderm and endoderm, as well as the liver specific factors. Though lots of works have been carried on, the molecular and cellular mechanism involved in early liver development is still poorly understood.In this study, we first confirmed that both zebrafish and human Def are localized in the nucleolus, and Def affected18S but not28S pre-rRNA processing in zebrafish. Loss-of-function of def in zebrafish defhi429mutant accumulated p53and△113p53protein in the nucleoli. Further studies showed both zebrafish and human Def selectively triggered the degradation of p53and its isoform△113p53/A133p53. More importantly, Def-mediated p53/△113p53/A133p53degradation was dependent on the activity of a specific cysteine proteinase Calpain3(CAPN3) rather than through the ubiquitination-26S proteasome pathway. Def and CAPN3could form a complex, and maximize their effect on p53/△113p53/A133p53degradation in nucleolus though a mutually dependent manner. Knockdown of Def or CAPN3expression significantly increased the endogenous p53protein level, and the elevated p53was not brought down by concomitantly knockdown of either RPL5or RPL11. The Def-CAPN3pathway played an important role in protecting embryos from the p53-induced survival reduction, apoptosis and transcriptional activation.Including the function of promoting p53/A113p53/A133p53degradation in nucleolus together with CAPN3, Def also regulated liver development in zebrafish through CAPN3a. This indicated that Def executed its multiple functions via different genes and pathways.All together, our findings define a novel nucleolar pathway Def-CAPN3-p53/△113p53/A133p53regulating p53/△113p53/△133p53protein level, as well as Def-CAPN3a pathway regulating zebrafish liver development. Also, Def as a nucleolus factor affects18S but not28S pre-rRNA processing in zebrafish. Our studies reveal how the new nucleolus factor Def coordinates certain intracellular protein level and liver development through distinct pathways. The results will no doubt advance our understanding of the function of nucleolus,as well as the development of endoderm including the digestive organs.