Construction Of HRSP12 Lentiviral Vector, Functional Study And Identification Of RYBP Nuclear Localization Signals

Heat-responsive protein12(HRSP12) has been first purified from the5%perchloric acid-soluble fraction of goat liver in1995. Studies have showed that it belongs to the new hypothetical protein family--YER057c/YIL051c/YjgF, which is highly conserved across the world range from prokaryotes to eukaryotes. Previous studies have indicated that the expression of HRSP12is tissue specific and the protein has been found to be most abundant in liver and kidney. As a kind of ribonuclease, HRSP12can cut the mRNA and inhibit the synthesis of protein, eventually inhibit cell proliferation. Growing data have showed that HRSP12is involved in the tumorigenesis, for example, HRSP12can inhibit the growth of mammary carcinomas induced in female Sprague-Dawley rats by DMBA. Latest studies have found that HRSP12was significantly down-regulated in most of hepatocellular carcinoma tissues compared with adjacent nontumor tissues at both mRNA and protein levels. Therefore, we hope to understand the molecular mechanism of HRSP12in tumorgenesis and apoptosis induction by further research.RYBP (Ring1and YY1Binding Protein) is a kind of PcG related protein, mainly participated in the construction of PRC1(Polycomb repressive complex1). RYBP is a multifunctional protein, broadly involved in epigenetic regulation and ontogeny of embryo, eye and central nervous system. Apart from this, RYBP also promoted apoptosis of cancer cells in cytoplasm and nucleus by interacting with apoptosis regulating proteins, such as FADD, pro-caspase8/10, Hippi, apoptin and DEDD. Other studies had demonstrated that RYBP could interact with MDM2and decrease MDM2-mediated p53ubiquitination, leading to stabilization of p53and an increase in p53activity, function as a tumor suppressor.Previous studies found that RYBP mainly located in the nucleus, and distributed a small amount in the cytoplasm. As a multifunctional protein, the localization of RYBP in the cell is of great significance. Firstly, we constructed the lentiviral vector pWPI-HRSP12, harvested the recombinant virus particles and infected Cervical Carcinoma Cell Line HeLa, and used Western blot to analyze the expression of Flag-HRSP12. It was showed that the lentiviral particles infected HeLa cells with high efficiency and the expressed fusion protein flag-HRSP12was also detected. In addition, the over-expressed HRSP12lead to the increase of cleaved-Caspase3in HeLa cells. What’s more, the over-expressed HRSP12can inhibit the proliferation of HeLa cell. The results showed that over-expressed HRSP12may induces HeLa cells apoptosis and inhibits their proliferation, and these will lay a solid foundation for the study of biological functions of HRSP12in the future.Secondly, according to the sequences characters of RYBP, we constructed a series of truncated deletion mutants and fused them to enhanced green fluorescent protein. Through transfection of them to some kinds of cell and fluorescence microscopy analyses, we were able to map the NLS region-6KKSPTRPKR14、47RKGTSTRKPR56and74PPPKKEKKEKVEKQDK89.Then we used site-directed mutagenesis and constructed the full-length mutant of RYBP which predominantly located in the cytoplasm, named it as RYBPmut. After analysis the function of mutant RYBPmut, we found that compared to RYBP, RYBPmut has a better ability to stabilize p53.But the detailed mechanism needs further research.In conclusion, we successfully constructed pWPI-HRSP12lentiviral vector and, for the first time, identified the Nuclear localization signal of RYBP, constructed the cytoplasm-located mutant RYBPmut, found the functional difference between RYBP and RYBPmut. They will allow us to gain further insight into the molecular of HRSP12and RYBP.