Identification Of Candidate Proteins Interacted With NLRP2 And The Research On Its Function In Early Mouse Embryos Development

In the previous research,NLRP2 protein complex was extracted from mouse ovaries and loaded by SDS-PAGE gel electrophoresis,then the differential bands were cutted following fast silver staining and carried out mass spectrometry.Four potential proteins were selected and further verified by co-immunoprecipitation and western blot.MYH11 and RPS3 protein were showed that they might interact with NLRP2 protein.In this study,Myh11,Rps3 and Nlrp2 expression plasmids were constructed and transfected into 293T cell.Co-immunoprecipitation and western blot were carried out and further validated the candidate proteins interacted with NLRP2.Then the function of candidate proteins interacted with NLRP2 was investigated in the early embryonic development by real-time quantitative PCR,immunohistochemistry,western blot,immunofluorescence and RNA interference.Result:RNA was extracted from mouse ovaries after Myh11,Rps3 and Nlrp2 primers were designed.cDNA was synthesized and CDs domain of Myh11,Rps3 and Nlrp2 were amplified by RT-PCR.Myh11,Rps3 and Nlrp2 eukaryotic expression vectors were successful constructed following enzyme digestion,fragment purification,connection,transformation,bacterial culture,plasmid minipreparation,restriction enzyme digestion and sequencing.The three expression plasmids were transfected into 293T cell respectively,in order to confirm whether these recombinant plasmids enable to express the corresponding protein.The results show that MYH11,RPS3 and NLRP2 proteins were expressed by Myh11,Rps3 and Nlrp2 eukaryotic expression vectors,respectively.To further determine whether NLRP2 and MYH11 or RPS3 proteins physically interact in the murine model,two recombinat plasmid(Nlrp2 and Myh11,Nlrp2 and Rps3)were transfected into 293T cell,then incubated 48h and confirmed by co-immunoprecipitation and western blot.The results show that MYH11 was not immunoprecipitated by anti-NLRP2 antibodies and was also detected by western blot using anti-MYH11 antibodies.A reciprocal experiment using antibodies raised against NLRP2 to immunoprecipitate MYH11 in whole ovarian lysates was also performed.NLRP2 was not detected by western blot using anti-NLRP2 antibodies,suggesting that there is no interaction between NLRP2 and MYH11 protein.Likewise,RPS3 was immunoprecipitated by anti-NLRP2 antibodies and was also detected by western blot using anti-RPS3 antibodies.A reciprocal experiment using antibodies raised against NLRP2 to immunoprecipitate RPS3 in whole ovarian lysates was also performed.NLRP2 was detected by western blot using anti-NLRP2 antibodies,implying that NLRP2 interacts with RPS3 protein.Moveover,NLRP2 and RPS3 protein co-localizd in mouse oocyte and early embryos by immunofluorescence and confocal microscopy and further validated the interaction between NLRP2 and RPS3 proteins.The aim of the present study was to analyze the expression and localization of RPS3 and its function in early embryonic development in the mouse.RPS3 protein were expressed in multiple mouse tissues.In the ovary,RPS3 protein was mainly expressed in oocytes and granulosa cells.After ovulation and fertilization,Rps3 mRNA and protein were detected in oocytes and preimplantation embryos.Furthermore,RPS3 protein was mainly localized in the cytoplasm of oocytes and preimplantation embryos.Using RNA interference,we knocked down Rps3 transcription specifically in mouse zygotes.The maternal depletion of Rps3 in zygotes led to early embryonic arrest.Conclusions:Myh11,Rps3 and Nlrp2 eukaryotic expression vectors were successful constructed and further confirmed the physical interaction between NLRP2 and RPS3 proteins.Furthermore,knockdown of Rps3 in zygotes led to early embryonic arrest in the mouse.The study have uncovered the physiological function of NLRP2 complex in the molecular level,provided information for elucidating the mechanism of early embryo development in mammals.