Interaction Of Prmt5 And Mep50 In Medaka

Prmt5 is a type ? protein arginine methyltransferase,Prmt5 has been shown to be involved in many biological processes such as cell-cycle,RNA processing,germ cell development,and oncogenesis.Mep50 is the cofactor of Prmt5.Mep50 directly binds Prmt5 and greatly enhances the methyltransferase ability of Prmt5.The analyses of the Prmt5-Mep50 complex had been done in human and In this study,we focus on the interaction of Prmt5 and Mep50 in medaka.At first,we have analyzed the expression of mep50 and prmt5 by in situ hybridization.mep50 and prmt5 were widely expressed during embryonic development of medaka.The signals of mep50 and prmt5 were strong in some tissues,such as the brain,pectoral fin and somite,which indicated that prmt5 and mep50 may have co-expression in medaka.In order to verify the assumption of in situ hybridization,we did co-immunoprecipitation and yeast two hybridization.The result of co-immunoprecipitation confirmed the interaction between Mep50 and Prmt5.But it was failed to detect the combination of the truncated Mep50 mutants with Prmt5 by co-immunoprecipitation.This could be due to the lost of the binding sites.Another possible reason is that the binding sites were encased because of the change in the structure of the tranctated protein.It needs further studies.The results of yeast two hybrid verify the interaction of Mep50 and Prmt5 again.According to the analysis of protein domain,we built a series of truncation and deletion mutants of Mep50 and Prmt5.The results showed that the shorter the Prmt5 was,the weaker the interaction of Mep50 and Prmt5 would be.When the length of the segment was truncated from 270 aa to 203 aa,the interaction of Mep50 and Prmt5 was decreased obviously.This indicated that the binding sites of Mep50 and Prmt5 existed in this region.Yeast two hybrid experiment also showed that the 3rd WD40 domain of Mep50 played a key role in the interaction of Mep50 and Prmt5.Finally,in order to obtain the purified protein for structure analysis of Mep50-Prmt5 complex and antibody production,we subcloned Mep50 and Prmt5 into pET vectors.Both of the proteins could be expressed in E.coli BL21 induced by IPTG.The Mep50 protein had been well purified through Ni-chelating affinity chromatography.The purification result of Prmt5 was bad.This might because the His or GST label could not exposed to chromatography column by the protein folding.The truncated Mep50 and/or Prmt5 were subcloned into pET vectors and were successfully expressed.The next step is to purify truncated Mep50 and/or Prmt5.All the above results were the base for the study of biological function and regulation mechanism of Mep50-Prmt5 complex in many biological processes of fish.