Modern Molecular Biology Techniques Analyze The Expression And Function Of Mammalian PUF Protein In Reproductive Development

The members of PUF family are conserved RNA binding proteins,and playing an important role in post-transcriptional regulation of eukaryotic cells.PUF homologs are crucial regulators during development of germline and gamete both in various animals.At present,there are many researches about Drosophila Pumilio and Caenorhabditis elegans FBF,but little study about expression of PUF proteins in mammals.The function of PUF proteins is not clear during mammalian growth and development.In this paper,we choose mice as experimental model and study expression of Puml and Pum2 in mouse tissues and various time points of postnatal testis development at mRNA and protein levels through Real time-PCR and Western Blot.The results show that Puml and Pum2 are expressed both in many tissues and different ages of mouse testes.We also analyze localization of Puml and Pum2 in high expression tissues from cell angle through Immunohistochemistry(IHC).RNA Binding Protein Immunoprecipitation(RIP)is a method that can find specific mRNAs interacting with PUF protein from biochemical level.The mRNAs pulled down by PUF antibodies may be target mRNAs of Puml and Pum2,the results of Real time-PCR and Western Blot are satisfactory.The main content and results are as following:1.Puml expression in mouse tissues and various time points of postnatal testis development was studied by Real-time-PCR and Western Blot analysis.Theresults displayed Puml were expressed both in many tissues and different ages of mouse testes(postnatal 3rd day to adult),and Pum1 expression level is highest in the testis,It suggested that Puml involves in mouse spermatogenesis.The result of IHC demonstrated that Pum1 is located in testicular spermatogonium and spermatocyte,ovarian granulosa cells of primary oocyte,secondary oocyte and corpus luteum,dermatic epidermis,hair follicle and sebaceous glands,and Puml protein was detected in cytoplasm of these cells.Human PUM1 was also high expressed in the testis,and located in testicular spermatocyte cytoplasm(73 years old man).2.Pum2 expression in mouse tissues and various time points of postnatal testis development was studied by Real-time-PCR and Western Blot analysis.The results were similar to expression of Puml,It displayed Pum2 were expressed both in many tissues and different ages of mouse testes(postnatal 3rd day to adult),and Pum2 expression level is highest in the testis,suggesting that Pum2 involves in mouse spermatogenesis.The result of IHC demonstrated that Pum2 is located in testicular spermatogonium,spermatocyte,round spermatid,elongated spermatid and sperm,ovarian ootid,granulosa cells of primary oocyte,secondary oocyte and corpus luteum,dermatic epidermis,hair follicle and sebaceous gland,and Pum2 protein was detected in cytoplasm of these cells.Human PUM2 was also high expressed in the testis,and located in cytoplasm of testicular spermatogonium and spermatocyte(73 years old man).3.mRNAs associated with Puml and Pum2 were studied in mouse testes by RNA Binding Protein Immunoprecipitation analysis,and the results of RT-PCR analysis indicated that Ypel3,Cdk1,Cdk2,cyclinE2,Mdm2,Piasl and Pias2 were enriched in Puml RIP while Ypel3,y Cdkl,Cdk2,cyclinE2,Map2k3,Piasl,Pias2 and p27.were enriched in Purn2 RIP.Therefore,these mRNAs may be relevant targets of Puml and Pum2.Western Blot analysis about relationship of Puml,Pum2 and other relevant proteins showed that Pxm2 and p27 were upregulated in Pum1-/-testis,and p27 was upregulated while Puml was not significantly changed in Pum2-/-testis.Thus,the fuction of PUF family maybe regulate p27 expression.