Phenotype Analysis Of Pum1 Knockout Mice On C57B6 Background

PUF family proteins are the highly conserved RNA-binding proteins that function as developmental regulators in animals.PUF proteins play important roles in post-transcriptional control through binding to the 3'UTR of target mRNAs to regulate mRNAs stability and translation.PUMILIO is a major branch of the PUF family and there are two members,Pumiliol(Puml)and Pumilio2(Pum2),in human and mouse.Recent studies in mice have shown that Puml played important roles in spermatogenesis,embryonic development and inhibition of SCA1-like neurodegener-ation and Pum2 was essential for maintaining the normal function of the nervous system of mice.Puml and Pum2 resisted virus infection in cells,but also promoted hematopoietic stem cells and human leukemia cell proliferation.In addition,PUM2 also had a positive effect on the proliferation of human adipose derived stem cells.Moreover,the PUF family proteins bound to interacting proteins and formed protein complexes that together maintained normal biological processes.Given that Puml knockout mice in mixed background were fertile and largely normal,we decided to generate mutant mice in pure background and hoped to uncover novel functions of Puml.Unexpectly,the birth rate of Puml homozygous mice was only 2%to 2.5%indicating that Puml deletion led to embryonic lethality.Unlike in mixed background,Puml null mice were infertile,exhibiting significantly reduced sperm counts,little sperm motility and abnormal sperm,similar to the phenotype of oligoasthenoteratozoospermia(OAT)in infertile men.Loss of Puml resulted in multiple sperm entanglement and sperm release was also delayed.Puml was necessary for the normal formation of mitochondria sheath in sperm tail using TEM.Moreover,absence of Puml led to abnormal formation of sperm head during shaping from Step9 of spermatid development.To investigate possible molecular mechanism,we chose mitochondrial outer membrane pore protein-VDAC2,which contained PUM binding motif.We found that VDAC2 was the candidate target of Puml.Skin inflammation were discovered in 5 to 6 month-old Puml null mice,and results of bone marrow transplantation from affected mice showed that skin lesions were not due to hematopoietic stem cell development defects.In order to determine whether Puml affected hematopoietic stem cell proliferation in vivo,bone marrow transplantation experiments were performed,and results indicated that Puml was not necessary for the proliferation and differentiation of hematopoietic stem cells.Analysis on the role of Puml in adipogenesis in adult Puml null mice indicated that white fat was significantly reduced and the size of adipocyte decreased significantly suggesting that Puml affected lipid storage which needs further verification.NIH3T3 and DU 145 cells were used to study proteins associated with PUM1 in mammals.PUM1 interacted with NDFIP2 proteins through immunoprecipitation and protein mass spectrometry analysis.In summary,we concluded:Firstly,loss of Puml resulted in some mouse embryonic lethality.Secondly,Pum1 was essential for spermiogenesis and maintenance of fertility.Thirdly,skin lesions in Puml null mice were not caused by hematopoietic stem cell development defects and Puml was not essential for hematopoietic stem cell proliferation and differentiation in vivo.Fourthly,Puml had an important role in the development of adipocytes.In addition,NDFIP2(NEDD4 family-interacting protein 2)was a candidate interacting protein of PUM1.