Isolation And Identification Of Pura-related Protein Complex And Preliminary Study On The Function Of Pura

When normal cells lost the control of differentiation,they might lose the normal physiological functions and might become into tumor cells.Therefore the study of cell differentiation mechanism is critical for the cell carcinoma research.In our previous studies,the histone methyltransferase,SET9,can physical interact with Pura.Because the histone methylation is important in cell differentiation,the study of the relations between Pura and the histone mehtylation has great meaning.Pura is a ubiquitous multifunctional protein strongly conserved throughout evolution,binding to the single-strand or double-strand DNA(or RNA)sequences specifically.Pura is a major player in the DNA replication,mRNA tansport,mRNA translation,cell cycle,DNA repair,and the cell carcinoma.Pura binds to several cellular regulatory proteins.The multi-functions of Pura are closely related with the component of its interacting proteins.Current research shows that Pura binds to several cellular regulatory proteins including the Rb,E2F-1,Spl,YB1,cyclinTl/Cdk9 and cyclinA/Cdk2.These proteins play important roles in the cell cycle and transcription regulation.Therefore,the research for the interaction proteins of Pura is important for revealing the mechanism of the functions of Pura.In this work,we constructed N-TAP-PURA vector,which can express N-terminal tandem affinity purification(TAP)tag Pura fusion protein in mammalian cells.By using TAP experiment and Mass spectrometry,we analyzed the components of Pura protein complex.In our experiments,38 proteins were identified,including Pura itself.These proteins can be divided different categories according to their functions.The first group includes 12 species of RPs(ribosomal proteins),8 hnRNPs,2 Splicing factors,2 Polyadenylate-binding proteins,and two other RNA binding protiens.These proteins are believed to be involved in mRNA transport and translation.The second group includes the proteins that play important roles in DNA repair.There're 6 helicases,and three proteins also participated in the DNA repiring process.The Ras GTPase-activating-like protein IQGAP1,which was believed to be involved in cell proliferation and differentiation,was also found.These proteins are all closely related with the function of Pura.There are no reports of special research on Pur?-related protein complex now,and the cell lines we used are different.Therefore our results have great meaning.In this work,the Pura down-regulated 293T and Hela cell lines was also constructed,by using RNAi technology.The effects of Pura on the cell viability versus MMS were also studied with MTT assay.Results showed that down-regulated Pura didn't affect the Hela cell growth rate apparently,but it made the sensitivity of Hela cells to MMS slightly enhanced.This result consisted with the function of Pura in DNA repair,also provided new clues on the further research for Pura function.