Expression And Purification Of RNA Processing Factor CPSF6 And Functional Study Of PQBP1 In Spliceosome Assembly

Alternative polyadenylation(APA)is the process that different poly(A)sites in the3'-untranslated region(3' UTR)of the precursor mRNAs are chosen for cleavage and polyadenylation,generating multiple mRNA subtypes with various lengths.Regulation of APA requires the cooperation of numerous cis-acting elements and trans-acting factors.The cleavage factor I(CFIm)complex,which is one of the key determinants of poly(A)sites' selection,recognizes the UGUA motif upstream of the poly A site and recruits other processing factors.In order to further study the function of the CFIm complex,we planned to recombinantly express CPSF6(CFIm68),one of the key components of the CFIm complex.However,human CPSF6 protein contains a special proline-rich region and failed to express in E.coli expression system.To overcome this difficulty,a Bac-to-Bac baculovirus expression system was constructed to express CPSF6 protein in insect cells.After infection with the baculovirus containing exogeneous CPSF6 gene,Sf9 cells successfully expressed full-length His-CPSF6 fusion protein that was detected by western blot analysis.The His-CPSF6 fusion protein was further purified through renaturation and dialysis process.The resulting high-purity proteins were tested in a GST pull-down experiment,and it specifically bound to its binding partner CPSF5,indicating that the recombinant CPSF6 retains its biological activity and can be used for subsequent biochemical analysis.Polyglutamine binding protein 1(PQBP1)is the causative gene of X-linked intellectual disability.It can bind to multiple splicing factors and participate in the regulation of pre-mRNA alternative splicing,however,the underlying molecular mechanism remains unknown.According to research papers and our laboratory's previous studies,PQBP1 may be a component of the NTC complex,which is involved in spliceosome activation.This study first explored the interaction between PQBP1 and the NTC complex core component PRPF19 through in vitro pull-down assay.To further explore the relationship between PQBP1 and different spliceosome complexes,we constructed an mRNA splicing substrate containing three MS2 stem-loops,and prepared nuclear extracts from He La cells,which can be used to isolate splicing complexes at different stages in vitro.Results showed that the splicing substrate binds to the protein complex in the nuclear extract and can be separated by MBP-MS2 binding protein.This study successfully constructed an in vitro system for spliceosome assembly and separation,and laid a solid foundation for future investigations on how PQBP1 affects the assembly of spliceosomes.