Study Of Function And Mechanism Of RNA Binding Protein GPKOW In Human Pre-mRNA Splicing Process

Background: Pre-mRNA splicing is a dynamic process through which snRNPs andtransacting proteins interact in an ordered manner in the spliceosome to removeintrons from pre-mRNA. Splicing of mRNA precursors comprises a series ofATP-dependent steps in which DExD/H-box proteins play a critical role. HumanDHX16is a DEAH-box protein associated with the spliceosome before the firsttransesterification reaction of pre-mRNA splicing. To further understand the functionof DHX16, we screened for its interacting proteins using yeast2-hybrid assays. Weidentified GPKOW as an interacting protein of DHX16, which contains a glycine-richdomain called G-patch as well as two KOW domains found in some RNA processingproteins. However, the role of GPKOW in splicing is not clear. We hypothesized thatGPKOW regulates or modulates the activity of DHX16in the splicing process.Methods: pET28a(+)-GPKOW-wild type (in frame) was constructed, which is fusedto His-tag at N-terminal. By using this pET28a(+)-GPKOW as a template, the N-terdeletion (Nd), C-ter deletion (Cd) and N-ter and C-ter double deletion (NC), themiddle part deletion of GPKOW (M) were amplified by PCR. All those PCR productswere then inserted into pET28a(+) vector as His-tag fusion proteins. The GW mutatedin G-patch domain and GK mutated in KOW1domain were introduced into GPKOWby using a site-directed mutagenesis kit. Recombinant proteins were expressed inE.coli Rosetta (DE3) pLysS cells and purified by cobalt affinity resins and poly (U)sepharose chromatography. GPKOW-RNA interaction was studies by Northwesternand EMSA assay. GPKOW-DHX16interaction was confirmed by pull-down andco-immunoprecipitation assay. The splicing defect caused by DHX16dominantmutant was introduced as a model to analyze GPKOW regulating DHX16functionduring splicing process by RT-PCR. GPKOW was associated with pre-mRNA,intermediates splicing products and snRNAs by anti-GPKOW co-immunoprecipitation and northern blot. GPKOW was immunodepleted fromHeLaS3nuclear extract by anti-GPKOW antibody, which causes splicing defect by invitro splicing assay and complex Bactaccumulation by spliceosomal complex analysis.RESULTS1. A database search with human GPKOW identified apparent orthologues in Xenla(Q6NU07), Zebrafish (Q90X38) and Mouse (Q56A08) which exhibited high degreeof homology with human GPKOW.2. pET28a(+)-GPKOW, EGFP-C1-GPKOW and truncated or point mutants wereconstructed and confirmed by restriction enzyme analysis and DNA sequencing.3. His6-GPKOW purified by cobalt affinity resins and poly U sepharosechromatography is pure without background proteins.4. By monitoring the nuclear and cytosolic protein fractions, DHX16and GPKOWare present in nucleus by Western blot; further, the EGFP fused DHX16or GPKOWshows the nuclear localization under microscopy.5. GPKOW can bind with U2, U4, U5, and U6snRNAs and splicing subtract pRG1by Northwestern blot analysis and EMSA assay. GK mutated in KOW1domain hadless RNA-binding activity than WT and GW mutated in G-patch domain.6. GPKOW-DHX16interaction was confirmed by pull down andco-immunoprecipitaton assay in vitro. GPKOW-GW lost the ability to bind withDHX16. Following RNase A treatment of the lysate, Flag-DHX16and GFP-GPKOWwere still co-immunoprecipitated. Co-immunoprecipitation at increasing saltconcentrations was performed to monitor the strength of interaction. GPKOW andDHX16can interact in0.15M NaCl and is dissociated with DHX16when NaClconcentrations were increased to0.3M or0.5M during binding reaction.7. The Pre-mRNA/mRNA ratio of BRD2and DNAJB1as well as minigene showedobvious decrease in cells with DHX16domain mutant and GPKOW overexpressionby RT-PCR, compared with MMS-treated cells with GPKOW overexpression.8. Overexpression GPKOW-GK or GW did not impair the splicing. GPKOW-GKfailed to rescue the splicing defect by DHX16-SL and GPKOW-GW can partiallyrescue but can't get to the level that GPKOW-WT did. 9. Anti-GPKOW serum was generated Anti-GPKOW serum was generated bysubcutaneous injection of New Zealand female rabbit using His6-GPKOW antigen.10. Anti-GPKOW can co-immunoprecipitate pre-mRNA, intermediates splicingproducts and snRNAs by co-immunoprecipitation and northern blot assay.11. GPKOW was depleted from HeLaS3by anti-GPKOW serum, which rendered theextract unable to support splicing by performing a kinetic in vitro splicing and led tocomplex Bactaccumulation. Addition of increasing His6-GPKOW to GPKOWimmunodepletion nuclear extract rescued splicing.12. Different amount of GPKOW-WT and mutant proteins–GW,-GK were added tothe GPKOW immunodepletion nuclear extract. GW can restore splicing to the levelsthat GPKOW WT did, but GK can not.Conclusion:1. pET28a(+)-GPKOW-wild type and truncated and point mutants were constructed.2. Pure His6-GPKOW recombinant protein can be purified by the two-step method ofcombining cobalt affinity resins with poly U sepharose chromatography.3. GPKOW is nuclear protein and localizes in the nucleus.4. GPKOW-WT and-GW mutant interact directly with snRNAs and pre-mRNA invitro, but GK mutants has less binding efficiency5. G-path domain of GPKOW interacts with DHX16and GPKOW-DHX16interaction is RNA independent and sensitive to salt.6. GPKOW-WT and-GW mutant suppresses DHX16mutant-caused splicing defect,but GPKOW-GK mutants does not.7. GPKOW is an essential splicing factor and associated with spliceosome.8. GPKOW palys a role during spliceosomal complex Bactto complex C.9. GPKOW modulates the function of DHX16during pre-mRNA splcing processthrough KOW1domain.