Characterization Of Molecular Mechanism Of A Transcriptional Coactivator-human P100 Protein In Pre-mRNA Splicing.

Objective:Eukaryotic gene expression is a complex stepwise process,in which transcription and pre-mRNA splicing are the key nuclear processes and both of them require multi-component complex to function.Transcription is regulated by protein complex composed of transcription factors,basal transcriptional machinery and a group of coactivators,while pre-mRNA splicing is carried out by spliceosome.Several studies have shown that pre-mRNA splicing occurred coordinately with the synthesization of nascent pre-mRNA.Therefore, transcription and splicing are not independent events,they are functionally coordinated.Co-transcriptional splicing appears as a new concept.Human p100 protein is a novel transcriptional coactivator,which was first identified as a transcriptional coactivator of Epstein-Barr virus nuclear antigen 2(EBNA-2). Since it can directly interact with RNA polymeraseâ…¡(RNA polâ…¡),p100 protein may facilitate the access of other transcription factor to the basal transcription machinery and form the pre-initiation complex of transcription.Thus,it could regulate the transcription in several signaling pathway,p100 consists of Staphylococcal nuclease(SN)-like and Tudor(TD)domains of which the SN-like domains have been shown to function in transcription,but the function of TD domain has remained elusive.However,our previous study showed that it could interact with U5 snRNP specific proteins,such as U5-220K(hPrp8), U5-200K(hBrr2)and U5-116K(hSnu114).p100 TD is homologous to SMN (survival of motor neuron protein)TD domain,which functions in the assembly of snRNP complexes and pre-mRNA splicing process.The purpose of this study was to validate the hypothesis that p100 protein functions in pre-mRNA splicing,and to reveal whether it could participate in the regulation of splicosome assembly.Methods:In this report,we demonstrate a novel function for p100 protein as a protein involves in pre-mRNA splicing and spliceosome assembly.Firstly,in the study of methodology of pre-mRNA splicing in vitro assay,two different splicing methods in vitro,³²P labeled and unlabeled pre-mRNA as the substrates in the reaction were investigated.The radiolabeled splicing reaction products were visualized by autoradiography,while the unlabeled products were observed by Ethidium Bromide(EB)staining.Secondly,the purified p100 protein and its functional domains were prepared by Flag-tag fusion protein purification system and GST(glutathione S transferase)fusion protein system respectively.The lysates of HeLa cells stable expressing Flag-tagged p100 and p100 over-expressed COS-7 cells were incubated with anti-Flag M2 agarose respectively.GST fusion proteins of p100-TD and p100-SN were bound on glutathione-Sepharose 4B beads when incubated with bacterial lysates.After the whole steps of purification,the purified p100 protein and its functional domains were detected by SDS-PAGE (polyacrylamide gel electrophoresis)with silver staining.Thirdly,to investigate the potential function of p100 protein in pre-mRNA splicing,the unlabeled in vitro splicing reaction was carried out.Different amount of purified p100 protein was added in the reaction system;and the time course of splicing reaction with addition of p100 was also examined.RNA interference was performed to examine whether the p100 protein is essential in the effective splicing reaction.The effect of p100 siRNA was examined by western blotting.All the results were analyzed by 8%denatured PAGE with 7M urea,and EB staining afterward.Fourthly,the in vitro splicing assay with radiolabel was carried out for the splicosome assembly in vitro reaction.Native PAGE analysis was performed and visualized by autoradiography and phosphorimager to investigate the roles of p100 protein and TD domain of it in the splicosome assembly.Results:Firstly,according to the comparison of the traditional radiolabeled pre-mRNA splicing assay with the new unlabeled one,the RNA products of in vitro splicing,such as mRNA,lariat-intermediate,etc,could be observed clearly, although there are more unspecific bands in the EB staining splicing assay than ³²P labeled one.Secondly,as a result of the purification of p110 protein,the only purified p100 protein could be obtained only from the p100 over-expressed COS-7 cells. The p100 protein prepared from the p100 stable expressing HeLa cells was found interacting with U5-220K(Prp8)å’ŒU5-200K(Brr2)stably.The p100 functional domains,p100-TD and p100-SN were purified by the GST fusion protein system successfully.Thirdly,to identify the effect of the p100 protein in the pre-mRNA splicing process,several splicing assays were carried out.The results indicated that the amount of mRNA was increased concomitantly with the increase in p100 protein level,while the BSA(bovine serum albumin)control did not affect the splicing activity;And the products of the first step of splicing was accumulated more quickly,p100 protein accelerated the mRNA accumulation in a time-dependent manner;To identify whether p100 is only functioning at the early stage of the splicing reaction,the addition of the purified p100 to the reaction 20 min after the initiation resulted in the same efficiency as pre-incubation of p100 with HeLa nuclear extract.In the study of RNAi(RNA interference),p100 siRNA efficiently reduced the amount of p100 protein in the p100-HeLa nuclear lysates. And the splicing reaction was significantly inhibited with p100 absence. However,the impaired splicing activity was recovered by addition of p100 protein.Fourthly,the results of the effect of the p100 protein and its functional domains in splicosomal complex formation were confirmed with the native gel analysis.Consistent with the splicing result,the native gel analysis demonstrated that TD domain alone had a similar function in splicing as the full-length p100 protein,that is,it accelerated the kinetics of all the spliceosomal complex formation,including complex A,B and C.Conclusion.The unlabeled pre-mRNA splicing assay still needs to be optimized, but it could be used as the alternative option,especially for the preliminary study,p100 protein functions as not only a transcriptional coactivator,but also a potential factor that involving in the regulation of pre-mRNA splicing.It could directly interact with RNA polâ…¡and U5 snRNP(small nuclear ribonucleoprotein)specific proteins(such as Prp8),and participate in the both events of transcription and pre-mRNA splicing.Thus our results suggest that p100 protein is a novel dual function regulator of gene expression that participates via distinct domains in both transcription and pre-mRNA splicing.