Identification Of U5-116 Protein As Specific Interaction Protein Of P100 Protein

Objective:Eukaryotic gene expression process is consisting of DNA transcription, pre-mRNA splicing and translation.In this processes transcription and pre-mRNA splicing are the key processes and both of them require multi-component complex to function,concluding of protein-DNA,protein-RNA and protein-protein.Transcription and splicing are not independent events.They are functionally coordinated.Some proteins have transcription and splicing function,so they are called cross-talk protein. Studying these cross-talk proteins will help us known much about transcription and splicing.Human p100 protein is a novel transcriptional coactivator.HCA(hydrophobic cluster analysis)show that p100 consists of staphylococcal nuclease(SN)-like and Tudor(TSN)domains.The SN-like domains have been shown to function in transcription,but the function of TSN domain in the assembly of snRNP complexes and pre-mRNA splicing process.Pre-mRNA splicing is happened in a big protein complex -splicosome,in which there is U5 snRNP specific proteins,such as U5-116.The purpose of this study was to validate the hypothesis that p100 protein functions in pre- mRNA splicing,and to reveal Splicing processes molecule mechanism.Methods:In this report,there are three parts.First,GST gene fusion proteins with various fragments of hPrp8 were used to pull down in vitro translated full length p100 or U5-116 labeled with³⁵S.Western Blot experiments were also used to confirm the specific interaction domains of hPrp8.In the second part,interactions of p100 and U5-116 proteins in cells.We constructed the recombinant plasmids of pEGFP-CI-U5-116 and pERFP-CI-p100. The two recombinant plasmids are used to studies in cells.We coimmunoprecipitation of pl00 and U5-116 proteins to confirm the interaction between them.We also studied the colocalization of p100 and U5-116 proteins by immunofluorescence microscopy.In the last part,identify the domain of p100 protein interacts with U5-116.GST gene fusions with various fragments of p100 were used to pull down U5-116 protein in cells to identify the interaction domains of p100.Results:In the first part,we found that p100 protein can bind to hPrp8 domain 2.2,not hPrp8 domain 2.1;U5-116 can bind to hPrp8 domain 2.1,2.2. In the second part,we constructed the following recombinant plasmids successfully:â‘ pEGFP-CI-U5-116â‘¡pERFP-CI-p100.Coimmunoprecipitation and Western Blot experiments also confirmed the interaction of over-express two proteins in cell.And we observed the colocalization of p100 protein and U5-116 protein by immunofluorescence microscopy.In the third part,p100-TSN fragment stably interacts with U5-116 protein.Conclusion:Our present study provides following views:p100 protein,U5-116 protein can binds to hPrp8 protein,but function domains of hPrp8 are different.p100 protein can interacts U5-116 protein.p100-TSN stably binds to U5-116 protein.