Identification Of Human Prp8and Snu114as Specific Interaction Proteins Of Human P100Protein

Objective:A novel coactivator, p100, was recently found to bind to the Epstein-Barr virus nuclear antigen2(EBNA-2) and coactivate gene expression mediated by the EBNA-2acidic domain. HCA method found that p100consisted of four repeat SN-like fragment and C-terminal Tudor-SN (TD) fragment. We found that p100participated in the gene transcriptional regulation as a coactivator, this kind of interaction depend on the SN-like fragment. But the function of p100-TD is not clear, but we found the structure and function of p100-TD are the same as SMN-Tudor, they have similar properties. SMN is two-function protein because it participates in gene transcriptional regulation and pre-mRNA splicing. Thus we presume that p100protein may be has splicing function because it has similar TD.Pre-messenger RNA (pre-mRNA) splicing takes place in the nucleus, catalyzed by a large RNA—protein complex called spliceosome. U5snRNP are important components of spliceosome. hPrp8(precursor mRNA processing) and hSnu114are core of U5snRNP because they bind to U5snRNA directly. We found that p100-TD interacted with a series of U5snRNP-specific proteins, include hPrp8and hSnu114. But we need to identify:(1) they bind directly or indirectly through bridge-protein.(2) which functional domain is to bind if they bind directly?Methods:The experiment is divided into three parts:in the first part, p100protein interacts with hPrp8in vivo. Firstly, p100-TD interacts with a series of U5snRNP-specific proteins, which is researched by GST pull down and Western Blot. Then HeLa nuclear lysis solution is extracted, incubates with GST-p100-TD and SDS-PAGE by Coomacia Blue or silver staining. The nuclear proteins bound to p100-TD are analyzed by MALDI-TOF. And then, whether p100protein and hPrp8 interact with each other in vivo, we utilize CO-IP and western blot. Then the HeLa-p100stable cell nuclear lysis solutions are extracted and detect proteins which bind to pi00protein. And purifying the complexes which bind to p100protein by sepharose beads and analyzing the result by SDS-PAGE and western blot. Another lysis solution is precipitated by anti-hPrp8antibody, the result is analyzed by the same method as above. The result indicates that p100protein interacts with hPrp8in vivo. In order to confirm the binding site of p100and hPrp8by GST pull down and western blot, GST-p100-SN/GST-p100-TD incubate with HeLa-p100stable cell nuclear lysis solution, and are tested by SDS-PAGE. The interactions between hPrp8and p100-TD or p100-SN are detected by anti-hPrp8antibody.In the second part, we constructed the pcDNA3.1(+/-)-hPrp8/hSnu114and pEGFP C I-hSnull4and pcDNA3.1(-)-hSnu114(His)6tail full-length. The full-length of hPrp8and hSnu114are amplified by RT-PCR. hPrp8was amplified through four overlapping fragments, hSnul14was amplified through two overlapping fragments. These fragments contain specific and sole enzyme-digestion sites in order to construct the full-length of hPrp8and hSnu114. Because hPrp8sequence is very long (7.2kb), overlap extension PCR method is used to ligate the four overlapping fragments into full-length, and the two large fragments which produced by overlap extension PCR were sub-cloned into pcDNA3.1(+). hSnull4sequence is2.9kb in size, the two RT-PCR fragments were ligated into full-length by the sole enzyme-digestion sites which it contains itself and were sub-cloned into pcDNA3.1(-). The two recombinant plasmids are used to studies in vitro. Furthermore, we constructed the pEGFP C I-hSnull4and pcDNA3.1(-)-hSnu114(His)6tail full-length, now it has been shown that hPrp8and hSnu114proteins are a complex in cell. So the two recombinant plasmids are used to study the interaction between their and p100protein. In addition, we constructed the pGEX-4T-1/pBluescript SK -hPrp8/hSnu114functional domains recombinant plasmids. hPrp8and hSnull4include different functional domains which play different roles in organism. So we designed the corresponding functional domains in order to further study the interaction domains between hPrp8and hSnu114and p100protein in vitro.In the third part, firstly hPrp8and hSnu114functional domains were expressed by GST gene fusion system in vitro. The synthesized protein were labeled35S.And then study the interaction domains between hPrp8and hSnu114and p100-TD by TNT T7Coupled Reticulocyte Lysate Systems,.Results:In the first part,(1)p100protein interacts with U5snRNP-specific proteins, these proteins include hPrp8, hBrr2and hSnull4, their molecular weighs are respectively220kDa、200kDa and116kDa;(2)p100protein interacts with hPrp8in vivo, but we can not confirm that they interact directly or not;(3)p100-TD interacts with hPrp8, p100-SN can not.In the second part, we constructed the following recombinant plasmids successfully in order to identify p100-TD interacts with which domain of hPrp8:①pcDNA3.1(+/-)-hPrp8/hSnu114;②pEGFP C I-hSnull4and pcDNA3.1(-)-hSnu114(His)6tail full-length;③PGEX-4T-1-hPrp8/hSnu114functional domains recombinant plasmids;④pBluescript SK-hPrp8/hSnu114functional domains recombinant plasmids. These functional plasmids are used to study the interaction mechanism between hPrp8and hSnu114a transcriptional coactivator p100protein.In the third part, p100-TD fragment stably interacts with hPrp8-Domain2in vitro. Conclusion:Our present study provides following views:①p100is a novel component of U5snRNP;②p100-TD stably interacts with hPrp8Domin2;③p100 may be involves in pre-mRNA splicing by interacting hPrp8Domain2.