Identification And Functional Exploration Of RNA Associated P53 Interacting Proteins

The main function of p53 is as a transcription factor bound to the target gene promoter region, which regulate m RNA transcription. Numerous studies show that p53 plays an important role in maintaining cell growth and inhibition of tumor cell proliferation. p53 can regulate gene expression according to various stress induced cell signals, similar to a signal molecule. So p53 is also regulated both at the levels of protein stability and biochemical activities by kinds of molecules. With the development of RNA study, p53 and RNA are closely related. Some studies show that p53 can bind RNA, and many p53 binding proteins are RNA binding proteins. Moreover, some RNAs are found to regulate p53 by binding to other proteins, such as p53 m RNA, lnc RNA Apela. These results suggest that RNA may participate or even play an important role in p53-protein interaction. Hense, we studied p53 interacting proteins to explore the function of these proteins and RNA in p53 interaction network.In order to identify RNA associated p53 interacting protein, we improved the condition of immunoprecipitation, adding RNase A or RNase Inhibitor in cell lysates to digest RNA or protect RNA. Then Flag-p53 immunoprecipitation was used to acquire some proteins that bound RNA to interact with p53 and some that were inhibited to interact with p53 by RNA. Using MS analysis of protein and experiments, we identified the interaction among RNA, protein and p53 and the specific domain of protein and p53 interaction. Then we used RNAi to knockdowned target gene expression and overexpressed target gene to explore p53 dependent function of these proteins in p53 interaction network. We found that RNA participated in protein-p53 interaction, and these proteins played an important role in p53 interaction network.In order to analyse RNA which participated in p53-proteins interaction, we chose HITS-CLIP method to screen and identify p53 binding RNA. We used bioinformatics to analyse a large number of p53 protein binding RNA obtained by HITS-CLIP, and verify the interaction between RNA and p53 by RIP.We also explore the function of lnc RNA MEG3 binding protein, in order to reveal biological functions of MEG3 in cell, we established a method called MTRAP-MS(MS2-tagged RNA affinity purification and Mass spectrometry), screened and obtained a series of MEG3 binding proteins. We used Gene Onology analysis to analyze the protein from three aspects, cell component, molecular function and biological process, for the establishment of functional networks of protein-MEG3 interactions.The main improvements:1. We improved the conditions of immunoprecipitation to identify whether RNA participate in p53-protein interactions. We overexpressed Flag-p53 in cells and used Flag-IP to enrich p53 complexes with RNase or RNase Inhibitor, in order to digest or protect cell RNA. Then we acquired hn RNPC, NCL, ILF3 and HSPD1 protein using Coomassie Blue Staining and MS analysis.2. We found the interaction of NCL, ILF3 and HSPD1 with p53 through RNA for the first time. We constructed eukaryotic expression vector of NCL, ILF3 and HSPD1 and identified the interaction of NCL, ILF3 and HSPD1 with p53 through RNA by Co-IP. We found NCL and HSPD1 regulate p53 transcriptional activity using dual luciferase reporter.3. We found that RNA interfered the interaction of hn RNPC and p53, and RNA for the first time. GST pulldown and other experiments were used to identify the direct interaction of RRM domain in hn RNPC and p53, and identify that RNA inhibited the interaction of hn RNPC and p53.4. Knockdown of hn RNPC increased p53 protein level and transcriptional acitivity, and also induced p53 dependent apoptosis. The results showed that hn RNPC inhibited p53 protein level and transcriptional activity.5. We used HITS-CLIP and RIP experiments to find that p53 could bind PLAGC1, TERC and mi R143 HG. Knockdown p53 expression could decrease PLAGC1 and TERC RNA level. The results suggested that p53 might regulate PLAGC1 and TERC expression through binding them.6. We used bioinformatics to analyze MEG3 binding proteins obtained by MTRAP-MS, and we found that MEG3 binding proteins mainly distributed in nucleus and mitochondria. Most proteins had the character of nucleotide binding and took part in macromolecular complexes assembly process.To sum up, we identified that RNA participated p53-protein interaction for the first time and successfully acquired RNA associated p53 interacting protein NCL, ILF3, HSPD1 and hn RNPC that was inhibited to bind p53 by RNA. The results explained the mechanism of the inhibition of hn RNPC to p53 and the function in cells. And we also obtained a serious of RNA associated p53 interacting proteins. All of these results showed that RNA participated in the interaction of p53 and other proteins to play an important role, and regulated p53 with p53 interacting proteins.