| Objectives: The multifunctional protein Tudor-SN is an RNA-binding protein that can participate in cellular stress,exert its protective effect on m RNA,and can participate in RNA interference and other processes by forming RISC complexes.It can be seen that there is a close relationship between Tudor-SN and RNA.The research team has used the Tudor-SN to perform Pulldown-on-chip m RNAs experiments.The purpose of this study is to further analyze the function and regulatory mechanism of the RNA bound by Tudor-SN protein.Methods: This topic is divided into three major parts,PART1: Bioinformatics analysis of Tudor-SN pulldown-on-chip m RNAs results was performed and the results were verified.Using the online online analysis platform of DAVID,GO analysis of the captured RNA results and KEGG signaling pathway analysis.In He La cells,the results were verified using RIP technology.PART2: Bioinformatics analysis was performed on the mass spectrometry results of Flag-Tudor-SN fishing protein and verified by binding.Using the online online analysis platform of DAVID,GO analysis and KEGG signaling pathway analysis were performed on the captured proteins.In He La cells,the results of Tudor-SN and protein were verified using co-IP and GST pull down techniques.PART3: To investigate the regulation of Tudor-SN on its binding to RNA and proteins.(1)The effect of Tudor-SN on the distribution of its binding RNA nucleoplasm.Under normal conditions,cytoplasmic cytoplasmic RNA was isolated from He La WT and He La Tudor-SN-/-cells(Hereinafter referred to as He La KO),and the distribution of RNA in the cells was detected by RT-PCR.(2)Study the effect of Tudor-SN on its binding RNA stability.He La WT and He La KO cells were treated with actinomycin D,an RNA synthesis inhibitor,and the degradation of the RNA was detected by RT-PCR at 3 hours,6 hours,9 hours,and 12 hours.(3)Under stress conditions,the distribution of three stress granules combined with Tudor-SN changes compared with normal nucleus distribution.Under the influence of IPZ(transport receptor importin-β small molecule inhibitor),the distribution of nuclear protein in three stress particle proteins was changed.Using RNA interference technology in He La WT cells,we knocked down IPO5(nuclear translocation receptor)to observe changes in the nucleoplasm distribution of stress granules.Results: 1.Bioinformatics analysis showed that the Tudor-SN-bound m RNA mainly involved biological processes including translation process,RNA processing related,cell adhesion,protein folding,Wnt signaling pathway,cell classification cycle ubiquitination protein ligase activity,T Cell receptor signaling pathways,m RNA stability control,antigen processing,NIK/NF-kappa B,DNA damage response,proteasome ubiquitin-dependent regulation of protein metabolism,fatty acid synthesis,and ATP synthesis.Analysis of the KEGG pathway revealed that Tudor-SN-bound m RNAs are mainly involved in ribosome,metabolic pathways,oxidative phosphorylation,proteasomes,RNA transport,terpenoid biosynthesis,lysosomes,etc.,and are involved in Parkinson’s syndrome,Alz In the signal pathways of related diseases such as Hymer disease and Huntington’s disease.Results for Tudor-SN fishing were partially verified in He La cells by RIP experiments.2.Bioinformatics analysis showed that the biological processes involved in the Tudor-SN binding proteins include poly(A)RNA binding,ATP binding,RNA helicase activity,ATP-dependent RNA helicase activity,and double-stranded RNA binding,Protein binding,helicase activity,ubiquitin-protein ligase binding,and cadherin binding are involved in intercellular adhesion.In both endogenous and exogenous IP binding experiments and GST-puldown experiments,the nuclear transporter receptor and m RNA stability-related proteins were confirmed to bind to Tudor-SN.3.Under normal conditions,the effect of Tudor-SN on the nucleoplasm distribution of the bound m RNA is not significant.In He La WT and He La KO,the RNA synthesis inhibitor actinomycin D was added.The results showed that in He La WT cells,the degradation rate of m RNA bound to Tudor-SN was lower than that in He La KO cells at 3 hours,6 hours,9 hours,and 12 hours after dosing.4.Under stress conditions,the proportions of strong nuclear signal of hn RNP A1,Hu R,and TIA1 cells in the three stress-granule proteins all decreased compared with the normal state,and the proportion of strong cytoplasmic cells increased.Under stress conditions,hn RNP A1 protein under the action of IPZ(small molecule inhibitor of the importin-β transporter receptor),the strong nuclear signal decreased,and the strong cytoplasm signal increased.After the IPO5(nuclear translocation receptor)protein was knocked out in He La cells,the nucleoplasm distribution of hn RNP A1 protein was similar to that of IPZ.Conclusions: 1.Integrate Tudor-SN with RNA data and use the online online analysis platform of DAVID to obtain GO analysis results: 9 molecular functions,28 biological processes,and 25 cell components.Analysis of the KEGG signaling pathway revealed that RNA is mainly involved in 12 different signaling pathways.For Tudor-SN fishing results,partial verification was performed by RIP experiments.Tudor-SN can bind to proteins such as EIF3 A,EIF4A,EIF4 G,PABP1,IPO5,CRM1,LRPPRC.2.The Tudor SN-binding protein data was analyzed using the online online analysis platform of DAVID to obtain GO analysis results: 9 molecular functions,72 biological processes,and 14 cell components.Analysis of the KEGG signaling pathway revealed that aggregate proteins are mainly involved in 2 different signaling pathways.In both endogenous and exogenous IP binding experiments and GST-puldown experiments,the nuclear transporter receptor and m RNA stability-related proteins were confirmed to bind to Tudor-SN.Tudor-SN has a protective effect on its bound m RNA and can inhibit m RNA degradation.3.Under normal conditions,the effect of Tudor-SN on the nucleoplasmic distribution of the bound m RNA is not significant,and the stability of the bound m RNA can be maintained.4.Under stress conditions,Tudor-SN forms complexes with stress granule proteins that may participate in the importin-β receptor mediated nuclear cargo transport and stress protection of the bound m RNA in the form of stress particles. |
