Enrichment Of The Nascent RNA At Specific Spatial Sies

Objective: The tumor is heterogeneous,which leads to the uneven distribution of cell subsets with different genes in space and time.The spatial heterogeneity of tumors has become a research focus,which may lead to the viability of cancer cells after malignant transformation and continue to develop and differentiate.At present,studies on tumor heterogeneity are mainly conducted by single-cell sequencing.However,single-cell sequencing isolates cells in the process of preparing single cells,which destroys tumor microenvironment and may result in changes in the expression of some genes.The information obtained from the spatial transcriptome of tumors can not only infer the function of corresponding unknown genes,reveal the mechanism of action of specific regulatory genes,but also identify cell types and heterogeneity,providing theoretical basis for tumor diagnosis.RNA is the executor of gene expression,and new RNA is the most direct reaction of cell state.We can predict the trajectory of cell differentiation and development by enriching nascent RNA.The purpose of this study is to develop a technique that can not only label and enrich nascent RNA at specific sites.Methods: 1.Selected genes with different localization in the cell,and constructed fusion plasmid with targeted localization together with APEX2.Western-blot and immunofluorescence were used to verify the expression of fusion protein and localization of fusion plasmid.2.Construct a new RNA labeling system mediated by 4s U for specific subcellular localization and verify the labeling efficiency.3.According to the constructed PCDH-MITO-APEX2-Flag plasmid,the systematic labeling efficiency of neighboring markers,the localization of the enriched RNA and whether it is a new RNA were verified.4.According to the constructed PCDH-G3BP1-APEX2-Flag plasmid,the systematic labeling efficiency of neighboring markers,the localization of the enriched RNA and whether it is a nascent RNA were verified.5.According to the constructed PCDH-PML-APEX2-Flag plasmid,the localization of the plasmid in cells,the systematic labeling efficiency of neighboring markers,the localization of the enriched RNA and whether it is a nascent RNA were verified.Results:(1)Western-blot proved that the constructed plasmid could express the fusion protein in 293 T cells by applying anti-specific site antibody and anti-Flag antibody,and immunofluorescence verified that the location of the fusion plasmid at specific spatial sites in the cytoplasm was correct.(2)EB glue verified that there was no significant difference between the RNA transcribed in vivo and in vitro after incorporation of 4s U and that without incorporation of 4s U.Dot blot verified that RNA transcribed in vivo and in vitro with 4s U could covalently react with Biotin-HPDP and be enriched by Streptavidin magnetic beads.Dot blot verified in vivo and in vitro transcription that RNA doped with 4s U could covalently react with Biotin-APEX2 and be enriched by Streptavidin magnetic beads.(3)Mitochondrial localization of the fusion plasmid EB gel verification,it was found that after APEX2 catalyzed,4s U marker enrichment of RNA was more,q PCR verification of the enrichment of RNA was identified as mitochondrial localization,iodoacetamide induced 4s U reaction,T>C count,proved that the enriched RNA was new RNA.(4)The fusion plasmid of G3BP1 in the cytoplasm was verified by EB glue,and given external stimulation,it was found that after the APEX2 catalyst,the RNA enriched by 4s U labeling was located more.The RNA enriched by q PCR was located as G3BP1.The 4s U reaction was induced by iodoacetamide,and T>C was counted.It was proved that the enriched RNA was nascent RNA.(5)The fusion plasmid with PML body location in the nucleus was verified by immunofluorescence and the plasmid expression was correct.EB gel verified that the RNA enriched with 4s U labeling was more,and q PCR verified that the RNA enriched was located as PML body location.The 4s U reaction was induced by iodoacetamide.It was proved that the enriched RNA was nascent RNA.Conclusions: Firstly,the fusion plasmid was constructed and the expression was verified.The cells were cultured with appropriate concentration of 4s U.During the synthesis of nascent RNA,4s U and UTP could synthesize RNA together,and the structure and function of the synthesized RNA were normal.The constructed fusion plasmid was transfected into cells,and biotin phenol labeled RNA was catalyzed by APEX2 at specific sites.We found that the labeling efficiency of Apex2-mediated neighborhood labeling was improved with the participation of 4s U,and 4s U became easier due to Apex2-mediated neighborhood labeling enrichment.