Knockout Of The RNA Methylase TRDMT1 Gene And Preliminary Study For Its Function

As an epigenetic marker,RNA methylation plays an important role in gene regulation.So far,there were more than 100 kinds of modification had been found in RNA,and methylation was the most prevalent one.6-methyladenine(m6A)and 5-methylcytosine(m5C)are two of the most representative modifications.Studies have shown that RNA m5C modification plays an important role in the development of organisms and the occurrence of cancer.TRDMT1(tRNA aspartic acid methyltransferase 1)was initially thought to be a DNA methyltransferase,which later proved to be RNA m5C methyltransferase a decade ago,and it catalyzed tRNA to produce 5-methylcytosine modification.Recent studies have found that the TRDMT1 gene affects the formation of organs,the development of organisms,and the occurrence of diseases.However,the mechanism managing these processes is still unclear.In this study,CRISPR/Cas9-induced homologous recombination technology was used to knock the transcription termination sequence BGH Ploy(A)into the transcription initiation site of the RNA methyltransferase TRDMT1 gene to terminate the expression of TRDMT1 Based on the TRDMT1 knockout HEK293 cell line,the effects of TRDMT1 knockout on the cell phenotype and genes expression profile were studied to understand the function of the TRDMT1 gene and provide a new insight for studying of the function of RNA m5C methylation.The main research methods and results showed as follows:1.Establishment of RNA methylase TRDMT1 gene knockout HEK293 cell lineFirst,the sgRNA expression vector with high gene editing efficiency was designed to target the first exon of TRDMT1.The homologous recombinant vector containing the resistance screening gene(PuroR and NeoR)and the transcriptional termination sequences BGH Ploy(A)was constructed.Secondly,the Donor sequences was knocked into the first exon of the TRDMT1 gene through specific targeted homologous recombination mediated induced by CRISPR/Cas9 system.Then,transfected HEK293 cells were screened by puromycin(Puromycin)and neomycin(Neomycin).Finally,RT-qPCR was used to detect the relative expression level of TRDMT1 mRNA in gene edited HEK293 cells compared with that in normal cells.The results showed that the transcription of TRDMT1 gene was terminated by poly A signal and the terminating efficiency reached 99%.In conclusion,we successfully established a TRDMTl knockout HEK293 cell line.2.The effect of TRDMT1 knockout and recovery on the cell phenotypeTo investigate the effect of TRDMT1 knockout on the phenotype of HEK293 cells,a series of experiments were carried out including cell cycle,cell growth curve,cell scratch test and cell clone formation experiments.The results showed that knockout of the TRDMT1 gene could affect the cell cycle of HEK293,decrease the rate of G1 cells and increase the amount of G2 cells.The results of cell growth curve experiments showed that TRDMTl knockout could inhibit the proliferation of HEK293 cells,and the number of cells reached significant difference in the first 2-3 days(P<0.05),and reached a very significant difference in the 4-7th day(P<0.01).However,the results of clone formation experiments demonstrated that knockout of the TRDMT1 gene had no effect on cell population dependence of HEK293 cells.The results of cell scratch test showed that TRDMT1 knockdown could inhibit the migration ability of HEK293 cells.To investifate whether the effect of TRDMT1 knockout could be restored by re-expression of TRDMT1.The TRDMT1 overexpression vector pcDNA3.1-TRDMT1 was constructed and identified.The pcDNA3.1-TRDMT1 plasmid was transiently transfected into HEK293-TKO cells using ExFect(?)transfection reagent.Then cell growth curve and cell scratch experiments were performed.The restoring of TRDMT1 expression in HEK293-TKO cells promoted cell proliferation and activity,and enhanced its migration ability.These results indicated that the TRDMT1 gene knockout inhibits cell proliferation and migration and can be restored by re-expressing the TRDMT1 gene.3.Transcriptional sequence and analysis verificationIn this chapter,RNA-Seq was performed to study the effect of the TRDMT1 gene knockout on the gene expression of HEK293 cells.We found that there were 2352 differentially expressed genes between HEK293-TKO cells and HEK293 cells,of which 1585 genes were up-regulated and 767 genes were down-regulated.These differentially expressed genes were mainly enriched in the G2/M transition of mitotic cell cycle,cell migration,cell-cell adhesion and Rho protein signal transduction through the GO enrichment.KEGG analysis showed that the differentially expressed genes were mainly enriched in RNA splicing,neural ligand receptor interaction,cAMP signal transduction pathway,TGF-beta signal,NOTCH signal,cell cycle,cancer and other related pathways.From these pathways,11 differentially expressed genes were selected for RT-qPCR to varify the relust of RNA-Seq.The results of RT-qPCR were consistent with RNA-Seq.After knockout of TRDMT1,NOTCH1,DLL4,CREBBP,and FN1 were down-regulated,and CIR1,CCNE2,CDK1,TGFB2,UPF3B,ROCK1,ROCK2,and APC were up-regulated,further indicating that TRDMT1 is closely related to cell cycle,cell proliferation,and migration.Furthermore,the same expression trends of CREBBPa,NOTCH1,and DLL4 were found in TRDMT1 knockout zebrafish,suggesting that TRDMT1 may play important roles in development through regulating the NOTCH signaling pathway,cAMP signaling pathway,and cancer-related pathways.