Mechanism Of FBXO24-induced Degradation Of Arginine Methyltransferase 6(PRMT6)through The Ubiquitin Proteasome Pathway

Objective:Arginine methyltransferase 6(PRMT6)methylates histone H3R2,H4R3,and non-histone proteins;The main functions of PRMT6 are transcriptional regulation,RNA-selective cleavage,DNA replication and cell cycle regulation.This topic clarifies that FBXO24 induces the degradation of PRMT6 via the ubiquitin-proteasome pathway by binding to the C-terminal VGGRF sequence of PRMT6.Methods and Results:H1299 cell line with stable knockout of PRMT6 was constructed and the stable and control cell lines were analyzed by cloning assay.The results showed that the number of clones of stable strain was far less than the control group;Cell migration and invasion assays revealed that the migration and invasion of H1299 cells was significantly reduced after knock-out of PRMT6.Based on the database,it was found that PRMT6 was more highly expressed in cancer tissue than paracancerous tissue.The survival curve analysis showed that the survival rate of cancer patients highly expressing PRMT6 was significantly lower than that of low expression.Cycloheximide(CHX)is an intracellular protein synthesis inhibitor.When CHX was used to treat H1299 cells at different times,the protein level of PRMT6 in the cells decreased continuously,and the half-life was about 6 h.The results showed that PMRT6 was unstable in the cells.Using the ubiquitin-proteasome inhibitor MG132 and CHX to deal with H1299 cells,the protein level of PRMT6 was almost unchanged.The result of co-immunoprecipitation experiments showed that PRMT6 has a polyubiquitylation modification in the cells.Overexpression of HA-Ub plasmid in H1299 cells can lead to the degradation of PRMT6;The above results reveal that PRMT6 is degraded via ubiquitin-proteasome pathway in H1299 cells.The results of work showed that FBXO24 could induce the degradation of PRMT6;we further found that PRMT6 interacts with FBXO24 through co-immunoprecipitation experiments;then overexpressing FBXO24 of different quality in H1299 cells,found that the protein level of PRMT6 is in dose The mode of dependence decreased;by transfecting different plasmids that knocked down FBXO24 in H1299 cells,plasmids with the best knockdown effect were selected;plasmids and sh RNA FBXO24 plasmids were transfected into cells and treated with CHX for different times.The level of PRMT6 protein knocked down in FBXO24 group was significantly higher than that in the control group;FBXO24 was then transfected into H1299 cells,and FBXO24 was found to increase ubiquitination of PRMT6 through IP experiments;these results strongly demonstrate that FBXO24 can induce PRMT6.The truncated mutants of PRMT6 is constructed,and cells transfected with the truncated mutants are treated with MG132,and the possible ubiquitination site is analyzed as K360,K367,K369;Construction of PRMT6 K360,K367,K369 point mutants,through the co-transfection experiments with FBXO24 found that PRMT6-K369 almost no degradation;cells were transfected with three point mutants,through the half-life experiments PRMT6-K369 is the most stable;these results indicate that the ubiquitination site of PRMT6 is K369.FBXO24 binds to the C-terminus of PRMT6;constructing truncated mutants of PRMT6,co-immunoprecipitation experiment is performed with truncated mutants and FBXO24 shows that the motif of FBXO24 to PRMT6 is VGGRF.The PRMT6-K360,PRMT6-K367 and PRMT6-K369 mutants were transfected into H1299 cells stably knocked out PRMT6.The experimental results showed that the protein level of the experimental group transfected with PRMT6-K369 is lowest;PRMT6-WT,K360,K367,and K369 point mutants were transfected in H1299 cells stably knocked out by PRMT6.After PI incubation,the cell cycle was detected by flow cytometry.The experimental results showed that the proportion of G0/G1 in the PRMT6-K369 group was the lowest,and the proportion in the S phase was the highest.The H1299 cell line stably expressing FBXO24 was constructed,compared with the blank cells,the PRMT6 level of the stable cell line was significantly lower;the H1299 cell line stably expressing FBXO24 was analyzed by cloning formation assay.Compared with the control group,the number of clones in the experimental group was also significantly lower;Through cell migration and invasion experiments,it was found that the reduction of PRMT6 can significantly reduce the migration and invasion of H1299 cells;The above results demonstrate that the level of PRMT6 is closely related to cell cycle,proliferation,migration,and invasion of H1299 cells.Conclusion: The combination of SCF-FBXO24 and PRMT6 motif VGRFR induces PRMT6 degradation in H1299 cells via the ubiquitin-proteasome pathway.The PRMT6 ubiquitination site is located at the K369 residue which decreases PRMT6 levels in cells can lead to the inhibition of cell proliferation,migration and invasion.