The Role And Mechanism Of M6A RNA Methylation In The α-synuclein Metabolism Prosses

Objective:To explore the role of m6A RNA methylation in the biological process of alpha-synclein(α-syn)and its potential mechanism,and to further explain the potential pathogenesis of Parkinson’s disease(PD),as well as provide a new therapeutic target for PD.Methods:Firstly,a Parkinson’s disease cell model was constructed by overexpressing α-syn in mouse dopaminergic nerve cell SN4741.Western blot and RT-qPCR were used to verify efficiency of transfection.Secondly,m6A ELISA was used to detect the m6A level of total RNA between α-syn group and vector group,and the expression of m6A methyltransferases,such as METTL3,METTL14 and WTAP,and demethylases including FTO and ALKBH5 between the two groups were determined by Western blot and RT-qPCR.Thirdly,after transfected with siRNA targeted METTL3,METTL14 or ALKBH5 in α-syn cells,the m6A level of total RNA was detected by m6A ELISA and the expression of a-syn was measured by Western blot and RT-qPCR.Next,after transfected with wild-type α-syn plasm in over-expressed METTL14 or ALKBH5 cells,the m6A level of total RNA was detected by m6A ELISA and the expression of a-syn was measured by Western blot and RT-qPCR.Moreover,SRAMP software was used to predict the m6A modification sites on the a-syn transcriptome,the m6A modification on a-syn was verified by MeRIP-qPCR,and also,the effect of METLL14 and ALKBH5 on m6A modification of α-syn was detected by MeRIP-qPCR.The expression of α-syn in over-expressed METTL14 transfected with wild-type α-syn plasm or mutant plasm(mutl and mut2)was measured by Western blot,RT-qPCR and immunofluorescence,to explore which m6A modification site on α-syn are the most important site for the expression of α-syn.Results:high expression of α-syn was observed in SN4741 transfected with α-syn lentivirus,which mean that PD cell model was established successfully.Compared with the Vector group,the m6A level of total RNA was significantly decreased in the a-syn cells.Besides,the expression of methyltransferase METTL3 and METTL14 decreased significantly,while the expression of demethylase ALKBH5 increased significantly in overexpressedα-syn cells.However,the expression of methyltransferase WTAP and demethylase FTO did not changed.Moreover,we found that low expression of METTL3 and METTL14 decreased the m6A level and increased the a-syn protein level.However,low expression of METTL14 increased the α-syn mRNA level,while low expression of METTL3 did not changed the a-syn mRNA level.Low expression of ALKBH5 increased the m6A level and decreased the expression of α-syn both in mRNA and protein.The high expression of METTL14 decreased the expression of α-syn mRNA and protein,and increased the m6A level.The high expression of ALKBH5 decreased the m6A level and increased the expression of α-syn mRNA and protein.Three m6A modification sites on the α-syn transcriptome predicted by SRAMP and MeRIP-qPCR showed that the anti-M6A antibody could enriched the α-syn mRNA.Silencing METTL14 or overexpression of ALKBH5 down-regulated m6A modification on a-syn while overexpressed METTL14 or low expression of ALKBH5 increased m6A modification on α-syn.The mRNA level and protein expression of α-syn were increased in overexpressing METTL14 cells transfected with mutant plasmid 2(mut2),while that did not change when transfected with mutant plasmid 1(mut1).Conclusion:In this study,methyltransferase METTL14 and demethylase ALKBH5 directly regulate the m6A modification on α-syn mRNA and participate in the biological metabolic process of α-syn,which may be one of the pathogenesis of PD.This study provides a new entry point for the study of the pathogenesis and a promising target for the diagnosis and therapy of PD.