N(6)-methyladenosine Is Regulated By METTL14 Phosphorylation And Its Role In Preeclampsia

N(6)-methyladenosine(m6A)is the most prevalent internal RNA modification in eukaryotic mRNAs and lncRNAs,and has been found to be highly conserved among different species.Dynamic and reversible changes of the m6A modification are mediated by the combined action of METTL3/METTL14 methyltransferase complex and demethyltransferase FTO/ALKBH5.The m6A modification plays important roles in diverse biological processes.Post translational modifications(PTMs)are critical for the dynamic regulation of enzyme activity in organisms,and may play a key role in the dynamic regulation of m6A,but few research has been reported.Dysregulation of m6A contributes to multiple human diseases including cancers.Preeclampsia is a serious disease for the pregnant women,but the role of m6A modification in preeclampsia has not been reported.The first part of this thesis was mainly focused on the functional study of m6A by the phosphorylation of serine 399(S399)of METL14.We identified that METTL14 S399 was phosphorylated by tandem affinity purification(TAP)combined with liquid chromatography tandem mass spectrometry(LC-MS/MS).METTL14 S399 is localized in the interface of the core enzyme METTL3 and METTL14 and is highly conserved among different species.We found that m6A level decreased to 40%in METTL14S399A mESCs and m6A RNA immunoprecipitation approach followed by high-throughput sequencing(meRIP-seq)analysis showed that m6 A peaks significantly decreased,suggesting that S399 phosphorylation regulated m6A modification in mESCs.We screened out CDK1 and CDK7 as possible kinases of METTL14 S399 by treating with inhibitors.The pluripotency of embryonic stem cells was reduced in METTL14S399A and cell cycle was not significantly affected.In mice,METTL14S399A lead to homozygous embryos lethal.This part of the study initially analyzed the effect of m6A by protein post-translational modification and it provided a new perspective for further research on dynamic regulation of RNA modification.The second part of this thesis is mainly about the role of m6A in preeclampsia(PE).Firstly,meRIP-seq was performed on normal and PE placental tissue samples.Data analysis found that compared with normal placental tissues,the level of m6A in PE was decreased.Among them,2196 m6A peaks were down-regulated and only 113 were upregulated.The expression of the demethylase ALKBH5 was increased.Therefore,we detected the gene expression of 18 normal placenta samples and 24 PE placenta samples,and the expression level of ALKBH5 in PE samples was significantly increased.Based on this,we constructed a mouse PE model using Mettl3 +/heterozygous.We found that when treated with NG-nitro-L-arginine methyl ester(LNAME)during pregnancy,Mettl3+/-heterozygous can develop PE symptoms such as hypertension.This research provided a theoretical basis for the function of m6A in preeclampsia,and provided a potential target for the diagnosis and treatment of preeclampsia.In summary,we have uncovered the role of protein phosphorylation in the regulation of N(6)-methyladenosine,suggesting the importance of protein posttranslational modification in the dynamic regulation of m6A.Furthermore,we have revealed the effect of m6A in preeclampsia,providing a potential target for the diagnosis and treatment of preeclampsia.