The Molecular Mechanism Of Milk Protein And Milk Fat Synthesis In Bovine Mammary Epithelial Cells Regulated By METTL3

The most important substance is the content and composition for milk protein and milk fat,but its is lower in China.Exploring the key signal molecus regulation of milk protein and milk fat which can providesan important experimental basis for improving dairy milk quality.METTL3(methyltransferase-like protein 3),also known as m~6A methyltransferase,catalyzingthe mRNA production of m~6A.A large number of studies have shown that METTL3 plays an important regulatory role by regulating the level of m~6A methylation in mRNA.In the synthesis of proteins in cells.Previous laboratory results showed that METTL3 affected the expression of milk protein and milk fat in BMECs by regulating the expression of mTOR and SREBP-1c genes.In this study,the expression and molecular mechanism of the expression of mTOR and SREBP-1c genes promoted by METTL3,and the molecular mechanism of p-GlyRS acting on METTL3 were studied.Glycyl tRNA synthetase(GlyRS)has a non-classical function and its analysis by protein co-immunoprecipitation mass spectrometry shows that GlyRS binds to its downstream protein METTL3 in the nucleus as p-GlyRS.Previous studies in the laboratory showed that p-GlyRS may have Kinase activity,p-GlyRS may phosphorylate METTL3.To activate METTL3,this study revealed that METTL3 regulates the molecular mechanism of milk protein and milk fat in BMECs and the molecular mechanism of p-GlyRS regulates METTL3.The BMECs were obtained by tissue block method.The expression of CK18(Cytokeratin 18)and?-casein in BMECs were detected by immunofluorescence and Western blot(WB).WB assay detected the expression of?-casein in BMECs,and detected the expression of triglyceride in BMECs using the triglyceride kit.The expression of mRNA m~6A in BMECs was detected by Dot blot.RIP assay detected mRNA m~6A expression of mTOR and SREBP-1c METTL3target proteins.FRET experiments to analyze the interaction between two proteins.By laser confocal co-localization experiments,we analyzed the changes in the interaction between p-GlyRS and METTL3 after the addition of methionine and estrogen.Other than,the ability of p-GlyRS to activate GST-METTL3was tested by an in vitro kinase assay.The experimental results showed that dairy cow mammary epithelial cells with lactating function were obtained.After overexpressing the METTL3,the expression of?-casein increased,the number of lipid droplets in the cells increased significantly,the volume of lipid droplets increased and the amount of TAG synthesis in cells increased.When METTL3 was inhibited,the expression of?-casein decreased,while the number of lipid droplets in the cells decreased,the lipid droplet volume became smallerand the amount of TAG synthesis in the cells decreased.The results of RIP showed that the expression of mTOR and SREBP-1c mRNA in MTEM3 overexpressed and interfered with METTL3 increased or decreased.FRET results showed that the value of p-GlyRS and METTL3 was greater than 20%.The interaction of p-GlyRS and METTL3 increased after adding methionine and estrogen by Laser co-localization experiments In vitro kinase experiments showed that p-GlyRS can phosphorylate METTL3.In summary,METTL3 is an important signaling molecule in BMECs,which positively regulates the synthesis of milk proteins and milk fat.METTL3 positively regulates mRNA m~6A levels and alsopositively regulates target genes of mTOR and SREBP-1c.p-GlyRS interacts directly with METTL3,and methionine and estrogen promote the binding of p-GlyRS to METTL3.In vitro kinase activity experiments showed that p-GlyRS can phosphorylate GST-METTL3.The above experiments help to reveal the molecular mechanism of METTL3 regulation of milk protein and milk fat in BMECs and p-GlyRS regulation of METTL3.