The Regulatory Role Of N6-methyladenosine In The Embryonic And Postnatal Development Of Skeletal Muscle In Pig

N6-methyladenosine(m~6A)is the most abundant post-transcription RNA modification in eucaryon,which is enriched near stop codons and in 3'UTRs of mRNAs.Recent studies exhibited that m6A regualates RNA fate through binding with m~6A reader protein,including RNA stability,export nucleus,alternative splicing,and translation,and involved in the regulation of broad biological processes.Previous researches revealed that m~6A participates in the regulation of adipogenesis and cortical development.However,the status and function of m~6A methylation in skeletal muscle development remains unclear now.Therefore,this research include following parts:The need of large amount of RNA limites the function research of m~6A,especially in the early stage of tissue formation.In this study,we developed an m~6A detecting method for limited RNA sample through combining SMART2 technology with MeRIP-seq,which realized transcriptome-wide m~6A detecting in 5?g RNA.It provided important method for m~6A identifation of low-input RNA sample.Knockdown METTL14 inhibit cell differentiation and promote proliferation.The binding genes of IGF2BP1 were enriched in muscle cell differentiation pathway and regulate C2C12 cell differentiation and proliferation.Based on this method,we firstly conduct the study of m~6A function in embryonic skeletal muscle development.These data exhibited that m~6A was highly dynamic change status.m~6A changes was strongly correlated with differential gene expression.Besides,we found m6A binding protein IGF2BP1 gene expression continuously decrease during the development process and demonstrate that IGF2BP1 regulate the differentiation of myoblast through siRNA-induced IGF2BP1.m~6A and IGF2BP1 RIP-seq analysis revealed 2281 gene was co-regulated with IGF2BP1 and m~6A,including MyoG and MYH2.These results suggest that m~6A play important role in embryonic skeletal muscle development and participate in the regulation of key gene metabolism with IGF2BP1.Dynamic change of m~6A was also observed during postnatal skeletal muscle development,when myofiber transition and maturation occur.Key regulatory factor of myofiber transition CAMK2A and MYOZ1 continuous significantly change.The m~6A level of CAMK2A significantly increase.MYOZ1m~6A level increase from 30d to 60d,then continuously reduce during postnatal skeletal muscle development.The expression of MYOZ1 persent opposite tendency.These results exhibite that m~6A also play crucial role in the regulation of myofiber transition.In this study,we developed a low-input m~6A identification method(R-MeRIP).Based on this method,we successfully identifed m~6A moditified gene in embryonic skeletal muscle and reveal a dynamic change of m~6A during skeletal muscle development in pig.The identification of m~6A methylation key genes revealed the crucial role of m~6A in myogenesis and myofiber transition period of skeletal muscle development.Our study provided direct evidence to elucidate the regulatory mechanism of m~6A in skeletal muscle development.