The Role Of MRNA M~6A Modification In The Proliferation And Differentiation Of Qinchuan Beef Cattle Skeletal Myoblast

The growth and development of skeletal muscle directly determine the meat production and quality of livestock and poultry,and the research on the mechanism of skeletal myogenesis is of great significance for the breeding and improvement of livestock and poultry.It has been shown that the process of skeletal myogenesis is regulated by a series of transcription factors,in addition to classical epigenetic modifications such as DNA methylation and histone methylation,which also play an important role in skeletal myogenesis.N~6-methyladenosine(m~6A)is the most abundant RNA methylation modification in eukaryotes.Several studies have shown that m~6A modification has a potential regulatory role in skeletal muscle growth and development.Transcriptome m~6A modification profiles in skeletal muscle development of mice,pigs,and geese have been revealed by m~6A-seq.However,the role of m~6A modification in skeletal myogenesis is still unclear,and the study of m~6A modification in cattle has not been reported.Therefore,in order to elucidate the functions and mechanisms of RNA m~6A methylation modification in bovine skeletal myogenesis and to provide a theoretical basis for genetic improvement of meat traits and molecular breeding in cattle,this study was conducted to investigate the mechanisms of m~6A modifications in myoblast proliferation and differentiation using primary myoblasts of Qinchuan cattle beef new strain(hereinafter referred to as"Qinchuan beef cattle")skeletal muscle as experimental materials,and to explore the preliminary relationships between m~6A methylation and other epigenetic modifications.interactions with other epigenetic modifications.The main findings of the study are as follows:1.The expression of m~6A methyltransferases METTL3,METTL14,and WTAP was significantly decreased in skeletal muscle of 24-month-old Qinchuan beef cattle compared with that of newborn cattle,while the expression of m~6A demethylases FTO and ALKBH5 was increased.In vitro studies of myoblasts found that FTO,METTL3 and METTL14 interference all promoted myoblast proliferation and inhibited differentiation.Correspondingly,overexpression of FTO,METTL3 and METTL14 promoted myoblast differentiation.In addition,both ALKBH5 and WTAP interference inhibited myoblast proliferation,but ALKBH5interference promoted myoblast differentiation,while WTAP had no significant effect on myoblast differentiation.These results suggest that m~6A methylation is involved in the regulation of skeletal myogenesis in Qinchuan beef cattle.2.The overall m RNA m~6A level of Qinchuan bovine skeletal myoblasts on day 4 of differentiation was significantly lower than that on day 0 of differentiation.Furthermore,m~6A-seq analysis showed that the motifs of m~6A modifications were very conserved,which were RRACH sequences(R=Guanine or Adenine,H=Adenine or Cytosine or Uracil);the distribution of m~6A modifications was mainly enriched near the stop codon,CDS and 3’UTR.A total of 24.2-33.8%of transcripts in all samples had m~6A modifications and each m~6A-modified transcript contained an average of about 1.6 m~6A peaks.GO and KEGG functional enrichment analysis showed that m~6A-modified genes were mainly clustered in transcriptional regulation-related GO terms,and enriched in muscle formation and developmental pathways.Meanwhile,the combined analysis of differential methylation and differential expression indicated that m~6A modification may be involved in the process of myoblast differentiation by regulating gene expression.Finally,this study screened and identified MEF2C,a key target gene of m~6A modification regulating skeletal muscle myoblast differentiation in Qinchuan beef cattle.Both m RNA expression and m~6A levels of MEF2C were significantly up-regulated during skeletal myoblast differentiation3.The present study carried out a synonymous mutation of the m~6A site in the MEF2C CDS region.The results showed that MEF2C-MUT inhibited myoblast differentiation compared to wild type,indicating that the m~6A site is required for the function of MEF2C to promote myogenic differentiation.The binding of the m~6A methyltransferase METTL3 and the m~6A reader protein YTHDF1 to MEF2C m RNA was verified,indicating that the expression of MEF2C in myoblast differentiation is positively regulated by the METTL3-m~6A-YTHDF1 axis,and METTL3 promote myoblast differentiation through this pathway.In turn,we found that MEF2C could direct bind to METTL3 promoter to promote METTL3 expression.It finally revealed the existence of a positive feedback loop in which METTL3 and MEF2C regulate each other in bovine skeletal myoblast differentiation.4 The expression of DNA demethylase TET1 decreased and its m~6A level increased during myoblast differentiation.TET1 promoted myogenic differentiation by reducing the DNA methylation levels of MYOD1,MYOG and itself.METTL3 promoted the degradation of TET1m RNA and inhibited its expression through m~6A-YTHDF2 dependent manner.TET1 could directly bind to METTL3 DNA to mediate the DNA demethylation of METTL3.Meanwhile,TET1 inhibited the levels of H3K4me3 and H3K27me3 after myoblast differentiation,and TET1 interference promoted the binding of H3K4me3 and H3K27me3 to METTL3 DNA.These results suggested that TET1 can regulate METTL3 expression by mediating DNA demethylation and histone methylation.These results indicate that there is an interaction between TET1 and METTL3 during bovine skeletal myoblast differentiation.In conclusion,the present study systematically investigated the role of m RNA m~6A methylation modification in the proliferation and differentiation of skeletal myoblast in Qinchuan beef cattle.According to the transcriptome m~6A modification profile of Qinchuan beef cattle skeletal myoblasts before and after differentiation,we identified two molecular regulatory networks,i.e.,METTL3/MEF2C and METTL3/TET1,in skeletal myogenesis.The present study preliminarily demonstrated that there is a regulatory network among m RNA m~6A methylation,DNA methylation and histone methylation in skeletal muscle differentiation.On the one hand,this study provides a new perspective for genetic improvement and molecular breeding in Qinchuan beef cattle.On the other hand,it provides a scientific basis for studying the function and molecular mechanism of m~6A methylation in regulating skeletal muscle growth and development,and provides a reference for understanding the interactions between epigenetic modifications.