Function And Mechanism Of METTL3 Regulating Bovine Myoblast Development Through M~6A Modification

In animal husbandry,the growth and development of skeletal muscle which is crucial for meat production,directly affects the quantity and quality of meat.Therefore,it has important guiding significance to explore the regulation mechanism of muscle development for the improvement of meat quality.At present,there are many studies on the regulatory function of muscle development with respect to functional genes,DNA methylation and non-coding RNA,but there are few studies about RNA modification in muscle development.N6-methyladenosine(m~6A)as one of the most common ways of RNA modification,plays an important biological role in the development of tissue and diseases,which has become a vital and fresh research hotspot.However,researches on m~6A modification in skeletal muscle development have only just begun,and its regulatory function and mechanism are still unclear.Furthermore,recent studies have uncovered that m~6A modification can affect the generation,translation and stability of circ RNA.However,the relationship between m~6A modification and circ RNA in skeletal muscle and the effect of m~6A-modified circ RNA on muscle development remains elusive.Therefore,this study firstly revealed the effect of METTL3 as m~6A methyltransferase on the development of bovine and mouse myoblasts,and screened out the target genes regulated by m~6A modification through m~6A-seq,then uncovered the regulatory mechanism.Meanwhile,the circ RNA regulated by METTL3 was identified,and its function and regulatory mechanism in muscle development were explored.The main results are as follows:1.Functional study of METTL3 regulating myoblasts development in a m~6A-dependent mannerBovine myoblast and mouse myoblast(C2C12)were used to verify the m~6A levels in GM(proliferation phase)and DM(differentiation phase)of myoblasts.The results showed that a markedly more abundant of the m6A levels in DM than GM,and METTL3 can affect m~6A deposition in bovine and mouse myoblasts.Function gain and loss experiments have revealed that METTL3 inhibited myoblast proliferation and promoted myoblast differentiation.Furthermore,through constructing the vector of METTL3 catalytic mutant and wild type,we found that the regulatory effects of METTL3 on bovine and mouse myoblasts rely on m~6A methyltransferase activity.Together,these data suggested that METTL3,as m~6A methyltransferase,inhibited myoblast proliferation and promoted myoblast differentiation.2.METTL3 regulates myoblast development by affecting the stability of TM4SF1M~6A-seq and RNA-seq were used to analyze the characteristics of m~6A modification and the change patterns of m~6A-regulated genes in bovine and mouse myoblasts during GM and DM.The most common motif GGAC was highly enriched in bovine and mouse myoblasts.Furthermore,results showed that m~6A peaks were especially enriched in the vicinity of stop codons and start codons,and were located in 3′UTR>exon regions>5′UTR.Next,we analyzed the transcripts with differences both in m RNA and m~6A levels in DM and GM of bovine and mouse myoblasts,and TM4SF1 was identified as the target gene regulated by m~6A modification during muscle development.Analysis of TM4SF1 stability showed that the half-life of TM4SF1 m RNA prolonged in METTL3-deletion myoblasts,and the expression of TM4SF1 was negatively regulated by METTL3.Meanwhile,we further verified that YTHDF2could destabilize the expression of TM4SF1 in an m~6A-dependent manner.3.Effect of METTL3 and TM4SF1 on muscle regeneration in vivoThe effect of METTL3 on muscle regeneration was investigated by constructing CTX-induced muscle injury in mice.The results showed that the expression profiles of METTL3were consistent with the marker genes of myogenesis including PAX7,Myo D and Myo G during the process of muscle damage repair.Importantly,overexpression of METTL3 in TA muscle promotes muscle regeneration.To verify whether TM4SF1 was involved in m~6A-regulated muscle regeneration,both METTL3 and TM4SF1 were overexpressed in TA muscle,and the results showed that TM4SF1 delayed skeletal muscle regeneration.However,overexpression of METTL3 can attenuate the suppression effect of TM4SF1 on muscle repair.4.Function and mechanism of circ CPE regulated by METTL3 on bovine myoblast developmentIn order to further explore the influence of m~6A on circ RNA,we found that the expression of circ CPE was regulated by METTL3 in bovine myoblasts,where loss of METTL3 increased circ CPE expression.Functionally,overexpression of circ CPE promoted bovine myoblast proliferation,and inhibited cell apoptosis and differentiation.Moreover,circ CPE could inhibit skeletal muscle regeneration in vivo.Bioinformatics prediction and dual-luciferase experiments confirmed that circ CPE could adsorb mi R-138.In addition,functional rescue results showed that circ CPE as a sponge of mi R-138 weakened the inhibitory effect of mi R-138 on bovine myoblast proliferation and the stimulative effect of mi R-138 on cell apoptosis and differentiation.And FOXC1 identified as a new target of mi R-138 during bovine muscle development,and deletion of FOXC1 can attenuates the promoting effect of circ CPE on cell proliferation and the inhibitory effect on cell differentiation and apoptosis.Taken together,this study provides a regulatory model of METTL3-mediated m~6A modification on muscle development,where METTL3/YTHDF2 m~6A axis inhibited myoblast proliferation and promoted cell differentiation by affecting the stability of TM4SF1.In addition,we found that the expression of circ CPE was negatively regulated by METTL3 in bovine myoblasts.And,circ CPE attenuates the inhibitory effect of mi R-138 on FOXC1,thus regulating the development of bovine myoblasts.These studies deeply analyzed the genetic mechanism of muscle growth and development from the epigenetics,which enriched the regulatory network of muscle development and provided a theoretical basis for improving the growth rate and meat quality of Chinese cattle.