Transcriptome-wide N~6-methyladenosine Methylome Profiling Of Porcine Muscle And Adipose Tissues Reveals A Potential Mechanism For Transcriptional Regulation And Differential Methylation Pattern

N~6-methyladenosine(m6A)is the most prevalent internal form of modification in polyadenylated mRNAs and long non-coding RNAs in higher eukaryotes,which is involved in regulation post-transcriptiona gene expression.In recent years,a few species of RNA m6A methylation maps have been constructed by using methylated RNA immunoprecipitation sequencing(MeRIP-seq)and have revealed some potential biological regulation fuctions.However,there has no report on the profile of porcine RNA m6A methylation.Some studies have revealed that RNA m6A methylation has been in existence of common and different regulatory functions in different tissues and populations.Nevertheless,up to date,there are no relevant reports on whether there is common or differential regulation function between muscle and adipose tissues and among different porcine breeds.In this paper,we successfully constructed the transcriptome-wide m6A methylation maps of three breed pigs(Wild boar,Landrace and Rongchang pigs)with different genetic backgrounds for the first time in muscle and adipose tissues by using the MeRIP-seq.This enabled us to compare the patterns of m6A distribution between muscle and adipose tissues and among porcine breeds,and investigated tissue and breed generality and selectivity of methylated genes and their functional implications.The obtained results as follows:1.We detected 5,872 m6A recurrent peaks among 4,544 expressed genes in Landrace muscle tissue(LM),and 2,826 m6A recurrent peaks among 2,137 expressed genes in Landrace adipose tissue(LA).We used this information to estimate that the porcine transcriptome contains 0.562 m6A peaks in LM and 0.254 m6A peaks in LA.The distribution of m6A methylation genes in swine transcriptase was mainly enriched in the CDS,stop codon and the 3'UTR region.The classical conserved sequence RRACH appears in porcine RNA m6A methylation region.2.We identified 1,957 common m6A peaks related to 1,615 co-expressed genes in muscles and adipose tissues.Gene ontology analysis revealed that common m6A peaks in nuclear genes are associated with transcriptional factors,suggesting there be a relationship between m6A mRNA methylation and nuclear genome transcription.3.We obtained 3,034 and 562 tissue-specific methylation modified genes(TSMGs)in muscle and adipose tissues,respectively.We found that differentially methylated genes of muscle tissue are mostly involved in the regulation of energy-dependent signaling pathways,while some of adipose tissues involved in the fatty acid metabolism-related signaling pathways.We also identified 319 tissue dynamic methylated genes(TDMGs)in the two tissues,and GO analysis showed that some TDMGs were involved in immune and disease-related signaling pathways.Besides,we searched for differential peaks in gene regions between the two tissues,and these genes are mainly enriched for the nuclear portions,and involved in the regulation of metabolic processes and protein binding.We also found that more m6A peaks were detected in the high expression gene of muscle than adipose tissues,and the results indicated that level of m6A methylation changes in genes of different tissues and it appears to be associated with gene activation.Furthermore,we researched the GPIHBP gene function,which was highly enriched in m6A methylation in adipose tissue,and found that the porcine GPIHBP1 mRNA levels were higher in adipose tissue compared to other tissues.Association analyses showed that four GPIHBP1 SNPs are genetic factors affecting adipose traits.These results confirmed that m6A modification is involved in the tissue-differential regulation process and also participate in diflferent biological functions.4.Approximately 6,500?7,500 m6A peak representing 5,500?6,000 expression genes were detected from wild boar(WB),Landrace(LD)and Rongchang pig(RC)breeds.WB showed the highest level of m6A,while the lowest level of m6A level was found in RC.The levels of m6A may be affected with the variety of living environment and differences in the genetic background.We identified 2,846 BSMGs and 171 BDMGs between WB and LD,3,186 BSMGs and 240 BDMGs between WB and RC.Similarly,2285 BSMGs and 89 BDMGs were obtained between RC and LD.Differential in the m6A methylaytion among breeds showed a connection to the functions of the transcripts required for a specific breed.These transcripts presents an extensive higher level of m6A methylation in LD which contribute in the growth and development of muscle and to adapt to lean LD breeding.However,some of the differential and extensively methylated transcripts in the fatty RC pig were involved in the development of the cardiovascular system.These results revealed the differential m6A methylation across the three pig breeds.In summary,we provide the first known porcine transcriptome-wide m6A map of differentiated tissues and breeds.Our map identifies tissue and breed generality as well as selectivity of methylated genes and their functional implications.This comprehensive map provides a solid basis for the determination of potential functional roles for RNA m6A modification in adipose deposition and muscle growth.