Analysis Of The Difference Of Liver Transcriptome And N6-methyladenosine In Different Growth Stages Of Pigs

N6-methyladenosine(m6A)is a ubiquitous reversible epigenetic RNA modification that plays an important role in the regulation of post-transcriptional protein coding gene expression.The dynamic and reversible patterns endow m6A flexible roles in various biological processes.In recent years,with the progress of high-throughput sequencing technologies,as well as the improvement of m6A-seq,detecting the transcriptome wide m6A modification is becoming more and more popular.Researches focusing on m6A modification in Arabidopsis,rice,human and mouse have been reported.However,information concerning the dynamic patterns of mRNA m6A methylation in pig that has been regarded as ideal model for researching human development and disease,has been long overdue.Liver is a vital organ undergoing a variety of internal and external environment regulation and plays a major role in metabolism with numerous functions.Also,the liver of human and pig share similar characteristics in anatomy and physiology,which can be used as an important model of human development and liver disease research.Another important member of the family of epigenetic regulation factors,long non-coding RNA(lncRNA)are also very popular in recent years.In view of this,we take advantage of the newest method of m6A detecting and lncRNA sequencing method,and explore the pig epigenetic regulation phenomenon during the porcine liver development.Livers in Newborn(birth day age),Suckling(21 days of age),and Adult(2 year)from Rongchang pig were collected for RNA-seq and m6A-seq,respectively.Here,we have acquired the mRNA,lncRNA and m6A modifications of the three developmental stages of liver.The main results were as follows:(1)The cytoplasm of the liver at Newborn represents obvious characteristics,such as edema,vacuoles and less cytoplasmic contents.Significantly reduced cell vacuoles was observed at Suckling with increased cytoplasmic contents.Liver cells at Adult did not show much vacuoles,rich in cytoplasmic content.(2)In nine RNA-seq library(Strand-Specific),a total of 118 Gb(gigabases)of raw data was obtained.After low quality data filtering,average 13.02 Gb high quality data was kept for further analysis.(3)After the assembly by stringtie,a total of 17,558 expressed encoding genes were identified(FPKM>0.1),covering about 69.33%(17,558/25,323)annotated encoding genes of the reference genome.About 69.12%of the protein-coding genes showed a FPKM greater than 1.(4)After five steps of filtering,a total of 6,743 the candidate lncRNA transcripts(FPKM>0.1)were obtained.According to the position relationship with adjacent genes,candidate IncRNAs can be divided into three groups,3,179 IncRNAs in encoding gene(Genic),2,586 lncRNAs in intergenic region(Intergenic),978 lncRNAs unable to determine the position relationship.About 69.33%of the lncRNA showed a FPKM less than 1.(5)The average expression level of lncRNA,with a relatively higher density,was significantly lower than the mRNA.(6)About 68%of the candidate lncRNA showed a length between 200 to 1,200 nt,at an average length of 615 nt.While the mRNA showed an average length of about 1,770 nt.In terms of the number of exons,lncRNA had an average of 2 exons with a small number of exon number more than 6.and an average number of exons of mRNA was 7.8.(7)There were 1,771 differential expressed genes(DEGs)between Newborn and Suckling,2418 DEGs between Newborn and Adult,1,212 DEGs between Suckling and Adult.Further analysis showed that there were 396 genes in Newborn,260 genes in Suckling,and more than 324 genes in Adult showed higher expression.Functional analysis showed that higher expression genes in Newborn showed an enrichment at protein related GO terms,gene transcription,translation,synthesis and catalysis of RNA.Higher expression of genes in Suckling enriched in cell cycle,DNA replication and recombination,nuclear mitosis and rapid cell proliferation.Genes with higher expression in Adult were enriched in the pathways related to the metabolism of nutrients such as oxidation-reduction process,as well as the process of drug metabolism.(8)There were 321 significantly differential expressed lncRNA between Newborn and Suckling,631 differential expressed lncRNA between Newborn and Adult,294 differential expressed lncRNA between Suckling and Adult.Further analysis showed that there were 87 lncRNA in Newborn,31 lncRNA in Suckling,and 120 lncRNA in Adult.Function predication of highly expressed lncRNA showed close relationship between the lncRNA regulation and liver growth and development in pig.lncRNA regulated genes involved in some very important signaling pathways related to regulation of body growth.Function predication of highly expressed of lncRNA in each of the three developmental stages,showed that some lncRNA are involved in the regulation of metabolic pathways.In addition,lncRNA regulated genes were also involved in some immune function related signaling pathways.(9)Using m6A-seq method,we acquired m6A methylation sequencing IP data and the corresponding Input data of nine samples,including Newborn,Suckling and Adult.After low quality data filtering of raw data,average 9.33 Gb high quality of IP data,7.67 Gb of Input data for each was obtained.The unique data mapping to the reference genome of IP was at an average of 6,70 Gb,Input was at an average of 5.86 Gb.(10)We finally detected 11,022,8,727 and 9,860 m6A peak in Newborn,Suckling and Adult,respectively.Over 80%of the identified peaks were consistently detected in at least two biological replicates of each stage.(11)About 33%of transcribed genes were modified by m6A,with 1.33 to 1.42 m6A peaks per modified gene.m6A was distributed predominantly around stop codons.The consensus motif sequence RRm6ACH was observed in 78.90%of m6A peaks.A negative correlation(average Pearson’s r =-0.45,P<10-16)was found between levels of m6A methylation and gene expression.(12)Functional enrichment analysis of genes consistently modified by m6A methylation at all three stages showed genes relevant to important functions,including regulation of growth and development,regulation of metabolic processes and protein catabolic processes.(13)Genes with higher m6A methylation and lower expression levels at any particular stage were associated with the biological processes required for or unique to that stage.