| Natural antisense transcripts(NATs)are a new class of RNAs identified in various species in recent years.Cis-natural antisense transcript(cis-NAT)has time and space advantage over tran-NAT on regulation of its sense gene expression.However,until now the biological function of cis-NATs remains unknown.As the largest tissue of pig,skeletal muscle donates almost 50% carcass weight.Skeletal muscle growth is an important factor which directly determines the production efficiency of the pig industry.Therefore,the study of regulation mechanism of pig skeletal muscle growth and development has been one of the key point of breeding work.In order to explore expression and regulation function of cis-NATs,we systematically analyzed the cis-NATs in landrace and Lantang pig skeletal muscle development.For further understanding of the regulation mechanism of cis-NATs,mining of published Ch IP-seq data was performed to accumulate evidence of transcription factor MYOD mediating regulation of cis-NATs.The main results are:(1)Cis-NATs were widespreadly detected in pig skeleton muscle development.Here,3561(sense-antisense,SA)genes were identified by investigating strand specific DGE RNA-seq data of 10 skeletal muscle development stage of Landrace and Lantang pigs.Based on that,transcript region of cis-NATs was identified,further modified and confirmed using strand specific d UTP RNA-seq data.Finally,3306 cis-NATs of 2,469 SA genes were identified.(2)Cis-NATs were primarily expressed at fetal stage and sharply decreased after birth.Expression pattern analysis results showed that the number of fetal stage expressed cis-NATs was obviously higher than that of postnatal stage and sharply decreased after birth.(3)At the transcriptional level,more than 1,300(about 40%)cis-NATs were correlated with their sense genes,and approximately 80% of them were co-expressed.Pearson correlation analysis and Range method analysis results showed 1,379(41.71%)and 1,386(41.92%)cis-NATs were correlated with their sense genes at the transcriptional level in LR and LT pigs respectively.Among them,75.05%(LR)and 81.53%(LT)were co-expressed.(4)Over 1200 cis-NATs were differentially expressed during pig skeletal muscle growth and development(|log2FC| ≥ 1 and FDR < 0.01).From different expressionanalysis,there were 1,281(LR)and 1,211(LT)cis-NATs differentially expressed during pig skeletal muscle development for fold change equal or higher than 2(|log2FC| ≥ 1)and FDR less than 0.01.(5)Cis-NATs are primarily related to energy metabolism-related pathway.Function annotation showed that the cis-NATs participated in muscle development through co-expressing with genes involved in energy metabolic pathways,including citrate cycle(TCA cycle),glycolysis or gluconeogenesis,mitochondrial activation and so on.(6)There was abrupt increase of energy metabolism pathway involved-genes and their correlated cis-NATs at the transition from the late fetal stages to the early postnatal stages followed by a decrease.The results of expression analysis showed that,in both LR and LT pigs,the energy metabolism pathway-involved genes and their correlated cis-NATs were up-regulated from fetal 63 days to postnatal 2 days.In the subsequent postnatal 2 days to postnatal 28 days,they were sharply down-regulated and continued until postnatal 90 days.In LR pigs the similar down-regulation were observed between postnatal 28 days to 90 days.(7)Cis-NATs involved in pig skeletal muscle development were potentially regulated by transcription factor MYOD.Myo D Ch IP-seq and RNA-seq data of C2C12 myotube were used to gain evidence of Myo D colocalized with SA genes.To compare with these SA genes in mouse C2C12 myotube,nearly 1/5(18.11%)of SA genes in pig skeletal muscle could match.The MYOD binding motif was significantly(P< 0.001)enriched in the sequence around the transcript start and terminated sites of the SA gene in pig skeletal muscle,and was significantly(P< 0.001)similar to the Myo D binding motif which identified in the C2C12 myotube Ch IP-seq data.Results of this study showed SA genes in pig skeletal muscle were potentially regulated by MYOD. |
