Identification And Functional Analysis Of Chimeric RNA Related To Porcine Skeletal Muscle

Chimeric RNA molecules possess exons from two or more independent genes.In the past research,chimeric RNA has always been considered as a result of chromosomal rearrangement,a special marker for malignant tumors,and has attracted much attention in the diagnosis,treatment and prognosis of cancer.With the development of bioinformatics and the reduction of the cost of Next-generation sequencing,the development of chimeric RNA has been greatly promoted.With the deepening of research,more and more chimeric RNAs have been identified in normal tissues and cells.Some of these chimeric RNAs can play an important regulatory role in normal physiological activities.Therefore,the identification of chimeric RNA from more normal tissues will help us fully understand the composition and function of the genome.Pigs are a crucial source of meat production worldwide and a potential medical model for human health issues.Muscle occupies a pivotal position in animal husbandry production.The quantity and quality of skeletal muscle are considered to be the one of main indicators for measuring meat quality.The growth and development of skeletal muscle is a very complex process,including the differentiation of muscle-derived stem cells into mononuclear myoblasts,fusion to form multinucleated myotubes,and mature muscle fibers.These processes involve the regulation of multiple myogenic factors.Therefore,research on the growth and development of skeletal muscle is great significance for improving the meat production and meat quality of livestock.Porcine Skeletal Muscle Satellite Cells(PSCs),as the progenitor cells of skeletal muscle,are considered to be the only source of stem cells for the myogenic differentiation of adult skeletal muscle cells,and they are generally in a resting state.After being stimulated,skeletal muscle satellite cells can activate,proliferate and differentiate into muscle cells through key signaling pathways,thereby participating in the formation and repair of skeletal muscle.In this study,the transcriptome data of 9 groups of pig muscles were deeply excavated by bioinformatics software,and the potential chimeric RNA in pig skeletal muscle was analyzed.Chimeric RNAs related to skeletal muscle growth and development were identified by molecular biology techniques and Sanger sequencing.PSCs were used as an in vitro experimental model to explore the effects of chimeric RNA and its parental genes on skeletal muscle development.The molecular mechanism of chimeric RNA-mediated skeletal muscle growth and development was discovered by high-throughput sequencing technology.The main results are as follows:1.Large white pig longissimus dorsi(LM),Min pig LM and Min pig biceps femoris RNA-Seq data were analyzed by STAR-fusion and Fusionmap.A total of 55 chimeric RNAs were predicted.Among them,49 were interchromosomal,accounting for 89%;6 were intrachromosomal,accounting for 11%.GO analysis showed that the parental gene enrichment functions were similar,mainly enriched in calmodulin binding,myosin fibers,myofibrils,cell motility and ATP binding.2.Using the c DNA of the pig,the chimeric RNA TNNI2-ACTA1 and its transcript variants were successfully identified by RT-PCR and named V1-V8,respectively.Structural analysis showed that the chimeric sites of TNNI2-ACTA1 V1-V8 were all on the exons of the parental genes.3.Each of TNNI2-ACTA1 V1-V8 contains a complete open reading frame(ORF)and has the potential to encode a protein.Western blot results showed that the chimeric proteins of V1,V2,V4,V7 and V8 could be detected,but the chimeric proteins of V3,V5 and V6 could not be detected.It indicates that V3,V5,and V6 may be non-coding RNA or be degraded by nonsense-mediated m RNA decay.4.In PSCs,TNNI2,ACTA1,and TNNI2-ACTA1 V1-V8 eukaryotic expression plasmids were transfected into the cells,and it was found that TNNI2-ACTA1 V1 could up-regulate the expression of Cyclin D1,arrest the cell cycle in G1 phase,and inhibit cell proliferation.The parental genes TNNI2,ACTA1 and other transcript variants V2-V8 have no significant effect on cell proliferation.It shows that the function of TNNI2-ACTA1 V1 to inhibit cell proliferation is not derived from the parental gene.5.After high-throughput sequencing,a total of 2669 differentially expressed genes(DEGs)were detected in the TNNI2 group compared with the control group(|log2Fold Change|≥1,q<0.05),of which 1592 genes were upregulated,and 1077 were downregulated;A total of 2128 DEGs(|log2Fold Change|≥1,q<0.05)were detected in the ACTA1 group,of which 1226 genes upregulated and 902 genes were downregulated;29 DEGs(|log2Fold Change|≥1,q<0.05)were detected in the TNNI2-ACTA1 V1 group,of which 13 genes upregulated and 16 genes were downregulated.GO analysis found that the DEGs of the two parental genes were mainly enriched in the functions of cellular processes,metabolic processes,biological regulation,cellular components,signal transduction activities and transcriptional regulatory activities;The enrichment results of the TNNI2-ACTA1 V1 group were similar to those of the parental group,and were mainly enriched in cellular processes,metabolic processes,signal transduction activities,and transcriptional regulatory activities.The KEGG enrichment results of the two parental genes mainly focused on the PI3K-Akt signaling pathway,the MAPK signaling pathway and the Fox O signaling pathway.6.Analysis of transcriptome data revealed that TNNI2-ACTA1 V1 specifically enriched three DEGs(Nuclear receptor coactivator 3(NCOA3),Radixin(RDX)and Discoidin domain receptor 2(DDR2))compared with the parental genes.The results of q PCR showed that after overexpression of TNNI2-ACTA1 V1,the expression level of NCOA3,RDX and DDR2 was inhibited,while the parental genes TNNI2 and ACTA1 had no significant effect on these genes,which indicates that NCOA3,RDX and DDR2 may be downstream effectors of TNNI2-ACTA1V1.7.After transfecting the eukaryotic expression vectors p CMV-HA-NCOA3,p CMV-HA-DDR2 and p CMV-HA-RDX into PSCs,it was found that NCOA3 can up-regulate the expression level of Cyclin D1,promote the cell cycle from G1 phase to S phase,and promote cell proliferation;while interfering NCOA3 can down-regulate the expression level of Cyclin D1,arrest the cell cycle in G1 phase,and inhibit cell proliferation.8.After co-transfection of p CMV-HA-TNNI2-ACTA1-V1 and p CMV-HA-NCOA3 into PSCs,it was found that overexpression of NCOA3 could rescue the inhibition of cell proliferation caused by overexpression of TNNI2-ACTA1 V1.9.Co-IP experiments demonstrated that TNNI2-ACTA1 V1 protein could interact with NCOA3 protein.In summary,this study jointly predict porcine skeletal muscle-related chimeric RNA through STAR-fusion and Fusionmap,and was successfully identified through RT-PCR.At the same time,it is found that TNNI2-ACTA1 V1 is different from the parental gene function,and has its unique biological function.TNNI2-ACTA1 V1 can interact with NCOA3,regulate the expression of NCOA3,and indirectly affect the expression of Cyclin D1,thereby regulating the proliferation of PSCs.This study provides strong evidence for the existence of chimeric RNA in normal tissues or cells,and provides a new idea for the growth and development regulatory network of skeletal muscle.