Cloning,expression And Function Analysis Of Porcine PRMT1 Gene

Protein arginine methyltransferases 1(PRMT1)is one member of protein methyltransferases family,which has the highest catalytic activity responsible for asymmetric dimethylation of arginin.PTMT1 is widely expressed in mammalian embryos and adult organs,and play a key role in biological processes such as cell signal transduction,RNA splicing,DNA repair,transcriptional regulation and so on.To reveal the characterization,expression and function of porcine PRMT1 gene,several technologies were used,such as RACE,qRT-PCR,immunoblotting,cell transfection,immunofluorescence and overexpression in this study.The main results are as follows: 1.Molecular characterization of porcine PRMT1The cDNA sequence of porcine PRMT1 gene was 1381 bp,containing 1116 bp CDS region,encoding 372 amino acids,the initiation codon was located at 25-27 bp,the stop codon was located at 1138-1140 bp,and there were 24 bp in the 5'UTR and 241 bp in the 3'UTR.Porcine PRMT1 is an acidic,unstable and hydrophilic protein.A 1807 bp promoter sequence containing the 5'flanking region and the exon 1 and the intronof PRMT1 gene was cloned.The sequence from-343 to-126 is highly GC-rich(65.2% GC).Promotor prediction analysis indicated that have many confirmed binding sites for cis-acting regulatory elements,including Myod1,Myog,MEF2 C,SP1,SP2,TBP,and so on.2.Temporal and spatial expression analysis of porcine PRMT1 geneThe expression of porcine PRMT1 were analysed in Meishan different tissues at 65 dpc,3 d and 4 m.The results showed that PRMT1 gene was widely expressed in all tested tissues,but especially highly expressed in liver at 65 dpc.After birth,the mRNA expression was the highest in pig spleen tissue and very low in longissimus dorsi muscle.The expression of porcine PRMT1 gene in the longissimus dorsi muscle of Large white pig and Meishan pig was studied by qRT-PCR.The results showed that the expression level of 65 dpc was significantly higher than that of postnatal 3 days(P < 0.05).Compared the two breeds,the expression of PRMT1 gene in Large white pig was higher than Meishan pig at the stage of 65 dpc,3 d,21 d and 1 m after birth(P < 0.05).However,the expression level of 4 m Large white pig was significantly lower than Meishan pig(P < 0.05).3.The effects of porcine PRMT1 on proliferation and differentiation of C2C12 myoblastThe PRMT1 was mainly expressed in the cytoplasm of C2C12 myoblast and the expression level was increased during C2C12 myoblast differentiation.The proliferation of C2C12 myoblast was inhibited by overexpression of porcine PRMT1.After over expression of porcine PRMT1 gene,the expression of myogenic transcription factor,such as MyoG,Mef2 a and Myo D was significantly higher than that in the control group(P > 0.05).Meanwhile,the expression of CDK inhibitor Cdkn1 a was also significantly up-regulated(P > 0.05),and the cell cycle regulators Ccnd1 was significantly down-regulated compared with the control(P < 0.05).It was suggested that PRMT1 could be inhibited the proliferation of C2C12 myoblast and promoted the C2C12 differentiation by direct or indirect regulation of MyoD expression.