The Cloning Of Grass Carp Prmt5 And Its Regulation On The Expression Of Ho1

Protein arginine methyltransferase 5(Prmt5)is a type ? methyltransferase and plays critical roles in transcriptional regulation,mRNA splicing,signal transduction and DNA repair.However,few studies concern about the functions of Prmt5 in hypoxia response of fish.Heme oxygenase 1(Ho1)is an enzyme that degrades heme in the first and rate-limiting reaction.It has protective effects through anti-inflammatory,anti-apoptosis and anti-oxidant stress in many species.Our previous studies have found that crucian carp and zebrafish ho1 are hypoxia-responsive genes and play protective roles in cells in response to hypoxic stress.However,its transcriptional regulation mechanism is unclear during hypoxia response.In this paper we cloned the ORFs of grass carp prmt5 and ho1,and then studied the regulation of Prmt5 on the expression of Ho1 in grass carp ovarian cells(CO cells).The results are as follows:We cloned the ORF of grass carp prmt5 using RT-PCR.The ORF of grass carp prmt5 contains 1896 bp which encodes a polypeptide of 631 amino acid residues.Comparisons of deduced amino acid sequences demonstrated that grass carp Prmt5 was highly homologous and shared 77%-97%similarity with those of other vertebrates.Moreover,grass carp Prmt5 contains a SAMBD domain.The tissue expression pattern analysis showed that grass carp Prmt5 was expressed ubiquitously in liver,brain,heart,spleen,kidney,gill,testis,and the expression in the liver was obviously higher than others.When CO cells exposed to hypoxia for 6h,12h,24h and 48h,we detected the expression of prmt5 by qRT-PCR.It was found that the level of prmt5 mRNA was induced significantly after 6h exposure but then decreased significantly after 12h,24h and 48h exposure by hypoxia.We cloned the ORF of grass carp ho1 using RT-PCR.The ORF of grass carp Ho1 contains 816 bp which encodes a polypeptide of 271 amino acid residues.Comparisons of deduced amino acid sequences demonstrated that grass carp Ho1 was highly homologous and shared 46%-90%similarity with those of other vertebrates.Moreover,grass carp Ho1 contains a heme oxygenase domain.The tissue expression pattern analysis showed that grass carp Ho1 was expressed ubiquitously in liver,brain,heart,spleen,kidney,gill,testis,and the expression in the liver was obviously higher than others.When CO cells exposed to hypoxia for 6h,12h,24h and 48h,we detected the expression of ho1 by qRT-PCR.It was found that the level of ho1 mRNA was induced significantly at 6h and 12h,but then reduced and was not different from control after 24h and 48h exposure by hypoxia.We construct the over-expression vector and shRNA-expression vector of prmt5 and transfected them into CO cells.Then we investigate the regulatory effect of Prmt5 on the expression of Ho1.The results showed that overexpression of Prmt5 could significantly increase the mRNA and protein levels of ho1 gene in CO under normoxia.Knockdown of Prmt5 significantly decrease the mRNA levels of ho1 gene in CO under normoxia.Moreover,overexpression of Prmt5 also significantly increased the mRNA levels of ho1 gene and knockdown of Prmt5 also significantly decrease the protein levels of ho1 gene in CO cells after 6h hypoxia treatment.These results indicated that Prmt5 regulated the expression of Ho1 in certain degree both under normoxia and hypoxia.We also investigated the regulatory effect of Prmt5 on the expression and transcriptional activity of Hif1a using overexpression and knockdown technique.The qRT-PCR analysis showed that the mRNA levels of hif1 gene and its target genes igfbpl and cited3 were upregulated after Prmt5 overexpressed both under normoxia and hypoxia.Dual luciferase reporter gene assay revealed that overexpressed Prmt5 increased significantly the dual luciferase activity.These results indicate that Prmt5 regulate the expression and transcriptional activity of Hif1?.Combined with the references,HIF signaling pathway may be involved in the regulation of Prmt5 on Ho1 expression.This need to be further investigated.In addition,we constructed the prokaryotic expression plasmid of Hif1? and induced its prokaryotic expression using IPTG.The western blot analysis revealed that the peptide fragment of Hif1? has been successfully induced to express.This work was useful for making Hif1? polyclonal antibodies and also made a good foundation for further investigation for the regulation machanism of Prmt5 on the expression of Ho1.