Regulation Of M~6A RNA Methylation On Transcription Of Porcine Germ Cells

The N~6-methyladenosine(m~6A)is a post-transcriptional modification that is prevalent in mammalian transcriptome.m~6A functions in transcription,alternative splicing,nuclear export,translation,and stability of m RNA,but its role in male germ cells remains obscure.Pigs(Sus Scrofa)are important for food production,and act as an important animal model for human biomedical research.Here,the methylated RNA immunoprecipitation sequencing(Me RIP-seq)was performed in porcine spermatogonia,spermatocytes,and round spermatids to establish the profile of m~6A modification during spermatogenesis.Liquid chromatography–mass spectrometry(LC–MS),RNA interference(RNAi),and immunoprecipitation were performed to reveal the regulatory mechanism of m~6A in porcine germ cells.The main results of this study are listed as follow:1.Spermatogonia,spermatocytes,and round spermatids were isolated from porcine testes using STA-PUT velocity sedimentation.The purity of the isolated cells was over 90%as determined by morphological observation and immunofluorescence analysis.The level of m~6A remained relatively stable(～0.3%)during the developmental stages as shown by LC-MS/MS.2.The profile of m~6A modification in porcine spermatogonia,spermatocytes,and round spermatids was established by Me RIP-seq.Bioinformatics analysis showed that the transcriptomic distribution of m~6A is dynamic.m~6A peaks were highly enriched near the start codon(start C),coding sequence(CDS),and stop codon(stop C).m~6A reads were distributed throughout the m RNA transcripts,in which the reads increased in the CDS and reached the peak at the 3’UTR.The GGACU was a top motif in porcine germ cells,which is consistent with RRACH,the m~6A consensus sequence.3.A paired analysis of differentially methylated genes(DMGs)and differentially expressed genes(DEGs)between each two adjacent stages was performed.The level of m~6A exhibited positive correlation with gene expression.The dynamics of m~6A are related to genes with spermatogenic function.Comparison with MeRIP-seq data from recent reports of murine germ cells showed that the overlapped m~6A-methylated transcripts were preferentially enriched and reported to be essential for spermatogenesis.4.The expression of METTL3,a m~6A methyltransferase,was knocked down in porcine(spermatogonial stem cell)SSCs by a small interfering RNA(si RNA).The level of m~6A was visibly decreased but the cell proliferation was not significantly altered.Me RIP-q PCR and q RT-PCR analysis showed that knockdown of METTL3 significantly reduced the relative level of m~6A and the relative gene expression of SETDB1,FOXO1,and FOXO3,which is associated with the fate of SSCs.These data suggest that m~6A mediates the expression of spermatogenic genes and further govern the process of spermatogenesis.The first m~6A transcriptome-wide map of porcine germ cells was established in this study.The findings in this study provide a roadmap for uncovering the mechanisms how m~6A methylation modulates the SSC proliferation and differentiation,and will provide information for the study of the other domestic germ cells as well.