Comprehensive Analysis of the Transcriptome-Wide m6A Methylome in Shaziling Pig Testicular Development.

RNA N6-methyladenosine (m6A) modification is one of the principal post-transcriptional modifications and plays a dynamic role in testicular development and spermatogenesis. However, the role of m6A in porcine testis is understudied. Here, we performed a comprehensive analysis of the m6A transcriptome-wide profile in Shaziling pig testes at birth, puberty, and maturity. We analyzed the total transcriptome m6A profile and found that the m6A patterns were highly distinct in terms of the modification of the transcriptomes during porcine testis development. We found that key m6A methylated genes (AURKC, OVOL, SOX8, ACVR2A, and SPATA46) were highly enriched during spermatogenesis and identified in spermatogenesis-related KEGG pathways, including Wnt, cAMP, mTOR, AMPK, PI3K-Akt, and spliceosome. Our findings indicated that m6A methylations are involved in the complex yet well-organized post-transcriptional regulation of porcine testicular development and spermatogenesis. We found that the m6A eraser ALKBH5 negatively regulated the proliferation of immature porcine Sertoli cells. Furthermore, we proposed a novel mechanism of m6A modification during testicular development: ALKBH5 regulated the RNA methylation level and gene expression of SOX9 mRNA. In addition to serving as a potential target for improving boar reproduction, our findings contributed to the further understanding of the regulation of m6A modifications in male reproduction.

Interactive regulation of DNA demethylase gene TET1 and m6A methyltransferase gene METTL3 in myoblast differentiation.

DNA methylation (5mC) and mRNA N6-methyladenosine (m6A) play an essential role in gene transcriptional regulation. DNA methylation has been well established to be involved in skeletal muscle development. Interacting regulatory mechanisms between DNA methylation and mRNA m6A modification have been identified in a variety of biological processes. However, the effect of m6A on skeletal muscle differentiation and the underlying mechanisms are still unclear. It is also unknown whether there is an interaction between DNA methylation and mRNA m6A modification in skeletal myogenesis. In the present study, we used m6A-IP-qPCR, LC-MS/MS and dot blot assays to determine that the DNA demethylase gene, TET1, exhibited increased m6A levels and decreased mRNA expression during bovine skeletal myoblast differentiation. Dual-luciferase reporter assays and RIP experiments demonstrated that METTL3 suppressed TET1 expression by regulating TET1 mRNA stability in a m6A-YTHDF2-dependent manner. Furthermore, TET1 mediated DNA demethylation of itself, MYOD1 and MYOG, thereby stimulating their expression to promote myogenic differentiation. Ectopic expression of TET1 rescued the effect of METTL3 knockdown on reduced myotubes. In contrast, TET1 knockdown impaired the myogenic differentiation promoted by METTL3 overexpression. Moreover, ChIP experiments found that TET1 could bind and demethylate METTL3 DNA, which enhanced METTL3 expression. In addition, TET1 knockdown decreased m6A levels. ChIP assays also showed that TET1 knockdown contributed to the binding of H3K4me3 and H3K27me3 to METTL3 DNA. Our results revealed a negative feedback regulatory loop between TET1 and METTL3 in myoblast differentiation, which unveiled the interplay among DNA methylation, RNA methylation and histone methylation in skeletal myogenesis.

m6A mRNA methylation-directed myeloid cell activation controls progression of NAFLD and obesity.

N6-methyladenosine (m6A) RNA modification is a fundamental determinant of mRNA metabolism, but its role in innate immunity-driven non-alcoholic fatty liver disease (NAFLD) and obesity is not known. Here, we show that myeloid lineage-restricted deletion of the m6A "writer" protein Methyltransferase Like 3 (METTL3) prevents age-related and diet-induced development of NAFLD and obesity in mice with improved inflammatory and metabolic phenotypes. Mechanistically, loss of METTL3 results in the differential expression of multiple mRNA transcripts marked with m6A, with a notable increase of DNA Damage Inducible Transcript 4 (DDIT4) mRNA level. In METTL3-deficient macrophages, there is a significant downregulation of mammalian target of rapamycin (mTOR) and nuclear factor κB (NF-κB) pathway activity in response to cellular stress and cytokine stimulation, which can be restored by knockdown of DDIT4. Taken together, our findings identify the contribution of METTL3-mediated m6A modification of Ddit4 mRNA to macrophage metabolic reprogramming in NAFLD and obesity.

RNA methylomes reveal the m6A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening.

BACKGROUND: Methylation of nucleotides, notably in the forms of 5-methylcytosine (5mC) in DNA and N6-methyladenosine (m6A) in mRNA, carries important information for gene regulation. 5mC has been elucidated to participate in the regulation of fruit ripening, whereas the function of m6A in this process and the interplay between 5mC and m6A remain uncharacterized. RESULTS: Here, we show that mRNA m6A methylation exhibits dynamic changes similar to DNA methylation during tomato fruit ripening. RNA methylome analysis reveals that m6A methylation is a prevalent modification in the mRNA of tomato fruit, and the m6A sites are enriched around the stop codons and within the 3' untranslated regions. In the fruit of the ripening-deficient epimutant Colorless non-ripening (Cnr) which harbors DNA hypermethylation, over 1100 transcripts display increased m6A levels, while only 134 transcripts show decreased m6A enrichment, suggesting a global increase in m6A. The m6A deposition is generally negatively correlated with transcript abundance. Further analysis demonstrates that the overall increase in m6A methylation in Cnr mutant fruit is associated with the decreased expression of RNA demethylase gene SlALKBH2, which is regulated by DNA methylation. Interestingly, SlALKBH2 has the ability to bind the transcript of SlDML2, a DNA demethylase gene required for tomato fruit ripening, and modulates its stability via m6A demethylation. Mutation of SlALKBH2 decreases the abundance of SlDML2 mRNA and delays fruit ripening. CONCLUSIONS: Our study identifies a novel layer of gene regulation for key ripening genes and establishes an essential molecular link between DNA methylation and mRNA m6A methylation during fruit ripening.