m ⁶A RNA Methylation Regulators Contribute to Eutopic Endometrium and Myometrium Dysfunction in Adenomyosis

N 6-methyladenosine (m6A) is enriched in 3′untranslated region (3′UTR) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. Herein we report a characterization of the full m6A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and we show that VIRMA mediates preferential mRNA methylation in 3′UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m6A enrichment in 3′UTR. Depletions of VIRMA and METTL3 induce 3′UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3′UTR of over 2800 mRNAs, 84% of which are modified with m6A and have increased m6A peak density in 3′UTR and near stop codon after CPSF5 knockdown. Together, our studies provide insights into m6A deposition specificity in 3′UTR and its correlation with alternative polyadenylation.

N6-methyladenosine (m6A) modification plays important roles in various cellular responses by regulating mRNA biology. However, how m6A modification is involved in innate immunity via affecting the translation of immune transcripts remains to be further investigated. Here we report that RNA methyltransferase Mettl3-mediated mRNA m6A methylation promotes dendritic cell (DC) activation and function. Specific depletion of Mettl3 in DC resulted in impaired phenotypic and functional maturation of DC, with decreased expression of co-stimulatory molecules CD40, CD80 and cytokine IL-12, and reduced ability to stimulate T cell responses both in vitro and in vivo. Mechanistically, Mettl3-mediated m6A of CD40, CD80 and TLR4 signaling adaptor Tirap transcripts enhanced their translation in DC for stimulating T cell activation, and strengthening TLR4/NF-κB signaling-induced cytokine production. Our findings identify a new role for Mettl3-mediated m6A modification in increasing translation of certain immune transcripts for physiological promotion of DC activation and DC-based T cell response.

BACKGROUND The existence of stem/progenitor cells in the endometrium was postulated many years ago, but the first functional evidence was only published in 2004. The identification of rare epithelial and stromal populations of clonogenic cells in human endometrium has opened an active area of research on endometrial stem/progenitor cells in the subsequent 10 years. METHODS The published literature was searched using the PubMed database with the search terms ‘endometrial stem cells and menstrual blood stem cells' until December 2014. RESULTS Endometrial epithelial stem/progenitor cells have been identified as clonogenic cells in human and as label-retaining or CD44+ cells in mouse endometrium, but their characterization has been modest. In contrast, endometrial mesenchymal stem/stromal cells (MSCs) have been well characterized and show similar properties to bone marrow MSCs. Specific markers for their enrichment have been identified, CD146+PDGFRβ+ (platelet-derived growth factor receptor beta) and SUSD2+ (sushi domain containing-2), which detected their perivascular location and likely pericyte identity in endometrial basalis and functionalis vessels. Transcriptomics and secretomics of SUSD2+ cells confirm their perivascular phenotype. Stromal fibroblasts cultured from endometrial tissue or menstrual blood also have some MSC characteristics and demonstrate broad multilineage differentiation potential for mesodermal, endodermal and ectodermal lineages, indicating their plasticity. Side population (SP) cells are a mixed population, although predominantly vascular cells, which exhibit adult stem cell properties, including tissue reconstitution. There is some evidence that bone marrow cells contribute a small population of endometrial epithelial and stromal cells. The discovery of specific markers for endometrial stem/progenitor cells has enabled the examination of their role in endometrial proliferative disorders, including endometriosis, adenomyosis and Asherman's syndrome. Endometrial MSCs (eMSCs) and menstrual blood stromal fibroblasts are an attractive source of MSCs for regenerative medicine because of their relative ease of acquisition with minimal morbidity. Their homologous and non-homologous use as autologous and allogeneic cells for therapeutic purposes is currently being assessed in preclinical animal models of pelvic organ prolapse and phase I/II clinical trials for cardiac failure. eMSCs and stromal fibroblasts also exhibit non-stem cell-associated immunomodulatory and anti-inflammatory properties, further emphasizing their desirable properties for cell-based therapies. CONCLUSIONS Much has been learnt about endometrial stem/progenitor cells in the 10 years since their discovery, although several unresolved issues remain. These include rationalizing the terminology and diagnostic characteristics used for distinguishing perivascular stem/progenitor cells from stromal fibroblasts, which also have considerable differentiation potential. The hierarchical relationship between clonogenic epithelial progenitor cells, endometrial and decidual SP cells, CD146+PDGFR-β+ and SUSD2+ cells and menstrual blood stromal fibroblasts still needs to be resolved. Developing more genetic animal models for investigating the role of endometrial stem/progenitor cells in endometrial disorders is required, as well as elucidating which bone marrow cells contribute to endometrial tissue. Deep sequencing and epigenetic profiling of enriched populations of endometrial stem/progenitor cells and their differentiated progeny at the population and single-cell level will shed new light on the regulation and function of endometrial stem/progenitor cells.