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Project description:Haematopoietic stem cells (HSCs) maintain balanced self-renewal and differentiation, but how these functions are precisely regulated is not fully understood. N6-methyladenosine (m6A) messenger RNA methylation has emerged as an important mode of epitranscriptional gene expression regulation affecting many biological processes. We show that deletion of the m6A methyltransferase Mettl3 from the adult haematopoietic system led to an accumulation of HSCs in the bone marrow and a marked reduction of reconstitution potential due to a blockage of HSC differentiation. Interestingly, deleting Mettl3 from myeloid cells using Lysm-cre did not impact myeloid cell number or function. RNA sequencing revealed 2,073 genes with significant m6A modifications in HSCs. Myc was identified as a direct target of m6A in HSCs. Mettl3-deficient HSCs failed to upregulate MYC expression following stimulation to differentiate and enforced expression of Myc rescued differentiation defects of Mettl3-deficient HSCs. Our results reveal a key role of m6A in governing HSC differentiation.
| S-EPMC6556891 | biostudies-literature
Project description:Stage-specific requirement for Mettl3-dependent m6A mRNA methylation during hematopoietic stem cell differentiation
| PRJNA509123 | ENA
Project description:BackgroundN6-methyladenosine (m6A) is the most prevalent epigenetic modification in eukaryotic messenger RNAs and plays a critical role in cell fate transition. However, it remains to be elucidated how m6A marks functionally impact the transcriptional cascades that orchestrate stem cell differentiation. The present study focuses on the biological function and mechanism of m6A methylation in dental pulp stem cell (DPSC) differentiation.Methodsm6A RNA immunoprecipitation sequencing was utilized to assess the m6A-mRNA landscape during DPSC differentiation. Ectopic transplantation of DPSCs in immunodeficient mice was conducted to verify the in vitro findings. RNA sequencing and m6A RNA immunoprecipitation sequencing were combined to identify the candidate targets. RNA immunoprecipitation and RNA/protein stability of Noggin (NOG) were evaluated. The alteration in poly(A) tail was measured by 3'-RACE and poly(A) tail length assays.ResultsWe characterized a dynamic m6A-mRNA landscape during DPSC mineralization with increasing enrichment in the 3' untranslated region (UTR). Methyltransferase-like 3 (METTL3) was identified as the key m6A player, and METTL3 knockdown disrupted functional DPSC differentiation. Moreover, METTL3 overexpression enhanced DPSC mineralization. Increasing m6A deposition in the 3' UTR restricted NOG expression, which is required for DPSC mineralization. This stage-specific m6A methylation and destabilization of NOG was suppressed by METTL3 knockdown only in differentiated DPSCs. Furthermore, METTL3 promotes the degradation of m6A-tagged NOG by shortening the poly(A) tail length in the differentiated stage.ConclusionsOur results address an essential role of dynamic m6A signaling in the temporal control of DPSC differentiation and provide new insight into epitranscriptomic mechanisms in stem cell-based therapy.
| S-EPMC9756505 | biostudies-literature
Project description:N6-methyladenosine (m6A) is the most abundant chemical modification in mRNA and plays important roles in human and mouse embryonic stem cell pluripotency, maintenance, and differentiation. We have recently reported, for the first time, the role of m6A in the postnatal control of β-cell function in physiological states and in Type 1 and 2 Diabetes. However, the precise mechanisms by which m6A acts to regulate the development of human and mouse pancreas are unexplored. Here, we show that the m6A landscape is dynamic during human pancreas development, and that METTL14, one of the m6A writer complex proteins, is essential for the early differentiation of both human and mouse pancreatic cells.
| S-SCDT-10_1038-S44318-024-00213-2 | biostudies-other
Project description:N6-Methyladenosine (m6A) messenger RNA methylation is a well-known epitranscriptional regulatory mechanism affecting central biological processes, but its function in human cellular senescence remains uninvestigated. Here, we found that levels of both m6A RNA methylation and the methyltransferase METTL3 were reduced in prematurely senescent human mesenchymal stem cell (hMSC) models of progeroid syndromes. Transcriptional profiling of m6A modifications further identified MIS12, for which m6A modifications were reduced in both prematurely senescent hMSCs and METTL3-deficient hMSCs. Knockout of METTL3 accelerated hMSC senescence whereas overexpression of METTL3 rescued the senescent phenotypes. Mechanistically, loss of m6A modifications accelerated the turnover and decreased the expression of MIS12 mRNA while knockout of MIS12 accelerated cellular senescence. Furthermore, m6A reader IGF2BP2 was identified as a key player in recognizing and stabilizing m6A-modified MIS12 mRNA. Taken together, we discovered that METTL3 alleviates hMSC senescence through m6A modification-dependent stabilization of the MIS12 transcript, representing a novel epitranscriptional mechanism in premature stem cell senescence.
| S-EPMC7641765 | biostudies-literature
Project description:N6-methyladenosine (m6A) is a highly prevalent mRNA modification that promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other pathways. METTL3 is the major methyltransferase that catalyzes the formation of m6A in mRNA. As 30% to 80% of m6A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m6A. Here, we reexamined the source of m6A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines that continue to have m6A in their mRNA after Mettl3 knockout. We show that these cells express alternatively spliced Mettl3 transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported METTL3 knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after METTL3 deletion, suggesting that reported METTL3 knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3's role in synthesizing m6A using an exon 4 deletion of Mettl3 and found that METTL3 is responsible for >95% of m6A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m6A in the transcriptome, and that remaining m6A in putative METTL3 knockout cell lines is due to the expression of altered but functional METTL3 isoforms.
| S-EPMC9295969 | biostudies-literature
Project description:As the most prevalent mammalian mRNA epigenetic modification, N6-methyladenosine (m6A) has been shown to possess important post-transcriptional regulatory functions. However, the regulatory mechanisms and functional circuits of m6A are still largely elusive. To help unveil the regulatory circuitry mediated by mRNA m6A methylation, we develop here m6A-Driver, an algorithm for predicting m6A-driven genes and associated networks, whose functional interactions are likely to be actively modulated by m6A methylation under a specific condition. Specifically, m6A-Driver integrates the PPI network and the predicted differential m6A methylation sites from methylated RNA immunoprecipitation sequencing (MeRIP-Seq) data using a Random Walk with Restart (RWR) algorithm and then builds a consensus m6A-driven network of m6A-driven genes. To evaluate the performance, we applied m6A-Driver to build the context-specific m6A-driven networks for 4 known m6A (de)methylases, i.e., FTO, METTL3, METTL14 and WTAP. Our results suggest that m6A-Driver can robustly and efficiently identify m6A-driven genes that are functionally more enriched and associated with higher degree of differential expression than differential m6A methylated genes. Pathway analysis of the constructed context-specific m6A-driven gene networks further revealed the regulatory circuitry underlying the dynamic interplays between the methyltransferases and demethylase at the epitranscriptomic layer of gene regulation.
| S-EPMC5226821 | biostudies-literature
Project description:N6-methyladenosine (m6A) is an abundant nucleotide modification in mRNA that is required for the differentiation of mouse embryonic stem cells. However, it remains unknown whether the m6A modification controls the differentiation of normal and/or malignant myeloid hematopoietic cells. Here we show that shRNA-mediated depletion of the m6A-forming enzyme METTL3 in human hematopoietic stem/progenitor cells (HSPCs) promotes cell differentiation, coupled with reduced cell proliferation. Conversely, overexpression of wild-type METTL3, but not of a catalytically inactive form of METTL3, inhibits cell differentiation and increases cell growth. METTL3 mRNA and protein are expressed more abundantly in acute myeloid leukemia (AML) cells than in healthy HSPCs or other types of tumor cells. Furthermore, METTL3 depletion in human myeloid leukemia cell lines induces cell differentiation and apoptosis and delays leukemia progression in recipient mice in vivo. Single-nucleotide-resolution mapping of m6A coupled with ribosome profiling reveals that m6A promotes the translation of c-MYC, BCL2 and PTEN mRNAs in the human acute myeloid leukemia MOLM-13 cell line. Moreover, loss of METTL3 leads to increased levels of phosphorylated AKT, which contributes to the differentiation-promoting effects of METTL3 depletion. Overall, these results provide a rationale for the therapeutic targeting of METTL3 in myeloid leukemia.
| S-EPMC5677536 | biostudies-literature
Project description:Myogenic progenitor/stem cells retain their skeletal muscle differentiation potential by maintaining myogenic transcription factors such as MyoD. However, the mechanism of how MyoD expression is maintained in proliferative progenitor cells has not been elucidated. Here, we found that MyoD expression was reduced at the mRNA level by cell cycle arrest in S and G2 phases, which in turn led to the absence of skeletal muscle differentiation. The reduction of MyoD mRNA was correlated with the reduced expression of factors regulating RNA metabolism, including methyltransferase like 3 (Mettl3), which induces N6-methyladenosine (m6A) modifications of RNA. Knockdown of Mettl3 revealed that MyoD RNA was specifically downregulated and that this was caused by a decrease in processed, but not unprocessed, mRNA. Potential m6A modification sites were profiled by m6A sequencing and identified within the 5' untranslated region (UTR) of MyoD mRNA. Deletion of the 5' UTR revealed that it has a role in MyoD mRNA processing. These data showed that Mettl3 is required for MyoD mRNA expression in proliferative myoblasts.
| S-EPMC5627051 | biostudies-literature
Project description:METTL3 catalyzes the formation of N6-methyl-adenosine (m6A) which has important roles in regulating various biological processes. However, the in vivo function of Mettl3 remains largely unknown in mammals. Here we generated germ cell-specific Mettl3 knockout mice and demonstrated that Mettl3 was essential for male fertility and spermatogenesis. The ablation of Mettl3 in germ cells severely inhibited spermatogonial differentiation and blocked the initiation of meiosis. Transcriptome and m6A profiling analysis revealed that genes functioning in spermatogenesis had altered profiles of expression and alternative splicing. Our findings provide novel insights into the function and regulatory mechanisms of Mettl3-mediated m6A modification in spermatogenesis and reproduction in mammals.
| S-EPMC5587845 | biostudies-literature