Project description:N6-methyladenosine (m6A) is the most abundant mRNA modification in mammalian cells, mediated co-transcriptionally by a methyltransferase ‘writer’ complex containing METTL3 as the S-adenosyl methionine (SAM)-binding subunit as well as adaptors such WTAP and ZC3H13
Project description:We have developed a modified eCLIP-based method (meCLIP) to identify m6A residues at single-nucleotide resolution. By coupling the improvements of eCLIP with an easy-to-use computational pipeline, we have successfully identified over 50,000 unique m6A residues in the two breast cancer cell lines that were analyzed (MCF-7 and MDA-MB-231) and over 8,000 unique residues in HEK-293 cells. We compared these residues to the sites called using the currently most utilized m6A identification method (miCLIP).
Project description:Toxoplasma gondii (T. gondii) is an opportunistic parasite. After infection, macrophages finely regulate the immune response to restrict parasite proliferation. It is well-known that N6-methyladenosine (m6A) plays a critical role in fine-tuning gene expression. To investigate whether m6A modification is involved in regulating the anti-infection immune response in macrophages against T. gondii, this study utilized T. gondii tachyzoites from the RH strain to infect human THP-1 macrophages. qPCR and ELISA results showed that T. gondii infection mounted the expression of TNF-α. RNA-seq profiling showed that T. gondii infection was associated with difference in genes from pathway associated with TNF signaling. Expression of m6A regulators were evaluated using qPCR and Western blotting. T. gondii infection increased the abundance of m6A methyltransferase WTAP and demethylase FTO. Joint analysis of RNA-seq and m6A-seq data was utilized for enriching differentially expressed genes with significantly altered m6A modifications. After T. gondii infection, the m6A levels of genes associated with TNF signaling were significantly altered. In this study, we found that m6A methylation involved in T. gondii infection induced TNF-α expression.
Project description:This SuperSeries is composed of the following subset Series: GSE36958: Gene expression profiles of WT and ime4-/- mutant yeast cells, under vegetative and meiosis-inducing conditions GSE37001: METTL3 KD in HepG2 cells GSE37002: m6A mapping in human RNA (with treatments) GSE37003: m6A mapping in human RNA (untreated) GSE37004: m6A mapping in mouse RNA (mouse liver and human brain) Refer to individual Series
Project description:By performing m6A-seq analysis on the peritoneal macrophages that derived from ALKBH5-/- mice and littermate mice infected with or without vesicular stomatitis virus (VSV), we want to investigate whether ALKBH5 deficiency-mediated m6A RNA methylation contributes to the regulation of its target genes expression. m6A-seq analysis revealed enriched and specific m6A peaks on the transcript of ALKBH5-targeted gene, which were substantially increased in ALKBH5-deficient peritoneal macrophages than that in wild-type cells whatever infected with or without VSV. Meanwhile we didn’t observe up-regulation of m6A signal on VSV in ALKBH5-deficient cells; and also didn't find significant difference of m6A signals on IFN-β mRNA between ALKBH5-deficient and wild type cells whatever infected with or without VSV. These demonstrated that deficiency of ALKBH5 controls viral replication by increasing the m6A modification of ALKBH5 target gene to regulate its expression.
Project description:To investigate the role of METTL3-mediated m6A modification in macrophage, we performed m6A-sequencing to map the m6A modification in bone-marrow-derived macrophages (BMDMs) in wild type (WT) and Mettl3-/- mice.
Project description:Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using a recently developed enhanced UV crosslinking and immunoprecipitation (eCLIP) approach, we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region- and binding site-level IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3'UTR-enriched targets. RNA Bind-N-Seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and an increase in cell death. For cell adhesion, in hPSCs we find IMP1 maintains levels of integrin mRNA, specifically regulating RNA stability of ITGB5. Additionally, we show IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. eCLIP-seq was performed in biological replicate for IGF2BP1/IMP1 and IGF2BP2/IMP2, as well as one replicate each for IGF2BP3/IMP3, RBFOX2, and an IgG control. Each sample has a size-matched input control for analysis