Project description:N6-methyladenosine (m6A), the most prevalent modification of mRNAs at the (G/A)(m6A)C motif installed by METTL3 that plays critical role in regulation of gene expression. METTL3 is essential for embryonic development, and its dysregulation is linked to various diseases including metabolic disorders and cancer. Here, we provide insight into this by generating Mettl3fl/fl; Alb-Cre (M3LKO) mice carrying a hepatocyte-specific deletion of Mettl3. RNA-seq and MeRIP-seq analyses of mRNA identified significant dysregulation of metabolic genes in M3LKO mice.
Project description:m6A-seq of mouse embryonic stem cell wildtype or mutant depleted of Mettl3 or Mettl14 m6A-mRNA library for mouse embryonic stem cell each having one biological replicate were generated using HiSeq2000 v3 flowcell (Illumina) and sequenced for 100 bases with separate 7 base indexing read in a single lane.
Project description:We performed m6A-RIPs in Ascl1-induced neurons (iNeurons) to investigate the neuronal m6A epitranscriptome. Immunoprecipitation was done twice using two different antibodies, acquired from Abcam and Synaptic Systems (SySy), allowing for a more robust detection of m6A modification marks. Additionally, RIP-seq was performed separately with intact and fragmented RNA. The former approach allowed to identify proportions of m6A-modified transcripts among the total number, while the latter approach provided the information to identify genomic coordinates of m6A peaks.
Project description:The intestinal microbiota modulates host physiology and gene expression via mechanisms that are not fully understood. A recently discovered layer of gene expression regulation is N6-methyladenosine (m6A) and N6,2′ -O-dimethyladenosine (m6Am) modifications of mRNA. To unveil if these epitranscriptomic marks are affected by the gut microbiota, we performed methylated RNA-immunoprecipitation and sequencing (MeRIP-seq) to examine m6A-modifications in transcripts of mice displaying either a conventional, or a modified, or no gut microbiota and discovered that the microbiota has a strong influence on m6A- modifications in the cecum, and also, albeit to a lesser extent, in the liver, affecting pathways related to metabolism, inflammatory and antimicrobial responses . We furthermore analysed expression levels of several known writer and eraser enzymes and found the methyltransferase Mettl16 to be downregulated in absence of a microbiota. As a consequence, one of its targets, the S-adenosyl methionine synthase Mat2a was less expressed in mice without gut flora. We furthermore show that distinct commensal bacteria, Akkermansia muciniphila, Lactobacillus plantarum can affect specific m6A modifications. Together, we report here epitranscriptomic modifications as an additional level of interaction in the complex interplay between commensal bacteria and their host.
Project description:XIST is a long non-coding RNA (lncRNA) that mediates transcriptional silencing of X chromosome genes. Here we show that XIST is highly methylated with at least 78 N6-methyladenosine (m6A) residues, a reversible base modification whose function in lncRNAs is unknown. We show that m6A formation in XIST, as well as cellular mRNAs, is mediated by RBM15 and its paralog RBM15B, which bind the m6A-methylation complex and recruit it to specific sites in RNA. This results in methylation of adenosines in adjacent m6A consensus motifs. Furthermore, knockdown of RBM15 and RBM15B, or knockdown of the m6A methyltransferase METTL3 impairs XIST-mediated gene silencing. A systematic comparison of m6A-binding proteins shows that YTHDC1 preferentially recognizes m6A in XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m6A. These data reveal a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression. Three to four biological HEK293T replicates were used to perform iCLIP of endogenous YTH proteins, RBM15, and RBM15B. Crosslinking induced truncations were identified using CIMS-CITS pipeline.
Project description:We report the application of sequencing technology for high-throughput profiling of mRNA m6A methylation in mice colonic epithelial cells with or without mettl14 deletion. By obtaining over thirty billion bases of sequence from m6A antibody immunoprecipitated mRNA, we generated m6A methylation maps of mouse colonic epithelial cells with or with mettl14 deletion. We find that GsdmC2, GsdmC3, GadmC3 mRNA methylation is robustly decreased in colonic epithelial cells upon mettl14 deletion, leading to the decrease of Gsdmc expression.
Project description:The purpose of this protocol is to develop a detailed MRI technique and haemodynamic maps enabling early detection of colorectal metastases in the liver.
Project description:Epigenetic modifications are known to profoundly affect the development and behavior of social insects. In the well-known caste differentiation process of honeybee (Apis mellifera), female larvae with identical genomes are fed royal jellydifferently and develop into either normal workers or into very large, long-lived, and extremely fecund queens, and the queen-worker asymmetry of honeybee is known to be result largely to differential genomic imprinting during larval development that involves DNA methylation-based regulation. The discovery of reversible N6-methyladenosine (m6A) RNA methylation modification has defined a new era for RNA-metabolism-related genetic regulation, yet much remains unknown about m6A-mediated post-transcriptional regulatory mechanisms. Here, we report the first honeybee RNA m6A methylome. Specifically, we used the m6A-seq technique to examine the RNA m6A methylomes of honeybee larvae, including queen and worker larvae at multiple instar stages. We identified multiple conserved features of m6A methylation machinery and transcriptome-wide m6A distribution trends among insect species, and observed that m6A marks exert functions in regulating caste differentiation, with apparently particularly strong functional impacts on fifth instar worker larvae. Functional annotation of differentially methylated candidate caste-differentiation-related transcripts revealed many known regulators of caste differentiation (e.g. ILP-2, p110, PI3K, and JHAMT etc.) as well as the widely-studied Vitellogenin gene, which has not previously been implicated in caste differentiation. As ever-more regulatory roles for m6A marks are discovered, honeybees may become an excellent model studying the biology of such epi-transcriptomic regulatory systems, from embryonic development through holometabolous caste-specific development and on towards behavior and the emergent social hierarchies underlying eusociality in animals.
Project description:m6A is a ubiquitous RNA modification in eukaryotes. Transcriptome-wide m6A patterns in Arabidopsis have been assayed recently. However, m6A differential patterns among organs have not been well characterized. The goal of the study is to comprehensively analyze m6A patterns of numerous types of RNAs, the relationship between transcript level and m6A methylation extent, and m6A differential patterns among organs in Arabidopsis. In total, 18 libraries were sequneced. For the 3 organs: leaf, flower and root, each organ has mRNA-Seq, m6A-Seq and Input sequenced. And each sequence has 2 replicats.
Project description:m6A profiling in two accessions of Arabidopsis thaliana (Can-0 and Hen-16) using the m6A-targeted antibody coupled with high-throughput sequencing m6A-seq in two accessions of Arabidopsis, two replicates for each sample