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RNA-seq and Ribo-seq analysis of the effects of METTL3/14 depletion on gene expression during the interferon response in Huh7 cells

ABSTRACT: The RNA modification N6-methyladenosine (m6A) regulates gene expression through various transcript-specific effects. The overall goal of these experiments was to determine the effects of the m6A methyltransferase complex proteins METTL3 and METTL14 on the expression of interferon stimulated genes. First, we depleted METTL3 and METTL14 in Huh7 cells using siRNAs and treated with Mock or IFN-β to analyze the regulatory effects of METTL3/14 on interferon-stimulated genes' transcript abundance. We then used Ribo-seq following METTL3/14 siRNA depletion and IFN treatment to quantify its effect on the translation of ISGs and other genes. Overall, we found that METTL3/14 exerts transcript-specific regulatory effects on a subset of interferon-stimulated genes. Overall, METTL3/14 appeared to enhance the translation of a subset of interferon-stimulated genes. We characterized the molecular effects of METTL3/14 and m6A on certain ISGs and determined that m6A augments the antiviral effects of the interferon response.

ORGANISM(S): Homo sapiens

PROVIDER: GSE155447 | GEO | 2021/02/19

REPOSITORIES: GEO

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MeRIP-seq mapping of m6A in the IFN-β induced transcriptome in Huh7 cells

Project description:The RNA modification N6-methyladenosine (m6A) regulates gene expression through various transcript-specific effects. The goal of this experiment was to identify m6A-modified transcripts during the type I interferon response, with interest in determining which interferon-stimulated genes are modified by m6A. We found that a high percentage of ISGs are m6A-modified, and in follow-up experiments we determined the effects of m6A on the expression of interferon-stimulated genes. We found that m6A regulates the translation of a subset of ISGs, which in turn augments the antiviral effects of the type I interferon respnose.

2021-02-19 | GSE155413 | GEO

Widespread N6-methyladenosine-dependent RNA Structural Switches Regulate RNA-Protein Interactions

Project description:We show that N6-methyladenosine (m6A), the most abundant internal modification in mRNA/lncRNA with still poorly characterized function, alters RNA structure to facilitate the access of RBM for heterogeneous nuclear ribonucleoprotein C (hnRNP C). We term this mechanism m6A-switch. Through combining PAR-CLIP with Me-RIP, we identify 39,060 m6A-switches among hnRNP C binding sites transcriptome-wide. We show that m6A-methyltransferases METTL3 or METTL14 knockdown decreases hnRNP C binding at 16,582 m6A-switches. Taken together, 2,798 m6A-switches of high confidence are identified to mediate RNA-hnRNP C interactions and affect diverse biological processes including cell cycle regulation. These findings reveal the biological importance of m6A and provide insights into the sophisticated regulation of RNA-RBP interactions through m6A-induced RNA structural remodeling. Measure the m6A methylated hnRNP C binding sites transcriptome-wide by PARCLIP-MeRIP; measure the differential hnRNP C occupancies upon METTL3/METTL14 knockdown by PAR-CLIP; measure RNA abundance and splicing level changes upon HNRNPC, METTL3 and METTL14 knockdown

2015-02-27 | E-GEOD-56010 | biostudies-arrayexpress

Primarily Dissecting the Autonomous and Cooperative Functions of METTL3 and METTL14 in mRNA Modification and Tumor Progression

Project description:METTL3 and METTL14 are considered to faithfully form the m6A writing complex in a 1:1 ratio, regulating the fate of mRNA by adding m6A modifications. However, recent studies have shown inconsistent expression and prognostic value of METTL3 and METTL14 in some tumors, suggesting that they may not be faithful in tumors. Pan-cancer analysis based on TCGA data reveals significant differences in expression, function, tumor burden correlation, and immune correlation between METTL3 and METTL14, especially in esophageal squamous cell carcinoma (ESCC). Knockdown of METTL3 significantly inhibits the cell proliferation in vitro and in vivo in ESCC EC109 cells, while the impact of METTL14 knockdown on proliferation is limited, and it cannot abolish the expression of METTL3 protein. mRNA-seq results indicate that METTL3 independently regulates the expression of 1615 genes, while only 776 genes are co-regulated by METTL3 and METTL14. Furthermore, through immunofluorescence co-localization, it is observed that METTL3 and METTL14 have certain inconsistencies in cellular localization. HPLC-MS results show that METTL3 independently binds to the Nop56p-associated pre-rRNA complex and mRNA splicing complex, separate from METTL14. Through bioinformatics and various omics studies, we have preliminarily discovered that METTL3 independently regulating tumor cell proliferation, and the participation in mRNA splicing may be a critical molecular mechanism. Our study provides an experimental basis and theoretical foundation for further understanding of the m6A writing complex and tumor therapy targeting METTL3.

2024-02-01 | PXD048886 | Pride

m6A independent genome-wide METTL3 and METTL14 redistribution drives senescence-associated secretory phenotype [ChIP-seq]

Project description:Methyltransferase-like 3 (METTL3) and 14 (METTL14) are core subunits of the methyltransferase complex (MTC) that catalyzes mRNA N6-methyladenosine (m6A) modification. Despite the expanding list of m6A-dependent function of the MTC, m6A independent function of the METTL3 and METTL14 complex remains poorly understood. Here we show that genome-wide redistribution of METTL3 and METTL14 drives senescence-associated secretory phenotype (SASP) in a m6A-independent manner. METTL3 and METTL14 are necessary for SASP. However, SASP is not regulated by m6A mRNA modification. METTL14 is redistributed to the enhancers, while METTL3 is localized to the pre-existing NF-B sites within the promoters of the SASP genes during senescence. METTL3 interacts with NF-B and they are mutually dependent on their associations with the promoters of SASP genes. METTL14 but not METTL3 is necessary for function of SASP gene enhancers. METTL3 and METTL14 are required for both the tumor-promoting and immune surveillance functions of senescent cells mediated by SASP in vivo in mouse models. In summary, our results report a m6A independent function of the METTL3 and METTL14 complex in promoting SASP through regulating transcription by genome-wide redistribution of METTL14 to enhancers and METTL3 to promoters of SASP genes during senescence.

2020-12-10 | GSE141992 | GEO

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m6A independent genome-wide METTL3 and METTL14 redistribution drives senescence-associated secretory phenotype [RNA-seq]

2020-12-10 | GSE141991 | GEO

m6A independent genome-wide METTL3 and METTL14 redistribution drives senescence-associated secretory phenotype [MeRIP-seq]

2020-12-10 | GSE141993 | GEO

RNA methylation destabilizes developmental regulators in murine embryonic stem cells (m6A)

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.

2014-01-06 | E-GEOD-46878 | biostudies-arrayexpress

Identification and Characterization of the Mammalian Nuclear RNA N6-Adenosine Methyltransferase Core Complex

Project description:N6-methyladenosine (m6A) is the most prevalent internal modification found in mammalian messenger and non-coding RNAs. The discoveries of functionally significant demethylases that reverse this methylation as well as the recently revealed m6A distributions in mammalian transcriptomes strongly indicate regulatory functions of this modification. Here we report the identification and characterization of the mammalian nuclear RNA N6-adenosine methyltransferase core (RNMTC) complex. Besides METTL3, a methyltransferase which was the only known component of RNMTC in the past, we discovered that a previously uncharacterized methyltransferase, METTL14, exhibits a N6-adenosine methyltransferase activity higher than METTL3. Together with WTAP, the third component that dramatically affects the cellular m6A level, these three proteins form the core complex that orchestrates m6A deposition on mammalian nuclear RNA. Biochemistry assays, imaging experiments, as well as transcriptome-wide analyses of the binding sites and their effects on m6A methylation support methylation function and reveal new insights of RNMTC. PAR-CLIP and m6A-seq in HeLa cells

2013-11-29 | E-GEOD-46705 | biostudies-arrayexpress

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