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Transcript-wide m6A methylation affects the cap-independent translation initiation [RNA-seq]

ABSTRACT: In this study, we generated transcriptome (RNA-Seq) and translatome (Ribo-Seq) profiles from the wild-type HEK293T cells and HEK293T cells with knockdown of METTL3 and METTL14 (HEK293T KD), cultivated in normal conditions or under Torin1 treatment. The data were produced to explore the effect of m6A methylation on cap-dependent translation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE309586 | GEO | 2025/12/01

REPOSITORIES: GEO

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Transcript-wide m6A methylation affects the cap-independent translation initiation [Ribo-seq]

Project description:In this study, we generated transcriptome (RNA-Seq) and translatome (Ribo-Seq) profiles from the wild-type HEK293T cells and HEK293T cells with knockdown of METTL3 and METTL14 (HEK293T KD), cultivated in normal conditions or under Torin1 treatment. The data were produced to explore the effect of m6A methylation on cap-dependent translation.

2025-12-01 | GSE309588 | GEO

Identify METTL3, 14 and 16-bound transcripts via RIP-seq

Project description:To identify the directly bound transcripts of METTL3, METTL14, and METTL16, RNA immunoprecipitation sequencing (RIP-seq) was conducted HEK293T stablely expressing METTL3, METTL14, and METTL16, respectively. Briefly, HEK293T cells were infected with lentivirus, pmiRNA1-3 x Flag-METTL3, pmiRNA1-3 x Flag-METTL14, and pmiRNA1-3 x Flag-METTL14, to overexpress the three METTL family members with 3 x Flag fused in the N-terminal. Only the GFP-positive cells were used for study and expanded in DMEM medium.

2021-10-28 | GSE156797 | GEO

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

Analysis of the eIF4E3-dependent translatome after Torin1-induced stress in N2a cells ctrl and eIF4E3 KO

Project description:The eIF4E are a family of initiation factors that bind the mRNA 5’ cap, regulating the proteome and the cellular phenotype. eIF4E1 mediates global translation and its activity is controlled via the PI3K/AKT/mTOR pathway. mTOR down-regulation results in eIF4E1 sequestration into an inactive complex with the 4E binding proteins (4EBPs). The second member, eIF4E2, regulates the translatome during hypoxia. However, the exact function of the third member, eIF4E3, has remained elusive. We have dissected its function using a range of techniques. Starting from the observation that it does not interact with 4EBP1, we demonstrate that eIF4E3 recruitment into an eIF4F complex occurs when Torin1 inhibits the mTOR pathway. Ribo-seq studies demonstrate that this complex (eIF4FS) is translationally active only during stress and that it selects specific mRNA populations based on 5’ TL (UTR) length. The interactome reveals that it associates with cellular proteins beyond the cognate initiation factors, suggesting that it may have “moon-lighting” functions. Finally, we provide evidence that cellular metabolism is altered in an eIF4E3 KO background but only upon Torin1 treatment. We propose that eIF4E3 acts as a second branch of the integrated stress response, re-programming the translatome to promote “stress resistance” and adaptation.

2021-02-24 | GSE167324 | GEO

Determine the methyltransferase activities of METTL3, METTL14, and METTL16 via MeRIP-seq (m6A-seq)

Project description:All the three enzymes, METTL3, METTL14, and METTL16, belong to methyltransferase like (METTL) family and possess the ability to deposit N6-methyladenosine (m6A) in mRNA. Via immunofluorescence staining and wertern blotting, we discovered either METTL3 or METTL14 mainly localize in nuclear, but METTL16 localizes in both cytosol and nuclei. To compare the m6A methyltransferase activities of the three m6A 'writers' and better understand their differences, MeRIP-seq (m6A-seq) was conducted with poly(A) RNAs isolaed from HEK293T cells with METTL3, 14 and 16 knockout.

2021-10-28 | GSE182607 | 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

Transcript-wide m6A methylation affects the cap-independent translation initiation [Ribo-seq]

Project description:Transcript-wide m6A methylation affects the cap-independent translation initiation [Ribo-seq]

| PRJNA1336295 | ENA

HEK293T cell translatome analysis upon Torin1 treatment or single amino acid withdrawal. Related to Supplementary Table 2 from An et al (2020)

Project description:HEK293T subject to Torin1 treatment or amino acid withdraw dataset and translatome analysis was determined using Azidohomoalanine (AHA) incorportation, Biotin-click, and streptavidin enrichment. TMT 10plex labelling was used. Three genotypes, WT (24 fractions), ATG7 KO (HA-9-53-1 single shot), RB1CC1 KO (HA-9-53-2 single shot).

2020-04-30 | PXD018252 | Pride

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

Project description: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.

2021-02-19 | GSE155447 | GEO

Cancer Mutations Rewire the RNA Methylation Specificity of METTL3-METTL14

Project description:Chemical modification of RNAs is important for post-transcriptional gene regulation. The METTL3-METTL14 complex generates most N6-methyladenosine (m6A) modifications in mRNAs, and dysregulated methyltransferase expression has been linked to numerous cancers. Here we show that m6A modification location, rather than the overall modification level, can impact oncogenesis. A gain-of-function missense mutation found in cancer patients, METTL14R298P, promotes malignant cell growth in culture and in transgenic mice. The mutant methyltransferase preferentially modifies noncanonical sites and transforms gene expression without increasing global m6A levels in mRNAs. The altered substrate specificity is intrinsic to METTL3-METTL14, helping us to propose a structural model for how the METTL3-METTL14 complex detects RNA sequences. Together, our work highlights that m6A location is important for function and that noncanonical methylation sites may impact aberrant gene expression and oncogenesis.

2024-12-21 | PXD039447 | Pride

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