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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE46705 | GEO | 2013/11/29

SECONDARY ACCESSION(S): PRJNA202057

REPOSITORIES: GEO

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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

m6A RNA modification controls cell fate transition in mammalian embryonic stem cells

Project description:N6-methyl-adenosine (m6A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m6A by mapping the m6A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m6A modification, including transcripts encoding core pluripotency transcription factors. m6A is enriched over 3M-bM-^@M-^Y untranslated regions at defined sequence motifs, and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m6A methylases, led to m6A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESCM-bM-^@M-^Ys exit from self-renewal towards differentiation into several lineages in vitro and in vivo. Thus, m6A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages. Examing m6A modification differences in two different cell types

2014-10-20 | E-GEOD-52600 | biostudies-arrayexpress

LC-MS/MS analysis of co-immunoprecipitation products of METTL3

Project description:N6-methyladenosine (m6A) methylation of mRNA by the methyltransferase complex (MTC), with core components including METTL3-METTL14 heterodimers and Wilms’ tumor 1-associated protein (WTAP), contributes to breast tumorigenesis, but the mechanism of MTC assembly remains elusive. Here, we identify a novel cleaved form METTL3a (residues 239-580 of METTL3), that is highly expressed in breast cancer. Furthermore, we find that both METTL3a and full-length METTL3 are required for MTC assembly, RNA m6A deposition, as well as cancer cell proliferation. Mechanistically, we find that METTL3a is required for METTL3-METTL3 interaction, which is a prerequisite step for recruitment of WTAP in MTC assembly. Analysis of m6A sequencing data shows that depletion of METTL3a globally disrupts m6A methylation, and METTL3a mediates mTOR activation via m6A-mediated suppression of TMEM127 expression. Consequently, we find that METTL3 cleavage is mediated by proteasome in an mTOR-dependent manner, revealing positive regulatory feedback between METTL3a and mTOR signaling. Our findings reveal METTL3a as an important component for MTC assembly, and suggest the METTL3a-mTOR axis as a potential therapeutic target for breast cancer.

2023-08-18 | PXD036899 | Pride

METTL3-mediated m6A modification is required for cerebellar development m6A modification and cerebellar development

Project description:N6-methyladenosine (m6A) RNA methylation is the most abundant modification on mRNAs and plays important roles in various biological processes. The formation of m6A is catalyzed by a methyltransferase complex including methyltransferase like 3 (METTL3) as a key factor. However, the in vivo functions of METTL3 and m6A modification in mammalian development remain unclear. Here we show that specific inactivation of Mettl3 in mouse nervous system causes severe developmental defects in the brain. Mettl3 conditional knockout mice manifest cerebellar hypoplasia caused by drastically enhanced apoptosis of new born cerebellar granule cells (CGCs) in the external granular layer (EGL). METTL3 depletion induced loss of m6A modification causes extended RNA half-lives and aberrant splicing events, consequently leading to dysregulation of transcriptome-wide gene expression and premature CGC death. Our findings reveal a critical role of METTL3-mediated m6A in regulating the development of mammalian cerebellum.

2018-07-15 | GSE100528 | GEO

Arabidopsis Messenger RNA N6-methyladenosine Demethylation Regulates Plant Development

Project description:N6-methyladenosine (m6A) is the most abundant internal modification in the messenger RNA (mRNA) of all higher eukaryotes. This modification has been shown to be reversible in mammals; it is installed by a methyltransferase heterodimer complex of METTL3 and METTL14 bound with WTAP, and reversed by iron(II)- and Î±-ketoglutarate-dependent demethylases FTO and ALKBH5. This modification exhibits significant functional roles in various biological processes. The m6A modification as a RNA mark is recognized by reader proteins, such as YTH domain family proteins and HNRNPA2B1; m6A can also act as a structure switch to affect RNA-protein interactions for biological regulation. In Arabidopsis thaliana, the methyltransferase subunit MTA (the plant orthologue of human METTL3, encoded by At4g10760) was well characterized and FIP37 (the plant orthologue of human WTAP) was first identified as the interacting partner of MTA. Here we report the discovery and characterization of reversible m6A methylation mediated by AtALKBH10B (encoded by At4g02940) in A. thaliana, and noticeable roles of this RNA demethylase in affecting plant development and floral transition. Our findings reveal potential broad functions of reversible mRNA methylation in plants. m6A peaks were identified from wild type Columbia-0 and atalkbh10b-1 mutant in two biological replicates

2016-03-24 | E-GEOD-79523 | biostudies-arrayexpress

Mapping of m6A and Its Regulatory Targets in Prostate Cancer Reveals a METTL3-low Induction of Therapy Resistance

Project description:Genomic and transcriptomic alterations are insufficient to explain the variance in protein expression seen in cancer. Recent evidence has highlighted the role of N6-methyladenosine (m6A) in the regulation of mRNA expression, stability and translation, supporting a potential role for post-transcriptional regulation mediated by m6A in cancer. Here we explore prostate cancer as an exemplar cancer and generate the first prostate m6A maps, and further examined how low levels of N6-adenosine-methyltransferase (METTL3) associates with advanced prostate cancer and results in altered expression at the level of transcription, translation, and protein. In particular extracellular matrix proteins have a high number of m6A sites and show significant changes in expression with METTL3 knock-down. We also discovered the upregulation of a hepatocyte nuclear factor-driven gene signature that is associated with therapy resistance in prostate cancer. Significantly, METTL3 knock-down rendered the cells resistant to androgen receptor antagonists, implicating changes in m6A as a mechanism for therapy resistance in metastatic prostate cancer.

2021-06-07 | PXD022348 | Pride

Gene expression in Mon-Mac-6 cells treated with METTL3 PROTAC, WD6305 and inhibitor, UZH2

Project description:N6-methyladenosine (m6A) methylation is the most prevalent RNA modification, which plays a critical role in various bioprocesses.1 This modification is predominantly catalyzed by the m6A methyltransferase complex (MTC), consisting of core subunits such as methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14), along with cofactors like Wilms tumor 1-associated protein (WTAP). We described the development of a first-in-class potent and selective METTL3-targeted degrader, WD6305, which can effectively degrade both METTL3 and its partner protein METTL14. WD6305 showed superior antiproliferative effects in multiple AML cell lines compared to its parent inhibitor, UZH2. Here, we want to compare the changes in genes between the two.

2023-11-29 | GSE247499 | GEO

Profiling of N6-Adenosine (m6A) Methylation in HepG2 cells

Project description:N6-methyladenosine (m6A) modification is the most abundant internal RNA modification in eukaryotes. Recent studies have shown that the dynamic and reversible regulation of m6A modifications in mRNAs or non-coding RNAs plays critical roles in tissue development, stem cell self-renewal and differentiation, control of heat shock response, and circadian clock controlling, as well as in RNA metabolism and processing. METTL14 is a major m6A writer which together with METTL3 forms the core of the methyltransferase complex that catalyzes the conversion of adenosine (A) to m6A. To investigate the effect of METTL14 on m6A modfication, we performed m6A-seq in HepG2 cells with stably expressed shRNAs against METTL14 (shMETTL14) or non-specific control shRNA (shNS).

2018-03-26 | GSE90642 | GEO

Non-canonical function of histone methyltransferase G9a in the translational regulation of chronic inflammation

Project description:We report a novel translation-regulatory function of G9a, a histone methyltransferase and well-understood transcriptional repressor, in promoting hyperinflammation and lymphopenia; two hallmarks of endotoxin tolerance (ET)-associated chronic inflammatory complications. Using multiple approaches, we demonstrate that G9a interacts with multiple translation regulators during ET, particularly the N6-methyladenosine (m6A) RNA methyltransferase METTL3, to co-upregulate expression of certain m6A-modified mRNAs that encode immune-checkpoint and anti-inflammatory proteins. Mechanistically, G9a promotes m6A methyltransferase activity of METTL3 at translational/post-translational level by regulating its expression, its methylation, and its cytosolic localization during ET. Additionally, from a broader view extended from the G9a-METTL3-m6A translation regulatory axis, our translatome proteomics approach identified numerous “G9a-translated” proteins that unite the networks associated with inflammation dysregulation, T cell dysfunction, and systemic cytokine response. In sum, we identified a previously unrecognized function of G9a in protein-specific translation that can be leveraged to treat ET-related chronic inflammatory diseases.

2023-10-20 | PXD044382 | Pride

METTL3 preferentially enhances non-m6A translation of epigenetic factors and promotes tumorigenesis (meRIP, RIP and polysome-seq)

Project description:Methyltransferase-like 3 (METTL3) is the best known m6A methyltransferase of the N6-adenosine-methyltransferase complex that functions in the reversible epi-transcriptome modulation of m6A modification. Besides acting as a m6A methyltransferase, METTL3 also regulates mRNA translation as well as other biological processes either through m6A “reader”-mediated downstream effect or directly binding to m6A sites, but the underlying mechanism and biological significance of these cytoplasmic events remain undefined. Here, we demonstrated that METTL3 inhibition significantly delayed gastric tumorigenesis in patient-derived xenograft model. Intriguingly, global METTL3-binding profiling revealed METTL3 occupied numerous oncogenic mRNAs without m6A signals, indicating a novel mechanism independent of m6A machinery. Furthermore, METTL3 was observed to translocate from nucleus to cytoplasm during gastric carcinogenesis, and its cytoplasm-to-nucleus ratio was correlated with cancer progression. In addition, the nuclear retention of METTL3 nearly abolished its tumor-promoting activity. Mechanistically, METTL3 interacted with PABPC1 to promote RNA looping, thus facilitating the translation of multiple oncogenic mRNAs. Taken together, our findings indicate an unexpected role of METTL3 as an important translational regulator independent of either m6A-“writer” or -“reader” activities and suggest METTL3 as a therapeutic target in gastric cancer.

2022-05-16 | GSE163310 | GEO

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