Transcriptome (RNA-Seq) of HEK293T and HEK293TΔALKBH5ΔFTO cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE198050 | GEO | 2022/03/09
REPOSITORIES: GEO
Similar Datasets
Project description:Since m6A demethylases (FTO and ALKBH5) have been reported to be involved in pre-mRNA splicing regulation, we hypothesized that dynamic m6A distribution during mRNA maturation might involve removal of m6A in internal exons by FTO or ALKBH5 accompanied by splicing factors. To explore this we performed pull-down assays coupled with protein mass spectrometry.
2022-12-09 | PXD038705 | Pride
Project description:N⁶-methyladenosine (m6A) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. To our knowledge, no systematic investigations have been conducted about the crosstalk between m6A and other modified nucleosides in RNA. Herein, we modified our recently established liquid chromatography-parallel-reaction monitoring (LC-PRM) method by incorporating stable isotope-labeled (SIL) peptides as internal or surrogate standards for profiling epitranscriptomic RWE proteins. We were able to detect reproducibly a total of 114 RWE proteins in HEK293T cells with the genes encoding m6A eraser proteins (i.e., ALKBH5, FTO) and the catalytic subunit of the major m6A writer complex (i.e., METTL3) being individually ablated. Notably, eight proteins were altered by more than 1.5-fold in the opposite directions in HEK293T cells depleted of METTL3 and ALKBH5. Analysis of published m6A mapping results revealed the presence of m6A in the corresponding mRNAs of four of these proteins. Together, we integrated SIL peptides into our LC-PRM method for quantifying epitranscriptomic RWE proteins, and our work revealed potential crosstalks between m6A and other epitranscriptomic modifications. Our modified LC-PRM method with the use of SIL peptides should be applicable for high-throughput profiling of epitranscriptomic RWE proteins in other cell types and in tissues.
2022-09-24 | PXD036032 | Pride
Project description:N6-methyladenosine (m6A) and N6,2′-O‐dimethyladenosine (m6Am) are two abundant modifications found in mRNAs and ncRNAs that can regulate multiple aspects of RNA biology and function mainly through regulation of interaction with specific RNA-binding proteins. Both modifications are linked to development, disease and stress response. To date three methylases and two demethylases have been identified that modify adenosines in mammalian (m)RNAs. Here we provide a comprehensive study of the in vivo protein-protein interactome of the key methyltransferases and demethylases using the BioID proximity biotinylation approach, which allows capturing of stable and short-lived interactions. Our results show that METTL3, METTL16, CAPAM, FTO and ALKBH5 occupy specific non overlapping protein interacting territories. Moreover, the individual interactomes help to understand the biological functions of these enzymes
2021-11-02 | PXD021566 | Pride
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 α-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
Project description:N6-methyladenosine (m6A) modification of messenger RNAs (mRNAs) is a pivotal mechanism controlling mRNA fate in cells. RNA m6A modification is regulated by the functional balance between methyltransferases and demethylases. Here we demonstrated that FTO-IT1 enhancer RNA (eRNA), a long non-coding RNA (lncRNA) transcribed from the last intron of FTO gene is significantly upregulated in CRPC and aggressive tumors compared to primary tumors. FTO-IT1 knockout by CRISPR/Cas9 almost completely blocks growth and G1-S cell cycle transition of both androgen-sensitive and castration-resistant prostate cancer cells. Meanwhile, the mRNA m6A was dramatically increased in FTO-IT knockout PCa cells and we identified FTO-IT1 as a binding partner of FTO. From m6A-seq, we unexpectedly found hypermethylated m6A associated with upregulated levels of the mRNAs for p53 signaling pathway genes in 22Rv1 prostate cancer cells. Mechanistic study showed that FTO-IT1 recruits FTO to the P53 target mRNA to promote their m6A demethylation, which leads to their degradation.
2023-07-09 | GSE189465 | GEO
Project description:We performed RNA-Seq and m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-Seq) to determine whether the altered gene expression and the regulatory functions in cancer immunotherapy could be a consequence of Alkbh5 or Fto-mediated m6A/m6Am demethylation. We used two m6A peak calling algorithms to find the common peaks between the two methods, and only kept the conserved peaks detected in all the biological replicates of each group to obtain the most stringent results. Our data suggest that the enzymatic activity and the altered m6A/m6Am peaks of Fto and Alkbh5 knockout had similarity but also had obvious distinct features, which may lead to the different mechanisms of the two protein to exert their functions in regulating immunotherapy. In addition, we performed single cell RNA-seq on a tumor obtained from a patient who had stage IV melanoma progressive disease and responded well to PD-1 blockage therapy. We observed substantial immune cell infiltration and very few residual melanoma cells, indicators of good immunotherapy responses. We then examined ALKBH5 expression and found that there were more melanoma cells expressing ALKBH5 than the surrounding normal control cells.
2020-08-10 | GSE134388 | GEO
Project description:Here we use MeRIP-Seq to analyze global adenosine methylation (m6A) in mRNAs in the midbrain and striatum of Fto-deficient mice. We find that Fto deficiency leads to increased methylation within a subset of mRNAs important for neuronal signaling, including many within the dopaminergic signaling pathway. Collectively, our results show that Fto regulates demethylation of specific mRNAs in vivo, and this activity relates to control of dopaminergic transmission. Profiling of m6A in midbrain and striatum from FTO knockout mice
2013-06-15 | E-GEOD-47216 | biostudies-arrayexpress
Project description:Various methylases and demethylases catalyze methylation and demethylation of N6-methyladenosine (m6A) and N6,2?-O-dimethyladenosine (m6Am) but precise methylomes uniquely mediated by each methylase/demethylase are still lacking. Here, we developed m6A-Crosslinking-Exonuclease-sequencing (m6ACE-seq) to map m6A and m6Am at transcriptome-wide single-base-resolution. m6ACE-seq's ability to quantify relative differences in methylation levels across samples enabled the generation of a comprehensive atlas of distinct methylomes uniquely mediated by every individual known methylase/demethylase. We determined METTL16 to indirectly impact manifold methylation targets beyond its consensus target motif, and highlighted the importance of precision in mapping PCIF1-dependent m6Am. Rather than reverse RNA methylation, we found that both ALKBH5 and FTO demethylases instead maintain their regulated sites in an unmethylated steady-state. In FTO's absence, anomalous m6Am disrupts snRNA interaction with nuclear export machinery, potentially causing aberrant pre-mRNA splicing events. We propose a model whereby RNA demethylases ensure normal RNA metabolism by suppressing disruptive RNA methylation in the nucleus.
2019-09-20 | GSE124509 | GEO
Project description:We engineered a new m6A editing system consisting of GCN4 (SunTag)-dCas13b, anti-GCN4 single chain variable fragment (scFv)-FTO or ALKBH5-GBI and sgRNA to provide an applicable method to remove m6A modification at specific loci.
2020-10-09 | GSE139647 | GEO
Project description:Objectives: To investigate the role of ALKBH5 in fibroblasts during post-myocardial infarction (MI) repair. Background: N6-methyladenosine (m6A) mRNA modification has been shown to play an important role in cardiovascular diseases. The RNA demethylase, AlkB homolog 5 (ALKBH5), is an m6A "eraser" that is responsible for decreased m6A methylation. However, its role in cardiac fibroblasts during the post-MI healing process remains elusive. Methods: MI was mimicked by permanent left anterior descending artery ligation in global ALKBH5-knockout, ALKBH5-knockin, and fibroblast-specific ALKBH5-knockout mice to study the function of ALKBH5 during post-MI collagen repair. Methylated RNA immunoprecipitation sequencing was performed to explore potential ALKBH5 targets. Results: Dramatic alterations in ALKBH5 expression were observed during the early stage post-MI and in hypoxic fibroblasts. Global ALKBH5 knockin reduced infarct size and improved cardiac function after MI. The global and fibroblast-specific ALKBH5-knockout mice both exhibited low survival rates along with poor collagen repair, impaired cardiac function, and cardiac rupture. Both in vivo and in vitro ALKBH5 loss led to impaired fibroblast activation and decreased collagen deposition. Additionally, hypoxia, but not TGF-β1 or Ang II, upregulated ALKBH5 expression in myofibroblasts in a HIF-1α-dependent transcriptional manner. Mechanistically, ALKBH5 promoted the stability of ErbB4 mRNA and the degradation of ST14 mRNA via m6A demethylation. Fibroblast-specific ErbB4 overexpression ameliorated the impaired fibroblast-to-myofibroblast transformation and poor post-MI repair due to ALKBH5 knockout. Conclusions: Fibroblast ALKBH5 positively regulates post-MI healing via post-transcriptional modification and stabilization of ErbB4 mRNA in an m6A-dependent manner. Targeting ALKBH5/ERBB4 may be a potential therapeutic option for post-MI cardiac rupture.
2023-10-01 | GSE226415 | GEO