RIP-seq of Ythdf3 in MEFs with or without nutrient starvation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE158660 | GEO | 2021/12/30
REPOSITORIES: GEO
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Project description:Oxaliplatin as a first-line drug frequently causes the chemo-resistance on colorectal cancer (CRC). N6-methyladenosine (m6A) methylation has been largely acknowledged in multiple biological functions. However, the molecular mechanisms underlying the m6A methylation in modulating anticancer drug resistance in CRC are still obscure. In present study, RIP-seq was conducted to investigate the occupancy of N6-methyladenosine RNA binding protein 3 (YTHDF3) served as “readers” that can recognize m6A modification site in HCT116 cells with oxaliplatin resistance (HCT116R). Then, YTHDF3 was knockdown by siRNA in HCT116 cells with oxaliplatin resistance, and RIP-seq was further conducted to investigate m6A methylation of HCT116, HCT116R and HCT116R cells with YTHDF3 knockdown.
2022-02-04 | E-MTAB-11321 | biostudies-arrayexpress
Project description:RIP-seq of Ythdf3 in MEFs with or without nutrient starvation
| PRJNA666149 | ENA
Project description:N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in cell differentiation and organism development. It regulates multiple steps throughout the RNA life cycle including RNA processing, translation, and metabolism, via the recognition by selective binding proteins. In cytoplasm, m6A binding protein YTHDF1 facilitates translation of m6A-modified mRNAs, and YTHDF2 accelerates the decay of m6A-modified transcripts. The biological function of YTHDF3, another cytoplasmic m6A binder of the YTH domain family, remains unknown. Here, we report that YTHDF3 promotes protein synthesis in synergy with YTHDF1, and affects methylated mRNA decay mediated by YTHDF2. Cells deficient in all of YTHDF proteins experience the most dramatic accumulation of the m6A-methylated transcripts. These results indicate that in cytoplasm, YTHDF proteins act in an integrated and cooperative network to accelerate metabolism of m6A-modified mRNAs. The combinative and dynamic nature of YTHDF proteins may collectively impact fundamental biological processes and diseases related to m6A RNA methylation.
2016-09-15 | GSE86214 | GEO
Project description:BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) induced by chronic liver damage is a major cause of cancer mortality, but its precise epigenetic mechanisms are severely under studied. In addition, the role of N6-methyladenine (m6A) reader YTHDF3 in human diseases remains poorly understood. METHODS: Liver injury and hepatocarcinogenesis in mice were induced by chemical. CRISPR/Cas9 technology was used to construct Ythdf3 and Mettl14 knockout mice. Hepatic cell population characteristics was determined by means of 10X single-cell RNA-seq and flow cytometry. Cell proliferation and DNA damage were evaluated by immunofluorescence, immunohistochemistry, and western blot. Liver organoids were cultured to examine liver stem cells function. MeRIP-seq was used to reveal alterations in m6A methylation patterns impacted by chemical-induced liver injury. RIP-seq and Ribo-seq were applied to identify YTHDF3 targets and determine translation efficiency. Small interfering RNAs and dCas13b-FTO-sgRNA plasmids were used to evaluate the function of YTHDF3 and CEBPA in vitro. RESULTS: YTHDF3 depletion exacerbated chemical-induced liver injury with a reduction in functional hepatocytes and stem cells. Furthermore, METTL14 and YTHDF3-dependent RNA m6A dysregulation induced DNA damage and promoted development of HCC. Mechanistically, knockout of Ythdf3 impeded the translation of CCAAT/enhancer-binding protein-alpha (CEBPA), subsequently inhibited expression of PARP1 and PRDX2 to promote DNA damage and induce genomic instability, finally leading to liver injury and HCC. CONCLUSIONS: m6A/YTHDF3/CEBPA regulatory axis plays an essential role in governing cell fates and genomic stability, thereby preventing liver injury and HCC, and offers potential therapeutic avenue for targeting YTHDF3 and CEBPA in the treatment of HCC.
2024-02-29 | GSE259225 | GEO
Project description:N6-methyladenosine (m6A) is the most prevalent modification of eukaryotic cells st post-transcriptional level. The goals of this study are to compare the N6-methyladenosine modification of transcripts in normal and senescent nucleus pulposus cells using Me-RIP-seq
2022-03-23 | GSE169484 | GEO
Project description:N6-methyladenosine (m6A) is the most prevalent internal modification present in the mRNA of all higher eukaryotes. Here we present that m6A is selectively recognized by human YTH domain family (YTHDF2) protein to regulate mRNA degradation. By using crosslinking and immunoprecipitation, we have identified over 4000 substrate RNA of YTHDF2 with conserved core motif of G(m6A)C. We further estabilshed the role of YTHDF2 in RNA metabolism by a combination of ribosome profiling, RNA sequencing, m6A level quantification and cell-based imaging: the C-terminal domain of YTHDF2 selectively binds to m6A of mRNA and the N-terminal domain is responsive for localizing mRNA from translatable pool to processing body where mRNA decay occurs. PAR-CLIP and RIP was used to identify YTHDF2 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF2 siRNA knock-down
2013-11-29 | E-GEOD-49339 | biostudies-arrayexpress
Project description:The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader. PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) was applied to human YTHDC1 protein to identify its binding sites.
2014-09-18 | E-GEOD-58352 | biostudies-arrayexpress
Project description:N6-methyladenosine RNA modification drives a rapid shift to cisplatin resistance in bladder cancer by regulating SLC7A11 expression via YTHDF3 [RIP-seq]
| PRJNA968001 | ENA
Project description:BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) induced by chronic liver damage is a major cause of cancer mortality, but its precise epigenetic mechanisms are severely under studied. In addition, the role of N6-methyladenine (m6A) reader YTHDF3 in human diseases remains poorly understood. METHODS: Liver injury and hepatocarcinogenesis in mice were induced by chemical. CRISPR/Cas9 technology was used to construct Ythdf3 and Mettl14 knockout mice. Hepatic cell population characteristics was determined by means of 10X single-cell RNA-seq and flow cytometry. Cell proliferation and DNA damage were evaluated by immunofluorescence, immunohistochemistry, and western blot. Liver organoids were cultured to examine liver stem cells function. MeRIP-seq was used to reveal alterations in m6A methylation patterns impacted by chemical-induced liver injury. RIP-seq and Ribo-seq were applied to identify YTHDF3 targets and determine translation efficiency. Small interfering RNAs and dCas13b-FTO-sgRNA plasmids were used to evaluate the function of YTHDF3 and CEBPA in vitro. RESULTS: YTHDF3 depletion exacerbated chemical-induced liver injury with a reduction in functional hepatocytes and stem cells. Furthermore, METTL14 and YTHDF3-dependent RNA m6A dysregulation induced DNA damage and promoted development of HCC. Mechanistically, knockout of Ythdf3 impeded the translation of CCAAT/enhancer-binding protein-alpha (CEBPA), subsequently inhibited expression of PARP1 and PRDX2 to promote DNA damage and induce genomic instability, finally leading to liver injury and HCC. CONCLUSIONS: m6A/YTHDF3/CEBPA regulatory axis plays an essential role in governing cell fates and genomic stability, thereby preventing liver injury and HCC, and offers potential therapeutic avenue for targeting YTHDF3 and CEBPA in the treatment of HCC.
2024-02-29 | GSE259229 | GEO
Project description:BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) induced by chronic liver damage is a major cause of cancer mortality, but its precise epigenetic mechanisms are severely under studied. In addition, the role of N6-methyladenine (m6A) reader YTHDF3 in human diseases remains poorly understood. METHODS: Liver injury and hepatocarcinogenesis in mice were induced by chemical. CRISPR/Cas9 technology was used to construct Ythdf3 and Mettl14 knockout mice. Hepatic cell population characteristics was determined by means of 10X single-cell RNA-seq and flow cytometry. Cell proliferation and DNA damage were evaluated by immunofluorescence, immunohistochemistry, and western blot. Liver organoids were cultured to examine liver stem cells function. MeRIP-seq was used to reveal alterations in m6A methylation patterns impacted by chemical-induced liver injury. RIP-seq and Ribo-seq were applied to identify YTHDF3 targets and determine translation efficiency. Small interfering RNAs and dCas13b-FTO-sgRNA plasmids were used to evaluate the function of YTHDF3 and CEBPA in vitro. RESULTS: YTHDF3 depletion exacerbated chemical-induced liver injury with a reduction in functional hepatocytes and stem cells. Furthermore, METTL14 and YTHDF3-dependent RNA m6A dysregulation induced DNA damage and promoted development of HCC. Mechanistically, knockout of Ythdf3 impeded the translation of CCAAT/enhancer-binding protein-alpha (CEBPA), subsequently inhibited expression of PARP1 and PRDX2 to promote DNA damage and induce genomic instability, finally leading to liver injury and HCC. CONCLUSIONS: m6A/YTHDF3/CEBPA regulatory axis plays an essential role in governing cell fates and genomic stability, thereby preventing liver injury and HCC, and offers potential therapeutic avenue for targeting YTHDF3 and CEBPA in the treatment of HCC.
2024-02-29 | GSE259226 | GEO