M5C MeRIP sequencing in PANC-1 cells to map the m5C methylome of PANC-1 cells treated with CAF-derived Evs
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE263948 | GEO | 2024/04/20
REPOSITORIES: GEO
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Project description:The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the embryo undergoes dramatic reprogramming to convert maternal environment to embryonic-driven programing. However, how the maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) in zebrafish early embryos, we show that m5C methylated maternal mRNAs display higher stability during MZT. We identify that the Y box-binding protein 1 (Ybx1) prefers to recognizing m5C-modified mRNAs through p-p interaction with a key residue Trp45 in its cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Cooperated with an mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates a novel mechanism of RNA m5C methylation-regulated maternal mRNA stability during zebrafish MZT, highlighting the critical role of m5C mRNA methylation in early development.
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Project description:The modification and recognition of 5-methylcytosine (m5C) are involved in the initiation and progression of various tumor types. However, the precise role and potential mechanism of Y-box-binding protein 1 (YBX1) in esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we found that YBX1 was frequently upregulated in ESCC compared with matched nontumor tissues. Gain- and loss-of-function assays showed that YBX1 promoted the proliferation and metastasis of ESCC cells both in vitro and in vivo. Functional studies revealed that NOP2/Sun RNA methyltransferase family member 2 (NSUN2) is a critical RNA methyltransferase that facilitates YBX1-mediated ESCC progression. Mechanistically, integrated analysis based on RNA immunoprecipitation sequencing (RIP-seq) and m5C methylated RNA immunoprecipitation and sequencing (MeRIP-seq) assays identified spermine oxidase (SMOX) as a target gene containing an m5C site in its coding sequence (CDS) region, which coincided well with the binding site of YBX1. Overexpression of SMOX-WT but not SMOX-Mut partially restored the proliferation and invasion ability of ESCC cells curbed by YBX1 knockdown. Moreover, YBX1 activated the mTORC1 signaling pathway by stabilizing SMOX mRNA. Our study revealed that YBX1 promotes ESCC development by stabilizing SMOX mRNA in an m5C-dependent manner, thus providing a valuable therapeutic target for ESCC.
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Project description:m5C MeRIP sequencing in PANC-1 cells to map the m5C methylome of PANC-1 cells treated with CAF-derived Evs
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Project description:The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the embryo undergoes dramatic reprogramming to convert maternal environment to embryonic-driven programing. However, how the maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) in zebrafish early embryos, we show that m5C methylated maternal mRNAs display higher stability during MZT. We identify that the Y box-binding protein 1 (Ybx1) prefers to recognizing m5C-modified mRNAs through p-p interaction with a key residue Trp45 in its cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Cooperated with an mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates a novel mechanism of RNA m5C methylation-regulated maternal mRNA stability during zebrafish MZT, highlighting the critical role of m5C mRNA methylation in early development.
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Project description:RNA m5C methylation profile of MCF10A and MDA486 by using MeRIP-Seq protocol Immunoprecipitation of Methylated mRNA at Cytosine (m5C) residues: Affinity purified of anti-methyl cytosine (m5C) polyclonal antibody 7ug (Zymo Research, Catalog#A3001-50) was conjugated with protein-A magnetic beads for 2 h at 4°C in end to end rotator. After that, conjugated beads were extensively washed with RNA immunoprecipitation (RIP) wash buffer to remove unbound antibody. Fragmented 25 ug polyA RNA (mRNA) was incubated with m5C conjugated beads for overnight at 4°C in in the rotating platform in RIP buffer. RIP was done using Megna RNA Immunoprecipitation kit (Millipore, Catalog#17-700). m5C mRNA-immune bead complex was treated with proteinase K buffer to release m5C mRNA from the conjugated antibody. To isolate m5C, mRNA was treated with phenol:chloroform:isoamyl and mixed with 400 ul of chloroform, which was centrifuged at 14000 rpm for 10 minutes to separate aqueous phase. The aqueous phase was ethanol precipitated at -80°C for overnight, to get m5C mRNA. This precipitated m5C mRNA pellet was washed twice with 70% ethanol and air dried. Finally, m5C mRNA pellet was dissolved in nuclease free Water. The m5C mRNA integrity and conentration was quantified by bioanalyzer (Agilent) and Qubit 2.0 flurometer (Invitrogen). The fragmented mRNA was used by following TruSeq RNA Sample Preparation Guide to develop RNA-Seq library for sequencing.
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Project description:Methylation of carbon-5 of cytosines (m5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. m5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. We recently showed that m5C is required for Caenorhabditis elegans development and fertility under heat stress. To contribute to the understanding of how m5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Our mass spectrometry analyses reveal unique binding proteins for each of the modifications. We validate our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m5C in vitro. The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m5C and its derivatives.
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