Identification of YTHDF1 targeted m6A modified transcripts in lung cancer cells
ABSTRACT: In addition to perform the m6A-seq in A549 cells, we sequenced RNA obtained from the immuno-purified complex of YTHDF1 (RIP-seq) to reveal YTHDF1 bound mRNAs, 3,676 genes were shared (m6A-seq+RIP-seq) as high-confident targets of YTHDF1 , which were mapped to cell cycle and tumor (including lung cancer) related signaling pathways in the KEGG pathway database Overall design: The transcriptome, m6A modified transcripts, and YTHDF1 binding transcripts were identified.
Project description:N6-methyladenosine (m6A), the most prevalent internal RNA modification on mammalian messenger RNAs, regulates the fates and functions of modified transcripts through m6A-specific binding proteins1-5. In the nervous system, m6A is abundant and modulates various neural functions6-11. Whereas m6A marks groups of mRNAs for coordinated degradation in various physiological processes12-15, the relevance of m6A for mRNA translation in vivo remains largely unknown. Here we show that, through its binding protein YTHDF1, m6A promotes protein translation of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory. Mice with genetic deletion of Ythdf1 show learning and memory defects as well as impaired hippocampal synaptic transmission and long-term potentiation. Re-expression of YTHDF1 in the hippocampus of adult Ythdf1-knockout mice rescues the behavioural and synaptic defects, whereas hippocampus-specific acute knockdown of Ythdf1 or Mettl3, which encodes the catalytic component of the m6A methyltransferase complex, recapitulates the hippocampal deficiency. Transcriptome-wide mapping of YTHDF1-binding sites and m6A sites on hippocampal mRNAs identified key neuronal genes. Nascent protein labelling and tether reporter assays in hippocampal neurons showed that YTHDF1 enhances protein synthesis in a neuronal-stimulus-dependent manner. In summary, YTHDF1 facilitates translation of m6A-methylated neuronal mRNAs in response to neuronal stimulation, and this process contributes to learning and memory.
Project description:There is growing evidence that tumour neoantigens have important roles in generating spontaneous antitumour immune responses and predicting clinical responses to immunotherapies1,2. Despite the presence of numerous neoantigens in patients, complete tumour elimination is rare, owing to failures in mounting a sufficient and lasting antitumour immune response3,4. Here we show that durable neoantigen-specific immunity is regulated by mRNA N6-methyadenosine (m6A) methylation through the m6A-binding protein YTHDF15. In contrast to wild-type mice, Ythdf1-deficient mice show an elevated antigen-specific CD8+ T cell antitumour response. Loss of YTHDF1 in classical dendritic cells enhanced the cross-presentation of tumour antigens and the cross-priming of CD8+ T cells in vivo. Mechanistically, transcripts encoding lysosomal proteases are marked by m6A and recognized by YTHDF1. Binding of YTHDF1 to these transcripts increases the translation of lysosomal cathepsins in dendritic cells, and inhibition of cathepsins markedly enhances cross-presentation of wild-type dendritic cells. Furthermore, the therapeutic efficacy of PD-L1 checkpoint blockade is enhanced in Ythdf1-/- mice, implicating YTHDF1 as a potential therapeutic target in anticancer immunotherapy.
Project description:Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, disease treatment, and organ transplantation. As the ethical issue of human ESCs and similarity of pig in human genome and physiological characteristics, the porcine iPSCs (piPSCs) have become an ideal alternative study model. N6-methyladenosine (m6A) methylation is the most prevalent modification in eukaryotic mRNAs, regulating the self-renewal and differentiation of pluripotency stem cells. However, the explicit m6A-regulating machinery remains controversial. Here, we demonstrate that m6A modification and its modulators play a crucial role in mediating piPSCs pluripotency. In brief, loss of METTL3 significantly impairs self-renewal and triggers differentiation of piPSCs by interfering JAK2 and SOCS3 expression, further inactivating JAK2-STAT3 pathway, which then blocks the transcription of KLF4 and SOX2. We identify that both of JAK2 and SOSC3 have m6A modification at 3'UTR by m6A-seq analysis. Dual-luciferase assay shows that METTL3 regulates JAK2 and SOCS3 expression in an m6A-dependent way. RIP-qPCR validates JAK2 and SOCS3 are the targets of YTHDF1 and YTHDF2, respectively. SiMETTL3 induced lower m6A levels of JAK2 and SOCS3 lead to the inhibition of YTHDF1-mediated JAK2 translation and the block of YTHDF2-dependent SOCS3 mRNA decay. Subsequently, the altered protein expressions of JAK2 and SOCS3 inhibit JAK2-STAT3 pathway and then the pluripotency of piPSCs. Collectively, our work uncovers the critical role of m6A modification and its modulators in regulating piPSCs pluripotency and provides insight into an orchestrated network linking the m6A methylation and SOCS3/JAK2/STAT3 pathway in pluripotency regulation.
Project description:N 6-Methyladenosine (m6A) is the most abundant RNA modification in mammal mRNAs and increasing evidence suggests the key roles of m6A in human tumorigenesis. However, whether m6A, especially its 'reader' YTHDF1, targets a gene involving in protein translation and thus affects overall protein production in cancer cells is largely unexplored. Here, using multi-omics analysis for ovarian cancer, we identified a novel mechanism involving EIF3C, a subunit of the protein translation initiation factor EIF3, as the direct target of the YTHDF1. YTHDF1 augments the translation of EIF3C in an m6A-dependent manner by binding to m6A-modified EIF3C mRNA and concomitantly promotes the overall translational output, thereby facilitating tumorigenesis and metastasis of ovarian cancer. YTHDF1 is frequently amplified in ovarian cancer and up-regulation of YTHDF1 is associated with the adverse prognosis of ovarian cancer patients. Furthermore, the protein but not the RNA abundance of EIF3C is increased in ovarian cancer and positively correlates with the protein expression of YTHDF1 in ovarian cancer patients, suggesting modification of EIF3C mRNA is more relevant to its role in cancer. Collectively, we identify the novel YTHDF1-EIF3C axis critical for ovarian cancer progression which can serve as a target to develop therapeutics for cancer treatment.
Project description:N 6-Methyladenosine (m6A) is a dynamic mRNA modification which regulates protein expression in various posttranscriptional levels. Functional studies of m6A in nervous system have focused on its writers and erasers so far, whether and how m6A readers mediate m6A functions through recognizing and binding their target mRNA remains poorly understood. Here, we find that the expression of axon guidance receptor Robo3.1 which plays important roles in midline crossing of spinal commissural axons is regulated precisely at translational level. The m6A reader YTHDF1 binds to and positively regulates translation of m6A-modified Robo3.1 mRNA. Either mutation of m6A sites in Robo3.1 mRNA or YTHDF1 knockdown or knockout leads to dramatic reduction of Robo3.1 protein without affecting Robo3.1 mRNA level. Specific ablation of Ythdf1 in spinal commissural neurons results in pre-crossing axon guidance defects. Our findings identify a mechanism that YTHDF1-mediated translation of m6A-modified Robo3.1 mRNA controls pre-crossing axon guidance in spinal cord.
Project description:N6-methyladenosine (m6A) is the most prevalent internal RNA modification in mammalian messenger RNAs (mRNAs). While m6A has been shown to mark groups of mRNAs for coordinated degradation in various physiological processes, the relevance of m6A in affecting translation remains to be determined in intact biological systems in vivo. Here we show that, through its reader protein Ythdf1, m6A promotes a pulse of protein synthesis of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory processes. Mice with genetic deletion of the Ythdf1 gene (Ythdf1-/-) exhibit learning and memory defects, as well as impaired hippocampal synaptic transmission and long-term potentiation (LTP). Selective re-expression of Ythdf1 in the hippocampus of adult Ythdf1-/- mice fully rescues the behavioral and synaptic defects, while hippocampus-specific knockdown of Ythdf1 or Mettl3, the catalytic component of m6A methyltransferase complex, recapitulates the hippocampal deficiency in adult mice. At the molecular level, transcriptome-wide mapping of m6A sites and RNA binding sites of Ythdf1 on hippocampal mRNAs using crosslinking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) uncovered key neuronal genes, including those involved in synaptic transmission and long-term potentiation. Nascent protein labelling and tether reporter assays in cultured hippocampal neurons revealed that Ythdf1 is critical for initiating a pulse of protein synthesis of target transcripts in a neuronal-stimulus-dependent manner. Collectively, our results uncover a pathway of mRNA m6A methylation in learning and memory, which is mediated through Ythdf1 in response to stimuli. Overall design: (1) The RNA binding targets of Ythdf1 were determined by anti-Ythdf1 antibody CLIP-seq in the adult mouse hippocampal tissue. (2) m6A-modified transcripts were mapped by anti-m6A antibody CLIP-seq using polyA RNA purified from the adult mouse hippocampus. (3) mRNA abundance in wild-type and Ythdf1-KO mouse hippocampus was determined by mRNA-seq. (4) Abundance of m6A-modified transcripts in mouse dentate gyrus before and after electroconvulsive treatment was determined by full length m6A-RIP-seq.
Project description:Recent studies that have emerged on the diversity of RNA modification in tumors suggest their eligibility as bona fide targets in diagnosis and drug discovery. N6-methyladenosine (m6A) was first reported and is most common in epitranscriptome modification of various RNAs. The YT521-B homology (YTH) domain family are representative m6A-binding proteins, but how the YTH domain family is involved in cancer remains to be clearly understood. Given that clinical sequence data in colorectal cancer indicate that overexpression of YTHDF1 is outstanding among other family members, we studied the role of Ythdf1 and the transcriptional control of YTHDF1. Immunostaining of Ythdf1 showed that its expression was associated with various malignant tumor behaviors, such as depth, lymph node metastasis, and poorer cancer stages. The study of patient survival indicated that patients with high Ythdf1 expression had significantly poorer overall survival. The results indicated that Ythdf1 expression is an independent prognostic factor of patients. The in vitro study showed that the knockdown of YTHDF1 resulted in the suppression of cancer proliferation and sensitization to the exposure of anticancer drugs such as fluorouracil and oxaliplatin. Importantly, the study upstream of the YTHDF1 gene indicated that an oncogenic transcription factor c-Myc was associated with YTHDF1 in both expression and chromatin immunoprecipitation data. Moreover, the knockdown experiments of c-Myc showed the inhibition of YTHDF1, supporting a notion of c-Myc-driven YTHDF1 axis significance. These data suggest that m6A reader Ythdf1 plays a significant role in colorectal cancer progression.
Project description:N6-Methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) that occurred on the N6 nitrogen of adenosine. However, the roles of m6A in oral squamous cell carcinoma (OSCC) are still elusive. Here, we investigate the function and mechanism of methyltransferase-like 3 (METTL3) in OSCC tumorigenesis. Clinically, METTL3 was significantly upregulated in tissue samples and correlated with the poor prognosis of OSCC patients. Functionally, loss and gain studies illustrated that METTL3 promoted the proliferation, invasion, and migration of OSCC cells in vitro, and METTL3 knockdown inhibited tumor growth in vivo. Mechanistically, methylated RNA immunoprecipitation sequencing (MeRIP-seq) illustrated that METTL3 targeted the 3' UTR (near to stop codon) of the c-Myc transcript to install the m6A modification, thereby enhancing its stability. Furthermore, results revealed that YTH N6-methyladenosine RNA binding protein 1 (YTH domain family, member 1 [YTHDF1]) mediated the m6A-increased stability of c-Myc mRNA catalyzed by METTL3. In conclusion, our findings herein identify that METTL3 accelerates the c-Myc stability via YTHDF1-mediated m6A modification, thereby giving rise to OSCC tumorigenesis.
Project description:BACKGROUND:Dynamic N6-methyladenosine (m6A) RNA modification generated and erased by N6-methyltransferases and demethylases regulates gene expression, alternative splicing and cell fate. Ocular melanoma, comprising uveal melanoma (UM) and conjunctival melanoma (CM), is the most common primary eye tumor in adults and the 2nd most common melanoma. However, the functional role of m6A modification in ocular melanoma remains unclear. METHODS:m6A assays and survival analysis were used to explore decreased global m6A levels, indicating a late stage of ocular melanoma and a poor prognosis. Multiomic analysis of miCLIP-seq, RNA-seq and Label-free MS data revealed that m6A RNA modification posttranscriptionally promoted HINT2 expression. RNA immunoprecipitation (RIP)-qPCR and dual luciferase assays revealed that HINT2 mRNA specifically interacted with YTHDF1. Furthermore, polysome profiling analysis indicated a greater amount of HINT2 mRNA in the translation pool in ocular melanoma cells with higher m6A methylation. RESULTS:Here, we show that RNA methylation significantly inhibits the progression of UM and CM. Ocular melanoma samples showed decreased m6A levels, indicating a poor prognosis. Changes in global m6A modification were highly associated with tumor progression in vitro and in vivo. Mechanistically, YTHDF1 promoted the translation of methylated HINT2 mRNA, a tumor suppressor in ocular melanoma. CONCLUSIONS:Our work uncovers a critical function for m6A methylation in ocular melanoma and provides additional insight into the understanding of m6A modification.
Project description:Merkel cell carcinoma is a deadly skin cancer, which in the majority of cases is caused by the Merkel cell polyomavirus (MCPyV). The viral small T antigen is regarded as the dominant oncoprotein expressed in the tumor cells. We used genomic screening of copy number aberrations along with transcriptomic analysis to investigate regions with amplification that harbor differentially expressed genes. We identified YTHDF1, a protein that is a reader of N6-methyladenosine (m6A) RNA modifications, to have high copy gains and to be highly expressed in Merkel cell carcinoma. Importantly, we identified the presence of m6A on small T antigen mRNA suggesting a relation between YTHDF1 amplification and MCPyV gene expression. Interestingly, knockdown of YTHDF1 in Merkel cell carcinoma (MCC) cell lines negatively affected the translation initiation factor eIF3 and reduced proliferation and clonogenic capacity in vitro. Furthermore, analysis of survival data revealed worse overall survival in YTHDF1high MCC patients compared to YTHDF1low patients. Our findings indicate a novel oncogenic role of YTHDF1 through m6A machinery in the tumorigenesis of MCC.