Similar Datasets
Project description:N6-methyladenosine-mediated structural changes are associated with transcript cleavage and stability in Arabidopsis
| PRJNA428948 | ENA
Project description:N(6)-methyladenosine (m(6)A) is the most prevalent internal (non-cap) modification present in the messenger RNA of all higher eukaryotes. Although essential to cell viability and development, the exact role of m(6)A modification remains to be determined. The recent discovery of two m(6)A demethylases in mammalian cells highlighted the importance of m(6)A in basic biological functions and disease. Here we show that m(6)A is selectively recognized by the human YTH domain family 2 (YTHDF2) 'reader' protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m(6)A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies. The carboxy-terminal domain of YTHDF2 selectively binds to m(6)A-containing mRNA, whereas the amino-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m(6)A modification is recognized by selectively binding proteins to affect the translation status and lifetime of mRNA.
| S-EPMC3877715 | biostudies-literature
Project description:Spermatogenesis is a differentiation process during which diploid spermatogonial stem cells (SSCs) produce haploid spermatozoa. This highly specialized process is precisely controlled at the transcriptional, posttranscriptional, and translational levels. Here we report that N6-methyladenosine (m6A), an epitranscriptomic mark regulating gene expression, plays essential roles during spermatogenesis. We present comprehensive m6A mRNA methylomes of mouse spermatogenic cells from five developmental stages: undifferentiated spermatogonia, type A1 spermatogonia, preleptotene spermatocytes, pachytene/diplotene spermatocytes, and round spermatids. Germ cell-specific inactivation of the m6A RNA methyltransferase Mettl3 or Mettl14 with Vasa-Cre causes loss of m6A and depletion of SSCs. m6A depletion dysregulates translation of transcripts that are required for SSC proliferation/differentiation. Combined deletion of Mettl3 and Mettl14 in advanced germ cells with Stra8-GFPCre disrupts spermiogenesis, whereas mice with single deletion of either Mettl3 or Mettl14 in advanced germ cells show normal spermatogenesis. The spermatids from double-mutant mice exhibit impaired translation of haploid-specific genes that are essential for spermiogenesis. This study highlights crucial roles of mRNA m6A modification in germline development, potentially ensuring coordinated translation at different stages of spermatogenesis.
| S-EPMC5630681 | biostudies-other
Project description:After transcription, a messenger RNA (mRNA) is further post-transcriptionally regulated by several features including RNA secondary structure and covalent RNA modifications (specifically N6-methyladenosine, m6A). Both RNA secondary structure and m6A have been demonstrated to regulate mRNA stability and translation as well as have been independently linked to plant response to excess salt concentrations in the soil. However, the effect of m6A on regulating RNA secondary structure and the combinatorial interplay between these two RNA features during salt stress response has yet to be studied. Here, we globally identify RNA-protein interactions and RNA secondary structure during systemic salt stress. This analysis reveals that RNA secondary structure is highly dynamic during salt stress, which is independent of changes in RNA-protein interactions. Conversely, we find that m6A is anti-correlated with RNA secondary structure in a condition-specific manner, with salt-specific m6A resulting in a decrease in mRNA secondary structure during salt stress. Remarkably, we show that the combination of salt-specific m6A deposition and the associated loss of RNA secondary structure results in increases in mRNA stability and translation of transcripts encoding proteins involved in responses to abiotic stresses. In total, our comprehensive analyses reveal an important epitranscriptome, secondary structure-mediated post-transcriptional regulatory mechanism involved in plant long-term salt stress response and adaptation.
2020-04-01 | GSE147812 | GEO
Project description:BackgroundBreast cancer (BC) displays striking genetic, epigenetic and phenotypic diversity. N6-methyladenosine (m6A) in mRNA has emerged as a crucial epitranscriptomic modification that controls cancer self-renewal and cell fate. However, the key enzymes of m6A expression and function in human breast carcinogenesis remain unclear.MethodsThe expression of m6A methylases (METTL3, METTL14 and WTAP) and demethylases (FTO and ALKBH5) were analyzed by using ONCOMINE and The Cancer Genome Atlas databases and in 36 pairs of BC and adjacent non-cancerous tissue. The level of m6A in BC patients was detected by ELISA, and the function of m6A was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay and transwell assay. The database of bc-GenExMiner v4.0, Kaplan-Meier Plotter and cBioPortal were queried for correlation, mutation and prognosis analysis of BC.ResultsThe m6A methylases and demethylases were dysregulated in several major malignant tumors. Specifically, the expression of all m6A methylases was reduced in BC as compared with normal controls, but the demethylase ALKBH5 was induced in ONCOMINE databases and confirmed in clinical patients. METTL14 expression was positively correlated with METTL3 expression, and both showed high expression in normal breast-like and luminal-A and -B BC. Functionally, reducing m6A expression by overexpressing METTL14 and/or knockdown of ALKBH5 could inhibit breast cell viability, colony formation and cell migration. Furthermore, Kaplan-Meier, meta-analysis and univariate Cox assay showed that the expression of m6A members including METTL3, METTL14, WTAP and FTO but not their gene mutation and amplification, was tightly associated with cancer progression and poor survival.ConclusionsChanges of m6A modulators reduced m6A may promote tumorigenesis and predict poor prognosis in BC.
| S-EPMC6451293 | biostudies-literature
Project description:We show that N6-methyladenosine (m6A), the most abundant internal modification in mRNA/lncRNA with still poorly characterized function, alters RNA structure to facilitate the access of RBM for heterogeneous nuclear ribonucleoprotein C (hnRNP C). We term this mechanism m6A-switch. Through combining PAR-CLIP with Me-RIP, we identify 39,060 m6A-switches among hnRNP C binding sites transcriptome-wide. We show that m6A-methyltransferases METTL3 or METTL14 knockdown decreases hnRNP C binding at 16,582 m6A-switches. Taken together, 2,798 m6A-switches of high confidence are identified to mediate RNA-hnRNP C interactions and affect diverse biological processes including cell cycle regulation. These findings reveal the biological importance of m6A and provide insights into the sophisticated regulation of RNA-RBP interactions through m6A-induced RNA structural remodeling. Measure the m6A methylated hnRNP C binding sites transcriptome-wide by PARCLIP-MeRIP; measure the differential hnRNP C occupancies upon METTL3/METTL14 knockdown by PAR-CLIP; measure RNA abundance and splicing level changes upon HNRNPC, METTL3 and METTL14 knockdown
2015-02-27 | E-GEOD-56010 | biostudies-arrayexpress
Project description:More than 100 types of chemical modifications in RNA have been well documented. Recently, several modifications, such as N6-methyladenosine (m6A), have been detected in mRNA, opening the window into the realm of epitranscriptomics. The m6A modification is the most abundant modification in mRNA and non-coding RNA (ncRNA). At the molecular level, m6A affects almost all aspects of mRNA metabolism, including splicing, translation, and stability, as well as microRNA (miRNA) maturation, playing essential roles in a range of cellular processes. The m6A modification is regulated by three classes of proteins generally referred to as the "writer" (adenosine methyltransferase), "eraser" (m6A demethylating enzyme), and "reader" (m6A-binding protein). The m6A modification is reversibly installed and removed by writers and erasers, respectively. Readers, which are members of the YT521-B homology (YTH) family proteins, selectively bind to RNA and affect its fate in an m6A-dependent manner. In this review, we summarize the structures of the functional proteins that modulate the m6A modification, and provide our insights into the m6A-mediated gene regulation.
| S-EPMC6112310 | biostudies-literature
Project description:The addition of a methyl group to the N6 position of adenosine (m6A) is the most common posttranscriptional RNA modification, and it regulates most steps of RNA metabolism including splicing, stability, translation, nuclear-export, and RNA structures. Besides cellular RNA, m6A modifications have also been detected on viral RNA. A range of recent studies have demonstrated the crucial roles of m6A in the virus-host interactions; however, m6A cellular machineries are only characterized in limited mammalian species. Herein, we aim to present comprehensive evolutionary insights into major m6A writers, erasers, and readers and draw a comparative structural analysis between avian and mammalian m6A-associated machineries. The comparative collinearity on the chromosomal scale revealed that the majority of m6A-related genes were found less syntenic even among avian species. Genetic analysis of avian m6A erasers revealed a distinct phylogenetic clustering compared to mammalian orthologs and shared a weak percent (55%) identity with mammalian species with low identity percentage (55%). The overall comparative three-dimensional (3D) structure analyses among different mammalian species were maintained through synonymous structural mutations. Unlike erasers, the putative 3D structures in the active sites as for the aromatic cage in YTH-domain of YTHDC1 and two pivotal loops in MTD-domains in METTL3 exhibited structural alterations in chicken. In conjunction with in silico investigations, influenza viruses significantly downregulated gene the transcription of m6A writers and erasers, whereas m6A readers were moderately regulated in chicken fibroblasts. In light of these findings, future detailed biochemical and crystallographic studies are warranted to define the roles of m6A machinery in regulating both viral and cellular RNA metabolism in avian species.
| S-EPMC7373739 | biostudies-literature
Project description:The fat mass and obesity-associated protein (FTO) is an N6-Methyladenosine (m6A) demethylase, which has been revealed to play critical roles in tumorigenesis. However, its role in the development and progression of prostate cancer (PCa) remains poorly understood. Here, we aimed to investigate the function and clinical relevance of FTO in PCa. Our results demonstrated that FTO was notably downregulated in PCa tissues compared with the paired normal tissues. In addition, the decreased expression of FTO was correlated with poor prognosis of PCa. Functional experiments showed that depletion of FTO promoted the proliferation and metastasis of PCa both in vitro and in vivo. Conversely, ectopic expression of FTO exhibited the opposite effects. Combined with RNA-sequencing, MeRIP-RT-qPCR, and mRNA stability assays indicated chloride intracellular channel 4(CLIC4) was a functional target of FTO-mediated m6A modification. FTO depletion significantly increased the m6A level of CLIC4 mRNA and then reduced the mRNA stability. In conclusion, our findings suggest that FTO suppresses PCa proliferation and metastasis through reducing the degradation of CLIC4 mRNA in an m6A dependent manner. FTO may be used as a promising novel therapeutic target and prognostic evaluation biomarker for PCa.
| S-EPMC8994758 | biostudies-literature
Project description:DNA base lesions, such as incorporation of uracil into DNA or base mismatches, can be mutagenic and toxic to replicating cells. To discover factors in repair of genomic uracil, we performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluoro-uracil into DNA. We identified known factors, such as uracil DNA N-glycosylase (UNG), but also unknown factors, such as the N6-adenosine methyltransferase, METTL3, as required to overcome floxuridine-driven cytotoxicity. Visualized with immunofluorescence, the product of METTL3 activity, N6-methyladenosine, formed nuclear foci in cells treated with floxuridine. The observed N6-methyladenosine was embedded in DNA, called 6mA, which was confirmed using mass spectrometry. METTL3 and 6mA were required for repair of lesions driven by additional base damaging agents, including raltitrexed, gemcitabine, and hydroxyurea. Our results establish a role for METTL3 and 6mA to promote genome stability in mammalian cells, specially in response to base damage.
2024-11-22 | GSE282260 | GEO