M6A and YTHDF1, YTHDF2, YTHDF3 mapping of IAV RNA with PAR-CLIP and pA-m6A-seq
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE98033 | GEO | 2017/10/17
SECONDARY ACCESSION(S): PRJNA383739
REPOSITORIES: GEO
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Project description:Polyomaviruses are a family of small DNA tumor viruses that includes several pathogenic human members, including Merkel cell polyomavirus, BK virus and JC virus. As is characteristic of DNA tumor viruses, gene expression in polyomaviruses is temporally regulated into an early phase, consisting of the viral regulatory proteins, and a late phase, consisting of the viral structural proteins. Previously, the late transcripts expressed by the prototypic polyomavirus simian virus 40 (SV40) were reported to contain several adenosines bearing methyl groups at the N6 position (m6A), although the precise location of these m6A residues, and their phenotypic effects, have not been investigated. Here, we first demonstrate that overexpression of the key m6A reader protein YTHDF2 induces more rapid viral replication, and larger viral plaques, in SV40 infected BSC40 cells, while mutational inactivation of the endogenous YTHDF2 gene, or the m6A methyltransferase METTL3, has the opposite effect, thus suggesting a positive role for m6A in the regulation of SV40 gene expression. To directly test this hypothesis, we mapped sites of m6A addition on SV40 transcripts and identified two m6A sites on the viral early transcripts and eleven m6A sites on the late mRNAs. Using synonymous mutations, we inactivated the majority of the m6A sites on the SV40 late mRNAs and observed that the resultant viral mutant replicated more slowly than wild type SV40. Alternative splicing of SV40 late mRNAs was unaffected by the reduction in m6A residues and our data instead suggest that m6A enhances the translation of viral late transcripts. Together, these data argue that the addition of m6A residues to the late transcripts encoded by SV40 plays an important role in enhancing viral gene expression and, hence, replication.
2018-02-08 | GSE106698 | GEO
Project description:To replicate in the host, HIV-1 must produce RNA and protein, and this depends cellular factors that also control gene expression. Recently, it has been shown that novel components of this machinery post-transcriptionally modify mRNAs with direct consequences for their structure and stability. The most common among these modifications is the addition of a methyl group to the N6 position of adenosine, termed m6A methylation. Here we are interested the interface of HIV-1 with this posttranscriptional mRNA modification pathway. We show that viral transcripts harbor a number of discrete methylation sites and are bound at those sites by these YTHDF proteins. These sites are conserved among distinct HIV-1 isolates and are observed during viral replication in numerous cell types. With reporter assays we also show that these sites stabilize mRNAs when compared to similar sequences mutated to be incapable of methylation. We further show that overexpression of these reader proteins boosts viral protein expression.
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Project description:The N6-methyladenosine (m6A) mRNA modification and the mitochondrial respiratory chain (MRC) hold paramount importance in the advancement of MASLD. This study thoroughly investigates the relationship and impact of m6A mRNA modification and mitochondrial function in the progression of MASLD. Here we report that the mRNA and protein levels of mitochondrial respiratory chain (MRC) subunits showed inconsistent trends in vivo experiments. Abnormal m6A modification and mitochondrial dysfunction in MASLD were attributed to the upregulation of methyltransferase like 3 (Mettl3) and the downregulation of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) induced by high-fat foods. Mettl3 promoted the MRC's function. However, knockout of the reader protein YTHDF1, which plays a crucial role in the m6A modification process, counteracted the effect of Mettl3 and suppressed MRC. In MASLD, damage to the MRC may be regulated by the Mettl3-m6A-YTHDF1 complex axis, especially by the role of YTHDF1. Our research has offered a novel perspective on the involvement of m6A mRNA methylation in the pathogenesis of MASLD.
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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.
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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.
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Project description:To identify the target mRNAs of the m6A reader protein YTHDF1 and YTHDF2, we carired out anti YTHDF1 and anti YTHDF2 RNA Immunoprecipitation (RIP) followed by RNA-sequencencing. Using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore), RNA from P6-P8 wild type mouse cerebellum was pulled down by rabbit polyclonal anti-YTHDF1 (proteintech) or polyclonal anti-YTHDF2 (proteintech) and then sequenced on Illumina HiSeq3000 platform. The filtered reads were mapped to the mouse reference genome (GRCm38) using STAR v2.5 with default parameters. The resulting bam files were fed to HTSeq tool to count the number of RNA-seq reads, which was further normalized to calculate FPKM. To determine which gene is enriched, we computed the FPKM from RIP elute to input and any fold change greater than 2 was considered enriched. Finally, Biological replicates of anti-YTHDF1 RIP-Seq and anti-YTHDF2 RIP-Seq identified 506 and 596 mRNAs transcripts, respectively. This study provides gene lists which shows mRNA binding with YTHDF1 and YTHDF2 in mouse cerebellum.
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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.
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Project description: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
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