Methylation of the N6 position of adenosine (m6A) is a posttranscriptional modification of RNA with poorly understood prevalence and physiological relevance. The recent discovery that FTO, an obesity ...risk gene, encodes an m6A demethylase implicates m6A as an important regulator of physiological processes. Here, we present a method for transcriptome-wide m6A localization, which combines m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-Seq). We use this method to identify mRNAs of 7,676 mammalian genes that contain m6A, indicating that m6A is a common base modification of mRNA. The m6A modification exhibits tissue-specific regulation and is markedly increased throughout brain development. We find that m6A sites are enriched near stop codons and in 3′ UTRs, and we uncover an association between m6A residues and microRNA-binding sites within 3′ UTRs. These findings provide a resource for identifying transcripts that are substrates for adenosine methylation and reveal insights into the epigenetic regulation of the mammalian transcriptome.
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► m6A is a widespread RNA modification in many tissues with high levels in the brain ► MeRIP-Seq identifies m6A in 7,913 genes encoding both coding and noncoding RNAs ► m6A is enriched near stop codons and within 3′ UTRs in both mouse and human mRNAs ► The transcriptome-wide landscape of m6A provides important insights into m6A function
Adenosine methylation (m6A) is a widespread RNA modification found in >7,000 mammalian genes, encoding both mRNAs and noncoding RNAs, with an especially high prevalence in the developing brain. In both mouse and human mRNAs, m6A is enriched within 3′ UTRs that also contain miRNA-binding sites.
Posttranscriptionally modified nucleosides in RNA play integral roles in the cellular control of biological information that is encoded in DNA. The modifications of RNA span all three phylogenetic ...domains (Archaea, Bacteria, and Eukarya) and are pervasive across RNA types, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and (less frequently) small nuclear RNA (snRNA) and microRNA (miRNA). Nucleotide modifications are also one of the most evolutionarily conserved properties of RNAs, and the sites of modification are under strong selective pressure. However, many of these modifications, as well as their prevalence and impact, have only recently been discovered. Here, we examine both labile and permanent modifications, from simple methylation to complex transcript alteration (RNA editing and intron retention); detail the models for their processing; and highlight remaining questions in the field of the epitranscriptome.
The RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove ...this modification, as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A methyltransferases or an m6A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determined that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Altogether, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.
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•The RNA genomes of HCV, ZIKV, DENV, YFV, and WNV contain m6A modification•The cellular m6A machinery regulates HCV infectious particle production•YTHDF proteins reduce HCV particle production and localize at viral assembly sites•m6A-abrogating mutations in HCV E1 increase infectious particle production
N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. Gokhale et al. demonstrate that the RNA genomes of HCV, ZIKV, DENV, YFV, and WNV are modified by m6A. Depletion of cellular machinery that regulates m6A or introduction of m6A-abrogating mutations within HCV RNA increase viral particle production, suggesting that m6A negatively regulates HCV.
Understanding the pathophysiology of SARS-CoV-2 infection is critical for therapeutic and public health strategies. Viral-host interactions can guide discovery of disease regulators, and protein ...structure function analysis points to several immune pathways, including complement and coagulation, as targets of coronaviruses. To determine whether conditions associated with dysregulated complement or coagulation systems impact disease, we performed a retrospective observational study and found that history of macular degeneration (a proxy for complement-activation disorders) and history of coagulation disorders (thrombocytopenia, thrombosis and hemorrhage) are risk factors for SARS-CoV-2-associated morbidity and mortality-effects that are independent of age, sex or history of smoking. Transcriptional profiling of nasopharyngeal swabs demonstrated that in addition to type-I interferon and interleukin-6-dependent inflammatory responses, infection results in robust engagement of the complement and coagulation pathways. Finally, in a candidate-driven genetic association study of severe SARS-CoV-2 disease, we identified putative complement and coagulation-associated loci including missense, eQTL and sQTL variants of critical complement and coagulation regulators. In addition to providing evidence that complement function modulates SARS-CoV-2 infection outcome, the data point to putative transcriptional genetic markers of susceptibility. The results highlight the value of using a multimodal analytical approach to reveal determinants and predictors of immunity, susceptibility and clinical outcome associated with infection.
Recent studies have provided insights into the pathology of and immune response to COVID-19
. However, a thorough investigation of the interplay between infected cells and the immune system at sites ...of infection has been lacking. Here we use high-parameter imaging mass cytometry
that targets the expression of 36 proteins to investigate the cellular composition and spatial architecture of acute lung injury in humans (including injuries derived from SARS-CoV-2 infection) at single-cell resolution. These spatially resolved single-cell data unravel the disordered structure of the infected and injured lung, alongside the distribution of extensive immune infiltration. Neutrophil and macrophage infiltration are hallmarks of bacterial pneumonia and COVID-19, respectively. We provide evidence that SARS-CoV-2 infects predominantly alveolar epithelial cells and induces a localized hyperinflammatory cell state that is associated with lung damage. We leverage the temporal range of fatal outcomes of COVID-19 in relation to the onset of symptoms, which reveals increased macrophage extravasation and increased numbers of mesenchymal cells and fibroblasts concomitant with increased proximity between these cell types as the disease progresses-possibly as a result of attempts to repair the damaged lung tissue. Our data enable us to develop a biologically interpretable landscape of lung pathology from a structural, immunological and clinical standpoint. We use this landscape to characterize the pathophysiology of the human lung from its macroscopic presentation to the single-cell level, which provides an important basis for understanding COVID-19 and lung pathology in general.
A SARS-CoV-2 surveillance program conducted by the National Basketball Association identified 173 newly infected persons. The viral kinetics were systematically studied and are described.
Genetic and epigenetic heterogeneity is emerging as a fundamental property of human cancers. Reflecting the genesis of tumors as an evolutionary process driven by clonal selection. The complexity of ...clonal architecture has been known for many years in the setting of acute myeloid leukemia (AML), based on karyotyping studies. However the true complexity of AMLs is only now being understood thanks to in depth genome sequencing studies in humans, which reveal that heterogeneity is a multilayered and involves not only the genome but also the epigenome. Here, we review recent advances in genetic and epigenetic heterogeneity and clonal dynamics in AML and their relevance to biology, clinical outcomes and therapeutic implications. Special attention is focused on somatic mutations affecting regulators of cytosine methylation, since these tend to occur early in disease evolution, reprogram the epigenome of hematopoietic stem cells, and are linked to unfavorable outcome.
Biological insights can be obtained through computational integration of genomics data sets consisting of diverse types of information. The integration is often hampered by a large variety of ...existing file formats, often containing similar information, and the necessity to use complicated tools to achieve the desired results. We have built an R package, genomation, to expedite the extraction of biological information from high throughput data. The package works with a variety of genomic interval file types and enables easy summarization and annotation of high throughput data sets with given genomic annotations.
The software is currently distributed under MIT artistic license and freely available at http://bioinformatics.mdc-berlin.de/genomation, and through the Bioconductor framework.
Transcriptional silencing by heritable cytosine-5 methylation is an ancient strategy to repress transposable elements. It was previously thought that mammals possess four DNA methyltransferase ...paralogs-Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l-that establish and maintain cytosine-5 methylation. Here we identify a fifth paralog, Dnmt3c, that is essential for retrotransposon methylation and repression in the mouse male germline. From a phenotype-based forward genetics screen, we isolated a mutant mouse called 'rahu', which displays severe defects in double-strand-break repair and homologous chromosome synapsis during male meiosis, resulting in sterility. rahu is an allele of a transcription unit (Gm14490, renamed Dnmt3c) that was previously mis-annotated as a Dnmt3-family pseudogene. Dnmt3c encodes a cytosine methyltransferase homolog, and Dnmt3crahu mutants harbor a non-synonymous mutation of a conserved residue within one of its cytosine methyltransferase motifs, similar to a mutation in human DNMT3B observed in patients with immunodeficiency, centromeric instability and facial anomalies syndrome. The rahu mutation lies at a potential dimerization interface and near the potential DNA binding interface, suggesting that it compromises protein-protein and/or protein-DNA interactions required for normal DNMT3C function. Dnmt3crahu mutant males fail to establish normal methylation within LINE and LTR retrotransposon sequences in the germline and accumulate higher levels of transposon-derived transcripts and proteins, particularly from distinct L1 and ERVK retrotransposon families. Phylogenetic analysis indicates that Dnmt3c arose during rodent evolution by tandem duplication of Dnmt3b, after the divergence of the Dipodoidea and Muroidea superfamilies. These findings provide insight into the evolutionary dynamics and functional specialization of the transposon suppression machinery critical for mammalian sexual reproduction and epigenetic regulation.
Many cellular mRNAs contain the modified base m6A, and recent studies have suggested that various stimuli can lead to changes in m6A. The most common method to map m6A and to predict changes in m6A ...between conditions is methylated RNA immunoprecipitation sequencing (MeRIP-seq), through which methylated regions are detected as peaks in transcript coverage from immunoprecipitated RNA relative to input RNA. Here, we generated replicate controls and reanalyzed published MeRIP-seq data to estimate reproducibility across experiments. We found that m6A peak overlap in mRNAs varies from ~30 to 60% between studies, even in the same cell type. We then assessed statistical methods to detect changes in m6A peaks as distinct from changes in gene expression. However, from these published data sets, we detected few changes under most conditions and were unable to detect consistent changes across studies of similar stimuli. Overall, our work identifies limits to MeRIP-seq reproducibility in the detection both of peaks and of peak changes and proposes improved approaches for analysis of peak changes.
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