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Effect of ISG20 expression (wild-type or M1 mutant) on total RNA expression.

ABSTRACT: ISG20 is an interferon-induced 3'-to-5' RNA exonuclease that acts as a broad antiviral innate defense factor. At present, the features that expose RNA to the action of ISG20 remain unclear. However recent studies have pinpointed to RNA epitranscriptomic modifications as modulators that either expose or protect viral RNAs from ISG20, raising the question as to how cellular RNAs, on which these modifications are abundant, cope with ISG20. To obtain an unbiased perspective on this issue, we used RNAseq to pinpoint to the RNA elements that regulate the balance between susceptibility and resistance to ISG20.

ORGANISM(S): Homo sapiens

PROVIDER: GSE233792 | GEO | 2023/06/01

REPOSITORIES: GEO

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The interferon-induced exonuclease, ISG20, exerts antiviral activity through upregulation of type I interferon response proteins

Project description:Type I interferon-stimulated genes (ISGs) have critical roles in inhibiting virus replication and dissemination. Despite advances in understanding the molecular basis of ISG restriction, the antiviral mechanisms of many remain unclear. The 20 kDa ISG, ISG20, is a nuclear 3'-5'exonuclease with preference for single stranded RNA (ssRNA) and has been implicated in the IFN-mediated restriction of several RNA viruses. Although the exonuclease activity of ISG20 has been shown to degrade viral RNA in vitro, evidence has yet to be presented that virus inhibition in cells requires this activity. Here, we utilized a combination of an inducible, ectopic expression system and newly generated Isg20−/− mice to investigate mechanisms and consequences of ISG20-mediated restriction. Ectopically expressed ISG20 localized primarily to Cajal bodies in the nucleus and restricted replication of chikungunya and Venezuelan equine encephalitis viruses. Although restriction by ISG20 was associated with inhibition of translation of infecting genomic RNA, degradation of viral RNAs was not observed. Instead, translation inhibition of viral RNA was associated with ISG20-induced upregulation of over 100 other genes, many of which encode known antiviral effectors. ISG20 modulated the production of IFIT1, an ISG that suppresses translation of alphavirus RNAs. Consistent with this observation, the pathogenicity of IFIT1-sensitive alphaviruses was increased in Isg20−/− mice compared to wild-type viruses, but not in ISG20 ectopic-expressing cells. Our findings establish an indirect role for ISG20 in the early restriction of RNA virus replication by regulating expressionof other ISGs that inhibit translation and possibly other activities in the replication cycle.

2018-07-01 | GSE115729 | GEO

Expression data from CD8alpha positive dendritic cells

2016-06-10 | GSE75690 | GEO

Expression data from CD8alpha positive dendritic cells

Project description:We screened a number of interferon inducible genes that may be involved in impeding HBV replication and found an anti-HBV activity in ISG20. ISG20 is an IFN-inducible 3â??- to 5â??-exonuclease, that degrades DNA and RNA and reduces antigen production in hepatocyte-derived cells A range of candidate genes whose expression was dependent on type I IFN stimulation were identified by gene arrays Total RNA from CD8Î±+ DCs was extracted from mouse spleens 8h after polyI:C (50 Î¼g) stimulation by RNeasyTM kit. Total RNA (10 Î¼g) from each sample was used to prepare cRNA. Gene expression was analyzed with GeneChipTM

2016-06-10 | E-GEOD-75690 | biostudies-arrayexpress

RNAseq analysis of the serotype M1 group A Streptococcus strain MGAS2221

Project description:We recovered RNA from exponential and stationary phase cell cultures of MGAS2221 and utilized two distinct methods to remove rRNA from our samples, the RiboZero kit from Epicenter, and the terminator- 5--Phosphate-Dependent Exonuclease (TEX). This gave us 4 distinct RNAs with which we performed RNAseq analysis. Both rRNA removal methods worked well, with only a small percentage of the final total read counts being derived from rRNA sequences.

2013-03-20 | GSE45325 | GEO

RNAseq analysis of the serotype M1 group A Streptococcus strain MGAS2221

2013-03-20 | E-GEOD-45325 | biostudies-arrayexpress

Temporal Profiling of Epitranscriptomic Reader, Writer and Eraser Proteins during Osteogenic Differentiation of Human Embryonic Stem Cells

Project description:Osteogenesis is the process of bone formation and is modulated by multiple regulatory networks. With the rapid development of the epitranscriptomics field, RNA modifications and their reader, writer, and eraser (RWE) proteins are shown to be involved in the regulation of various biological processes. Few studies, however, were conducted to investigate the functions of RNA modifications and their RWE proteins in osteogenesis. By using a parallel-reaction monitoring (PRM)-based targeted proteomics method, we performed a comprehensive quantitative assessment of 154 epitranscriptomic RWE proteins during the time course of osteogenic differentiation of H9 human embryonic stem cells (ESCs). We found that approximately half of the 126 detected RWE proteins were downregulated during osteogenic differentiation, and they included mainly those proteins involved in RNA methylation and pseudouridine synthesis. Protein-protein interaction (PPI) network analysis revealed a high connectivity between the downregulated epitranscriptomic RWE proteins and osteogenesis-related proteins. Gene set enrichment analysis of previously published RNA-seq data from osteogenesis imperfecta patients suggested a potential role of METTL1, the top-ranked hub protein of downregulated RWE proteins, in osteogenesis through the cytokine network. Together, this is the first targeted profiling of epitranscriptomic RWE proteins during osteogenic differentiation of human ESCs and our work unveiled potential regulatory roles of these proteins in osteogenesis.

2023-05-23 | PXD039249 | Pride

Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

Project description:Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m5C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC), RNA-protein enrichment, and quantitative proteomics. We profile m5C dioxygenases in human cells including ALKBH1 and TET2 and use quantitative nucleoside LC-MS to show that ALKBH1 is the major hm5C and f5C-forming enzyme in RNA, including upon polyadenylated RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-based iCLIP analysis to show that ALKBH1 oxidizes m5C in a variety of tRNA anticodon stem loops (ASL), as well as on mRNA and lncRNA, and analyze its substrate specificity using in vitro enzymatic assays. Finally, we apply pyridine borane-mediated sequencing to identify f5C sites in tRNA ASLs. Our work provides powerful chemical approaches for studying RNA m5C dioxygenases and mapping oxidative m5C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.

2022-07-16 | PXD029955 | Pride

The interferon-induced exonuclease, ISG20, exerts antiviral activity through upregulation of type I interferon response proteins

Project description:The interferon-induced exonuclease, ISG20, exerts antiviral activity through upregulation of type I interferon response proteins

| PRJNA475885 | ENA

N6-methyladenosine modification is not a general trait of positive-sense RNA viral genomes

Project description:<p>Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-seq, an antibody-dependent technique with a high rate of false positives. Here, we addressed the presence of m6A in CHIKV and DENV RNAs. For this, we combined m6A-seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we found no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery did not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection had no effect on the m6A machinery’s localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.</p>

2024-01-25 | MTBLS6978 | MetaboLights

Impact of gut microbiota on the m6A epitranscriptome in mouse cecum and liver tissues

Project description:The intestinal microbiota modulates host physiology and gene expression via mechanisms that are not fully understood. A recently discovered layer of gene expression regulation is N6-methyladenosine (m6A) and N6,2′ -O-dimethyladenosine (m6Am) modifications of mRNA. To unveil if these epitranscriptomic marks are affected by the gut microbiota, we performed methylated RNA-immunoprecipitation and sequencing (MeRIP-seq) to examine m6A-modifications in transcripts of mice displaying either a conventional, or a modified, or no gut microbiota and discovered that the microbiota has a strong influence on m6A- modifications in the cecum, and also, albeit to a lesser extent, in the liver, affecting pathways related to metabolism, inflammatory and antimicrobial responses . We furthermore analysed expression levels of several known writer and eraser enzymes and found the methyltransferase Mettl16 to be downregulated in absence of a microbiota. As a consequence, one of its targets, the S-adenosyl methionine synthase Mat2a was less expressed in mice without gut flora. We furthermore show that distinct commensal bacteria, Akkermansia muciniphila, Lactobacillus plantarum can affect specific m6A modifications. Together, we report here epitranscriptomic modifications as an additional level of interaction in the complex interplay between commensal bacteria and their host.

2020-02-17 | PXD016099 | Pride

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