In response to infection, invertebrates process replicating viral RNA genomes into siRNAs of discrete sizes to guide virus clearance by RNA interference. Here, we show that viral siRNAs sequenced from fruit fly, mosquito, and nematode cells were all overlapping in sequence, suggesting a possibility of using siRNAs for viral genome assembly and virus discovery. To test this idea, we examined contigs assembled from published small RNA libraries and discovered five previously undescribed viruses from cultured Drosophila cells and adult mosquitoes, including three with a positive-strand RNA genome and two with a dsRNA genome. Notably, four of the identified viruses exhibited only low sequence similarities to known viruses, such that none could be assigned into an existing virus genus. We also report detection of virus-derived PIWI-interacting RNAs (piRNAs) in Drosophila melanogaster that have not been previously described in any other host species and demonstrate viral genome assembly from viral piRNAs in the absence of viral siRNAs. Thus, this study provides a powerful culture-independent approach for virus discovery in invertebrates by assembling viral genomes directly from host immune response products without prior virus enrichment or amplification. We propose that invertebrate viruses discovered by this approach may include previously undescribed human and vertebrate viral pathogens that are transmitted by arthropod vectors.arboviruses | piRNAs | siRNAs | viral immunity | massively parallel sequencing T he Dicer family of host immune receptors mediates antiviral immunity in fungi, plants, and invertebrate animals by RNA interference (RNAi) or RNA silencing (1-3). In this immunity, a viral dsRNA is recognized by Dicer and diced into siRNAs. These virus-derived siRNAs are then loaded into an RNA silencing complex to act as specificity determinants and to guide slicing of the target viral RNAs by an Argonaute protein (AGO) present in the complex. Dicer proteins typically contain an RNA helicase domain, a PAZ domain shared with AGOs, and two tandem type III endoribonuclease (RNase III) domains. Dicer cleaves dsRNA with a simple preference toward a terminus of dsRNA, producing duplex small RNA fragments of discrete sizes progressively from the terminus (4).In addition to siRNAs, micro-RNAs (miRNAs) and PIWIinteracting RNAs (piRNAs) guide RNA silencing in similar complexes but with distinct AGOs (4-6). In Drosophila melanogaster, miRNAs and siRNAs are predominantly 22 and 21 nucleotides in length, dependent on Dicer-1 (DCR1) and DCR2 for their biogenesis, and act in silencing complexes containing AGO1 and AGO2 in the AGO subfamily, respectively (4-6). In contrast, ∼24-30-nt piRNAs are Dicer-independent and require AGO3, Aubergine (AUB), and PIWI in the PIWI subfamily for their biogenesis (4-6). Genetic analyses (7-10) have clearly demonstrated a role for D. melanogaster DCR2 in the immunity and biogenesis of viral siRNAs targeting diverse positive-strand (+) RNA viruses, including Flock house virus (FHV), cricket par...
Replication of viral RNA genomes in fruit flies and mosquitoes induces the production of virus-derived small interfering RNAs (siRNAs) to specifically reduce virus accumulation by RNA interference (RNAi). However, it is unknown whether the RNA-based antiviral immunity (RVI) is sufficiently potent to terminate infection in adult insects as occurs in cell culture. We show here that, in contrast to robust infection by Flock house virus (FHV), infection with an FHV mutant (FHV⌬B2) unable to express its RNAi suppressor protein B2 was rapidly terminated in adult flies. FHV⌬B2 replicated to high levels and induced high mortality rates in dicer-2 and argonaute-2 mutant flies that are RNAi defective, demonstrating that successful infection of adult Drosophila requires a virus-encoded activity to suppress RVI. Drosophila RVI may depend on the RNAi activity of viral siRNAs since efficient FHV⌬B2 infection occurred in argonaute-2 and r2d2 mutant flies despite massive production of viral siRNAs. However, RVI appears to be insensitive to the relative abundance of viral siRNAs since FHV⌬B2 infection was terminated in flies carrying a partial loss-of-function mutation in loquacious required for viral siRNA biogenesis. Deep sequencing revealed a low-abundance population of Dicer-2-dependent viral siRNAs accompanying FHV⌬B2 infection arrest in RVI-competent flies that included an approximately equal ratio of positive and negative strands. Surprisingly, viral small RNAs became strongly biased for positive strands at later stages of infection in RVI-compromised flies due to genetic or viral suppression of RNAi. We propose that degradation of the asymmetrically produced viral positive-strand RNAs associated with abundant virus accumulation contributes to the positive-strand bias of viral small RNAs.Innate immunity includes distinct mechanisms that provide immediate and broad-spectrum protection against microbial infection and often are conserved across different kingdoms (56). The fruit fly Drosophila melanogaster is a powerful model for studying innate immunity (21). Drosophila innate immunity against bacteria and fungi involves recognition of microbial molecular patterns by germ line-encoded pattern recognition receptors and the production of potent antimicrobial peptides via closely related Toll and IMD signaling pathways (21). In contrast, protection of Drosophila against viruses is mainly mediated by the RNA interference (RNAi) pathway (24,40,67,72,74) as found in fungi and plants (13,18,32).RNAi and related RNA silencing pathways operate in diverse eukaryotes, including fungi, plants, invertebrates, and mammals, and recruit small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) to guide specific silencing of genes by an Argonaute protein in an RNA-induced silencing complex (RISC) or related effector complexes (2, 10, 34). Available data indicate that in Drosophila, doublestranded RNA (dsRNA) replicative intermediates of viruses with an RNA genome are recognized by Dicer-2 (Dcr-2) and further processed into v...
Chromatin-associated RNA (caRNA) has been proposed as a type of epigenomic modifier. Here, we test whether environmental stress can induce cellular dysfunction through modulating RNA-chromatin interactions. We induce endothelial cell (EC) dysfunction with high glucose and TNFα (H + T), that mimic the common stress in diabetes mellitus. We characterize the H + T-induced changes in gene expression by single cell (sc)RNA-seq, DNA interactions by Hi-C, and RNA-chromatin interactions by iMARGI. H + T induce inter-chromosomal RNA-chromatin interactions, particularly among the super enhancers. To test the causal relationship between H + T-induced RNA-chromatin interactions and the expression of EC dysfunction-related genes, we suppress the LINC00607 RNA. This suppression attenuates the expression of SERPINE1, a critical pro-inflammatory and pro-fibrotic gene. Furthermore, the changes of the co-expression gene network between diabetic and healthy donor-derived ECs corroborate the H + T-induced RNA-chromatin interactions. Taken together, caRNA-mediated dysregulation of gene expression modulates EC dysfunction, a crucial mechanism underlying numerous diseases.
Background: Metabolic disorders such as obesity and diabetes mellitus can cause dysfunction of endothelial cells (ECs) and vascular rarefaction in adipose tissues. However, the modulatory role of ECs in adipose tissue function is not fully understood. Other than vascular endothelial growth factor–vascular endothelial growth factor receptor-mediated angiogenic signaling, little is known about the EC-derived signals in adipose tissue regulation. We previously identified Argonaute 1 (AGO1; a key component of microRNA-induced silencing complex) as a crucial regulator in hypoxia-induced angiogenesis. In this study, we intend to determine the AGO1-mediated EC transcriptome, the functional importance of AGO1-regulated endothelial function in vivo, and the relevance to adipose tissue function and obesity. Methods: We generated and subjected mice with EC-AGO1 deletion (EC-AGO1-knockout [KO]) and their wild-type littermates to a fast food–mimicking, high-fat high-sucrose diet and profiled the metabolic phenotypes. We used crosslinking immunoprecipitation- and RNA-sequencing to identify the AGO1-mediated mechanisms underlying the observed metabolic phenotype of EC-AGO1-KO. We further leveraged cell cultures and mouse models to validate the functional importance of the identified molecular pathway, for which the translational relevance was explored using human endothelium isolated from healthy donors and donors with obesity/type 2 diabetes mellitus. Results: We identified an antiobesity phenotype of EC-AGO1-KO, evident by lower body weight and body fat, improved insulin sensitivity, and enhanced energy expenditure. At the organ level, we observed the most significant phenotype in the subcutaneous and brown adipose tissues of KO mice, with greater vascularity and enhanced browning and thermogenesis. Mechanistically, EC-AGO1 suppression results in inhibition of thrombospondin-1 ( THBS1 /TSP1), an antiangiogenic and proinflammatory cytokine that promotes insulin resistance. In EC-AGO1-KO mice, overexpression of TSP1 substantially attenuated the beneficial phenotype. In human endothelium isolated from donors with obesity or type 2 diabetes mellitus, AGO1 and THBS1 are expressed at higher levels than the healthy controls, supporting a pathological role of this pathway. Conclusions: Our study suggests a novel mechanism by which ECs, through the AGO1-TSP1 pathway, control vascularization and function of adipose tissues, insulin sensitivity, and whole-body metabolic state.
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