RNA circles with minimized immunogenicity as potent PKR inhibitors.

Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces ∼74 nt td or ∼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 103- to 106-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.

N6-Methyladenosine Modification Controls Circular RNA Immunity.

Circular RNAs (circRNAs) are prevalent in eukaryotic cells and viral genomes. Mammalian cells possess innate immunity to detect foreign circRNAs, but the molecular basis of self versus foreign identity in circRNA immunity is unknown. Here, we show that N6-methyladenosine (m6A) RNA modification on human circRNAs inhibits innate immunity. Foreign circRNAs are potent adjuvants to induce antigen-specific T cell activation, antibody production, and anti-tumor immunity in vivo, and m6A modification abrogates immune gene activation and adjuvant activity. m6A reader YTHDF2 sequesters m6A-circRNA and is essential for suppression of innate immunity. Unmodified circRNA, but not m6A-modified circRNA, directly activates RNA pattern recognition receptor RIG-I in the presence of lysine-63-linked polyubiquitin chain to cause filamentation of the adaptor protein MAVS and activation of the downstream transcription factor IRF3. CircRNA immunity has considerable parallel to prokaryotic DNA restriction modification system that transforms nucleic acid chemical modification into organismal innate immunity.

Circular RNAs in Immune Response and Viral Infection.

Circular RNAs (circRNAs) are a diverse class of RNAs with varying sizes, cellular abundance, and biological functions. Investigations from the past decade have revealed that circRNAs are ubiquitously found in eukaryotes and have defined the different biological roles of circRNAs to illuminate this previously unrecognized class of molecules. In the context of the immune system, immune responses and immune-related diseases alter circRNA expression. More recently, several oncogenic double-stranded DNA viruses have been found to encode circRNAs. In this review, we summarize the current understanding of circRNAs and their emerging functions in immune regulation and autoimmune disorders, and discuss the identification and potential roles of viral circRNAs during infections. Finally, we present promising areas for future investigations in the nascent field of circRNAs.

Viral non-coding RNAs: Stealth strategies in the tug-of-war between humans and herpesviruses.

Oncogenic DNA viruses establish lifelong infections in humans, and they cause cancers, often in immunocompromised patients, despite anti-viral immune surveillance targeted against viral antigens. High-throughput sequencing techniques allowed the field to identify novel viral non-coding RNAs (ncRNAs). ncRNAs are ideal factors for DNA viruses to exploit; they are non-immunogenic to T cells, thus viral ncRNAs can manipulate host cells without evoking adaptive immune responses. Viral ncRNAs may still trigger the host innate immune response, but many viruses encode decoys/inhibitors to counter-act and evade recognition. In addition, ncRNAs can be secreted to the extracellular space and influence adjacent cells to create a pro-viral microenvironment. In this review, we present recent progress in understanding interactions between oncoviruses and ncRNAs including small and long ncRNAs, microRNAs, and recently identified viral circular RNAs. In addition, potential clinical applications for ncRNA will be discussed. Extracellular ncRNAs are suggested to be diagnostic and prognostic biomarkers and, with the realization of the importance of viral ncRNAs in tumorigenesis, approaches to target critical viral ncRNAs are emerging. Further understanding of viral utilization of ncRNAs will advance anti-viral therapeutics beyond conventional medication and vaccination.

Circular RNA circDtx1 regulates IRF3-mediated antiviral immune responses through suppression of miR-15a-5p-dependent TRIF downregulation in teleost fish.

Circular RNAs (circRNAs) represent a class of widespread and diverse covalently closed circular endogenous RNAs that exert crucial functions in regulating gene expression in mammals. However, the function and regulation mechanism of circRNAs in lower vertebrates are still unknown. Here, we discovered a novel circRNA derived from Deltex E3 ubiquitin ligase 1 (Dtx1) gene, namely, circDtx1, which was related to the antiviral responses in teleost fish. Results indicated that circDtx1 played essential roles in host antiviral immunity and inhibition of SCRV replication. Our study also found a microRNA miR-15a-5p, which could inhibit antiviral immune response and promote viral replication by targeting TRIF. Moreover, we also found that the antiviral effect inhibited by miR-15a-5p could be reversed with the circDtx1. In mechanism, our data revealed that circDtx1 was a competing endogenous RNA (ceRNA) of TRIF by sponging miR-15a-5p, leading to activation of the NF-κB/IRF3 pathway, and then enhancing the innate antiviral responses. Our results indicated that circRNAs played a regulatory role in immune responses in teleost fish.

Circular RNA circSamd4a Regulates Antiviral Immunity in Teleost Fish by Upregulating STING through Sponging miR-29a-3p.

Circular RNAs (circRNAs) are a subgroup of endogenous noncoding RNA that is covalently closed rings and widely expressed. In recent years, there is accumulating evidence indicating that circRNAs are a class of important regulators, which play an important role in various biological processes. However, the biological functions and regulation mechanism of circRNAs in lower vertebrates are little known. In this study, we discovered a circRNA Samd4a (circSamd4a) that is related to the antiviral immune response of teleost fish. It can act as a key regulator of the host's antiviral response and play a key role in inhibiting Sininiperca chuatsi rhabdovirus replication. Further studies have shown that circSamd4a may act as a competing endogenous RNA, which can enhance the STING-mediated NF-κB/IRF3 signaling pathway by adsorbing miR-29a-3p, thereby enhancing the antiviral immune response. Therefore, circSamd4a plays an active regulatory role in the antiviral immune response of bony fish. Our research results provide a strong foundation for circular RNA to play a regulatory role in the antiviral immune response of teleost fish.

The circular RNA circBCL2L1 regulates innate immune responses via microRNA-mediated downregulation of TRAF6 in teleost fish.

Growing numbers of studies have shown that circular RNAs (circRNAs) can function as regulatory factors to regulate the innate immune response, cell proliferation, cell migration, and other important processes in mammals. However, the function and regulatory mechanism of circRNAs in lower vertebrates are still unclear. Here, we discovered a novel circRNA derived from the gene encoding Bcl-2-like protein 1 (BCL2L1) gene, named circBCL2L1, which was related to the innate immune responses in teleost fish. Results indicated that circBCL2L1 played essential roles in host antiviral immunity and antibacterial immunity. Our study also identified a microRNA, miR-30c-3-3p, which could inhibit the innate immune response by targeting inflammatory mediator TRAF6. And TRAF6 is a key signal transduction factor in innate immune response mediated by TLRs. Moreover, we also found that the antiviral and antibacterial effects inhibited by miR-30c-3-3p could be reversed with the expression of circBCL2L1. Our data revealed that circBCL2L1 functioned as a competing endogenous RNA (ceRNA) of TRAF6 by competing for binding with miR-30c-3-3p, leading to activation of the NF-κB/IRF3 inflammatory pathway and then enhancing the innate immune responses. Our results suggest that circRNAs can play an important role in the innate immune response of teleost fish.

The emerging role and significance of circular RNAs in viral infections and antiviral immune responses: possible implication as theranostic agents.

Circular RNAs (circRNAs) are ubiquitously expressed, covalently closed rings, produced by pre-mRNA splicing in a reversed order during post-transcriptional processing. Circularity endows 3'-5'-linked circRNAs with stability and resistance to exonucleolytic degradation which raises the question whether circRNAs may be relevant as potential therapeutic targets or agents. High stability in biological systems is the most remarkable property and a major criterion for why circRNAs could be exploited for a range of RNA-centred medical applications. Even though various biological roles and regulatory functions of circRNAs have been reported, their in-depth study is challenging because of their circular structure and sequence-overlap with linear mRNA counterparts. Moreover, little is known about their role in viral infections and in antiviral immune responses. We believe that an in-depth and detailed understanding of circRNA mediated viral protein regulations will increase our knowledge of the biology of these novel molecules. In this review, we aimed to provide a comprehensive basis and overview on the biogenesis, significance and regulatory roles of circRNAs in the context of antiviral immune responses and viral infections including hepatitis C virus infection, hepatitis B virus infection, hepatitis delta virus infection, influenza A virus infection, Epstein-Barr virus infection, kaposi's sarcoma herpesvirus infection, human cytomegalovirus infection, herpes simplex virus infection, human immunodeficiency virus infection, porcine epidemic diarrhoea virus infection, ORF virus infection, avian leukosis virus infection, simian vacuolating virus 40 infection, transmissible gastroenteritis coronavirus infection, and bovine viral diarrhoea virus infection. We have also discussed the critical regulatory role of circRNAs in provoking antiviral immunity, providing evidence for implications as therapeutic agents and as diagnostic markers.

Integrative analysis of circRNA/miRNA/mRNA regulatory network reveals the potential immune function of circRNAs in the Bombyx mori fat body.

Bombyx mori nucleopolyhedrosis virus (BmNPV) is one of the greatest threats to sustainable development of the sericulture industry. Circular RNA (circRNA), a type of non-coding RNA, has been shown to play important roles in gene expression regulation, immune response, and diseases. The fat body is a tissue with both metabolic and immune functions. To explore the potential immune function of circRNAs, we analyzed differentially expressed (DE)circRNAs, microRNAs(miRNAs), and mRNAs in the B. mori fat body in response to BmNPV infection using high-throughput RNA sequencing. A total of 77 DEcircRNAs, 32 DEmiRNAs, and 730 DEmRNAs that are associated with BmNPV infection were identified. We constructed a DEcircRNA/DEmiRNA/DEmRNA and DEcircRNA/DEmiRNA/BmNPV gene regulatory network and validated the differential expression of circ_0001432 and its corresponding miRNA (miR-2774c and miR-3406-5p) and mRNA (778467 and 101745232) in the network. Tissue-specific expression of circ_0001432 and its expression at different time points were also examined. KEGG pathway analysis of DEmRNAs, target genes of DEmiRNAs, and host genes of DEcircRNAs in the network showed that these genes were enriched in several metabolic pathways and signaling pathways, which could play important roles in insect immune responses. Our results suggest that circRNA could be involved in immune responses of the B. mori fat body and help in understanding the molecular mechanisms underlying silkworm-pathogen interactions.

Circular RNA circ-RELL1 regulates inflammatory response by miR-6873-3p/MyD88/NF-κB axis in endothelial cells.

Endothelial inflammation is an important contributor to the pathology of atherosclerotic cardiovascular disease (ASCVD). Circular RNAs (circRNAs) function and role in endothelium inflammation still unknown. In our present study, we firstly identified that circ-RELL1 plays a proinflammatory role in ox-LDL-induced HUVECs through high-throughput circRNA microarray assays. Knockdown circ-RELL1 can reduce the expression of ICAM1 and VCAM1 in ox-LDL induced endothelium inflammation. Mechanistically, circ-RELL1 directly bound to miR-6873-3p in cytoplasm. Subsequently miR-6873-3p reduced MyD88 (myeloid differentiation primary response 88) protein expression and alleviated MyD88 medicated NF-κB activation. Furthermore, circ-RELL1 can abolish the inhibition of inflammation response by miR-6873-3p. Our findings illustrate a novel regulatory pathway that circ-RELL1 modulate inflammatory response by miR-6873-3p/MyD88/NF-κB axis in ox-LDL induced endothelial cells, which provides a potential therapeutic candidate for endothelium inflammation in atherosclerotic cardiovascular disease.

Circular RNA hsa_circ_0004812 impairs IFN-induced immune response by sponging miR-1287-5p to regulate FSTL1 in chronic hepatitis B.

BACKGROUND: The present study aims to explore the functions of circular RNA hsa_circ_0004812 in chronic hepatitis B (CHB) and its underlying molecular mechanisms. METHODS: The expression of circular RNA (circRNA)_0004812 was examined using qRT-PCR and Western blot in blood and liver tissues from CHB patients and healthy volunteers. In the in vitro study, the expression levels of circular RNA hsa_circ_0004812, miR-1287-5p, interferon (IFN)-α, IFN-ß were determined using qRT-PCR and Western blotting in HBV-infected hepatoma cells, respectively. Luciferase and biotin pull-down assays were used to investigate the interactions between miR-1287-5p and circ_0004812. RESULTS: Levels of circ_0004812 were upregulated in CHB patients and HBV-infected hepatoma cells. Knockdown of circ_0004812 increased the expression of IFN-α and IFN-ß in HBV-infected Huh7 cells. MiR-1287-5p was identified as a target of circ_0004812 whose overexpression inhibited the expression of miR-1287-5p. Additionally, circ_0004812 promoted the expression of Follistatin-related protein (FSTL) 1 through inhibiting miR-1287-5p. Circ_0004812/miR-1287-5p/FSTL1 axis regulated HBV-induced immune suppression. CONCLUSION: Circ_0004812 was identified as a potential target for CHB infection. Circ_0004812 promoted the expression of FSTL1 by inhibiting miR-1287-5p.

Single molecule real-time sequencing and RNA-seq unravel the role of long non-coding and circular RNA in the regulatory network during Nile tilapia (Oreochromis niloticus) infection with Streptococcus agalactiae.

BACKGROUND: The tilapia aquaculture industry is facing heavy economic losses due to Streptococcus agalactiae (S. agalactiae) infections. While progress has been made in past years, the lack of a high-quality tilapia genome and transcript annotations makes systematic and comprehensive exploration for a non-coding RNA regulatory network associated with the infection process unfeasible, and it stunts further research focused on disease defense and treatment. Herein, single molecular real time sequencing (SMRT-Seq) and RNA-seq data were utilized to generate a high-quality transcript annotation. In addition, Changes in mRNA and non-coding RNA expression were also analyzed during a S. agalactiae infection in tilapia. FINDINGS: In total, 16.79 Gb of clean data were obtained by sequencing on six SMRT cells, with 712,294 inserts (326,645 full-length non-chimeric reads and 354,188 non-full-length reads). A total of 197,952 consensus transcripts were obtained. Additionally, 55,857 transcript sequences were acquired, with 12,297 previously annotated and 43,560 newly identified transcripts. To further examine the immune response in Oreochromis niloticus following a S. agalactiae infection, a total of 470.62 Gb of clean data was generated by sequencing a library containing 18 S. agalactiae infected tilapia samples. Of the identified genes, 9911 were newly exploited, of which 7102 were functional annotated. Furthermore, 7874 mRNAs, 1281 long non-coding RNAs (out of 21,860 long non-coding RNAs), and 61 circular RNAs (out of 1026 circular RNAs) were found to be differentially expressed during infection, with the 1026 circRNAs not previously identified in tilapia. Moreover, k-means clustering and WGCNA analyses revealed that the immune response of tilapia to a S. agalactiae infection can be divided into three stages: cytokines driven rapid immune response, energy metabolism promotion, and the production of lysosomes and phagosomes. During this response, the head kidney and spleen have synergistic effects, while maintaining independent characteristics. Finally, lncRNA-mRNA (trans and cis), lncRNA-miRNA-mRNA, and circRNA-miRNA-mRNA regulatory networks were constructed and revealed that non-coding RNA is involved in the regulation of immune-related genes. CONCLUSIONS: This study generated a greatly-improved transcript annotation for tilapia using long-read PacBio sequencing technology, and revealed the presence of a regulatory network comprised of non-coding RNAs in Nile tilapia infected with S. agalactiae.

Circ_0075932 in adipocyte-derived exosomes induces inflammation and apoptosis in human dermal keratinocytes by directly binding with PUM2 and promoting PUM2-mediated activation of AuroraA/NF-κB pathway.

It remains unclear why obese persons displayed a slower wound healing rate than the normal. In this study, we found that has_circ_0075932, a single-exon circular RNA, was outstandingly expressed in human normal adipose tissue and overexpressed in burned skin of obese persons compared with that of non-obese persons. Circ_0075932 overexpression or silencing in dermal keratinocytes had no obvious effect on cell behaviors, unless dozens of times overexpression, since its basal expression level in keratinocytes is too low. However, the exosome released from circ_0075932-overexpressing adipocytes displayed a significantly promoting effect on inflammation and apoptosis in dermal keratinocytes. Then, in our mechanism exploration, we found that circ_0075932 directly bound with the RNA-binding protein PUM2, which was reported to positively regulated AuroraA kinase, thus activating the NF-κB pathway. Moreover, either silencing PUM2, silencing AuroraA, or blockade of NF-κB activation, could abrogate the promoting effect of adipocyte-derived exosomal circ_0075932 on cell inflammation and apoptosis.

Hsa_circ_0005519 increases IL-13/IL-6 by regulating hsa-let-7a-5p in CD4+ T cells to affect asthma.

BACKGROUND: Circular RNAs (circRNAs) are a class of non-coding RNAs that could serve as novel biomarkers for the diagnosis and treatment of diseases. We hypothesized that circRNAs of CD4+ T cells are involved in asthma. OBJECTIVE: In this study, we investigated the circRNA expression profile and the possible mechanism by which hsa_circ_0005519 participates in asthma. METHODS: The expression profiles of circRNAs in CD4+ T cells were revealed by circRNA microarray. Hsa_circ_0005519 expression in CD4+ T cells was confirmed in asthmatic patients (n = 65) and healthy subjects (n = 30). Hsa-let-7a-5p, the target of hsa_circ_0005519, was predicted by online algorithms and verified by a dual-luciferase reporter assay. Correlation assays between the expression of hsa_circ_0005519 and hsa-let-7a-5p, the mRNA levels of interleukin (IL)-13 and IL-6 in CD4+ T cells, and the clinical characteristics of asthmatic patients were performed. The role of hsa_circ_0005519 in proinflammatory cytokine expression was investigated in CD4+ T cells from asthmatic patients in vitro. Hsa_circ_0005519 expression in PBMCs was determined in another cohort including 30 asthmatic patients and 24 controls. Correlation assays of hsa_circ_0005519 expressions between CD4+ T cells and PBMCs were performed. RESULTS: Hsa_circ_0005519 was up-regulated and negatively correlated with hsa-let-7a-5p expression in CD4+ T cells of asthmatic patients. Both the fraction of exhaled nitric oxide (FeNO) and the peripheral blood eosinophil ratio were positively correlated with hsa_circ_0005519 expression in CD4+ T cells. These outcomes were also different in asthmatic patients with low vs high hsa_circ_0005519 levels. Hsa_circ_0005519 expressions between CD4+ T cells and PBMCs were concordant in asthmatic patients. Mechanistically, hsa_circ_0005519 might bind to hsa-let-7a-5p and relieve suppression for IL-13/IL-6 in CD4+ T cells. CONCLUSIONS AND CLINICAL RELEVANCE: Our data suggest that hsa_circ_0005519 may induce IL-13 and IL-6 expression by regulating hsa-let-7a-5p in CD4+ T cells to affect asthma. And hsa_circ_0005519 may be a potential biomarker of asthma.

Identification and characterization of novel type of RNAs, circRNAs in crucian carp Carassius auratus gibelio.

Circular RNAs (circRNAs) with regulatory potency activity was identified from varieties of species. Crucian carp (Carassius auratus gibelio) is one of the most freshwater aquaculture species in China. Every year, huge economic damage to the farming was caused by the virus and bacterial infection. Until now, there is any information about circRNA reported from the Crucian carp. In this study, the expression pattern of circRNA in Crucian carp was investigated with transcriptomic analysis. The results showed that only 37 circRNAs were identified from the Crucian carp, and these circRNAs biogenesis was formed with canonical GU-AG splicing mechanism with unevenly distributed on the chromosomes. Wherein, most of the circRNAs were derived from the sense overlapping strategy. Reverse transcript PCR and Sanger sequencing data indicated that these circRNAs were existed authenticity in Crucian carp. The bioinformatics analysis indicated that circRNAs identified from the Crucian carp with potential miRNA sponge regulate the expression level of mRNAs. GO annotation and KEGG pathway analysis of these circRNAs showed that more than 20% circRNAs were related with catalytic activity and binding in the category of molecular function, and these circRNAs were enriched in 9 signaling pathways, such as, Wnt signaling pathway, MAPK signaling pathway, Ubiquitin mediated proteolysis et al. 220 mRNAs would be regulated by the circRNAs via miRNAs mediation. These target mRNAs were further analyzed with functional annotation and KEGG analysis. GO annotation analysis showed that several genes were related with function of nucleotide binding, transcription regulatory activity. KEGG pathway analysis showed that 5 genes were enriched in the pathway of Endocytosis. The circRNA-miRNA-mRNA regulation network indicated that one miRNA can link one or more circRNA and one or more mRNA. Overall, these results will not only help us to further understand the novel RNA transcripts in Crucian carp, but also provide the novel clue to investigate the interaction between host and pathogens from this novel circRNA molecule.

CircPTPN22 modulates T-cell activation by sponging miR-4689 to regulate S1PR1 expression in patients with systemic lupus erythematosus.

BACKGROUND: Circular RNAs are involved in autoimmune disease pathogenesis. Our previous study indicated that circPTPN22 is involved in autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, but the underlying mechanisms remain unclear. METHODS: First, the expression of circPTPN22 was detected by real-time PCR and western blotting. After overexpression or knockdown of circPTPN22, the proliferation of Jurkat cells was detected by the CCK-8 assay, and the apoptosis of Jurkat cells was detected by flow cytometry. In addition, the relationship between circPTPN22-miR-4689-S1PR1 was confirmed by bioinformatic analyses, fluorescence in situ hybridization assays, RNA-binding protein immunoprecipitation, and dual luciferase reporter assays. RESULTS: We found that circPTPN22 expression was downregulated in the PBMCs of SLE patients compared to those of healthy controls. Overexpression of circPTPN22 increased proliferation and inhibited apoptosis of Jurkat T cells, whereas knockdown of circPTPN22 exerted the opposite effects. CircPTPN22 acts as a miR-4689 sponge, and S1PR1 is a direct target of miR-4689. Importantly, the circPTPN22/miR-4689/S1PR1 axis inhibited the secretion of TNF-α and IL-6 in Jurkat T cells. CONCLUSIONS: CircPTPN22 acts as a miR-4689 sponge to regulate T-cell activation by targeting S1PR1, providing a novel mechanism for the pathogenesis of SLE.

circCsnk1g3- and circAnkib1-regulated interferon responses in sarcoma promote tumorigenesis by shaping the immune microenvironment.

Exonic circular RNAs (circRNAs) produce predominantly non-coding RNA species that have been recently profiled in many tumors. However, their functional contribution to cancer progression is still poorly understood. Here, we identify the circRNAs expressed in soft tissue sarcoma cells and explore how the circRNAs regulate sarcoma growth in vivo. We show that circCsnk1g3 and circAnkib1 promote tumor growth by shaping a pro-tumorigenic microenvironment, possibly due to their capabilities to regulate tumor-promoting elements extrinsic to the tumor cells. Accordingly, circCsnk1g3 and circAnkib1 can control the expression of interferon-related genes and pro-inflammatory factors in the sarcoma cells, thus directing immune cell recruitment into the tumor mass, and hence their activation. Mechanistically, circRNAs may repress pro-inflammatory elements by buffering activation of the pathways mediated by RIG-I, the cytosolic viral RNA sensor. The current findings suggest that the targeting of specific circRNAs could augment the efficacy of tumor and immune response to mainstay therapies.

Emerging roles of circular RNAs in innate immunity.

The proper function of the innate immune system depends on an intricate network of regulation that promotes effective responses to pathogens while avoiding autoimmunity. Circular RNAs (circRNAs), a class of RNAs that lack 5' and 3' ends, have emerged as key actors in these networks. Recent studies have demonstrated that endogenous circRNAs in eukaryotes regulate the activation of innate immune proteins and cells through diverse modes of action. Some DNA viruses also encode circRNAs, and foreign circRNAs have been found to stimulate an innate immune response. This review summarizes recent investigations that reveal the critical roles that circRNAs play in innate immunity and points to future areas of study in this emerging field.

A Previously Undiscovered Circular RNA, circTNFAIP3, and Its Role in Coronavirus Replication.

Circular RNAs (circRNAs) are a newly discovered class of noncoding RNAs (ncRNAs) present in various tissues and cells. However, the functions of most circRNAs have not been verified experimentally. Here, using deltacoronavirus as a model, differentially expressed circRNAs in cells with or without deltacoronavirus infection were analyzed by RNA sequencing to characterize the cellular responses to RNA virus infection. More than 57,000 circRNA candidates were detected, and seven significantly dysregulated circRNAs were quantitated by real-time reverse transcription-PCR. We discovered a previously unidentified circRNA derived from the TNFAIP3 gene, named circTNFAIP3, which is distributed and expressed widely in various tissues. RNA viruses, including deltacoronaviruses, rather than DNA viruses tend to activate the expression of endogenous circTNFAIP3. Overexpression of circTNFAIP3 promoted deltacoronavirus replication by reducing the apoptosis, while silencing of circTNFAIP3 inhibited deltacoronavirus replication by enhancing the apoptosis. In summary, our work provides useful circRNA-related information to facilitate investigation of the underlying mechanism of deltacoronavirus infection and identifies a novel circTNFAIP3 that promotes deltacoronavirus replication via regulating apoptosis. IMPORTANCE CircRNAs, a new class of ncRNAs, play important roles in cell growth, neural development, carcinogenesis, and anticarcinogenesis. Porcine deltacoronavirus is an emerging enteropathogenic coronavirus that causes diarrhea, but the role of host circRNAs in regulating its infection is unknown. Here, we performed expression profiling of circRNAs in mock- and deltacoronavirus- infected cells and identified the novel differentially expressed circular RNA circTNFAIP3. We demonstrate that circTNFAIP3 promotes deltacoronavirus replication by inhibiting apoptosis. Our findings first illustrate that circRNA can act as an apoptosis negative regulator during RNA virus infection and help to explore the underlying mechanism of deltacoronavirus infection.

Systematic Identification and Analysis of Circular RNAs of Japanese Flounder (Paralichthys olivaceus) in Response to Vibrio anguillarum Infection.

Circular RNA (circRNA) is a new class of non-coding RNA that is structured into a closed loop without polyadenylation. Recent studies showed that circRNAs are involved in the host immune response to pathogen infection. Japanese flounder (Paralichthys olivaceus), an important economical marine fish cultured in north Asia, is affected by Vibrio anguillarum, a pathogenic bacterium that can infect a large number of fish. In this study, we systematically explored the circRNAs in the spleen of V. anguillarum-infected flounder at different infection time points. A total of 6581 circRNAs were identified, 148 of which showed differential expression patterns after V. anguillarum infection and were named DEcirs. Most of the DEcirs were strongly time-specific. The parental genes of the DEcirs were identified and functionally classified into diverse pathways, including immune-related pathways. Among the immune-related DEcirs, seven were predicted to sponge 18 targeted miRNAs that were differentially expressed during V. anguillarum infection (named DETmiRs). Further analysis showed that the DEcirs and their corresponding DETmiRs intertwined into complicated immune related networks. These results indicate that in flounder, circRNAs are regulated by V. anguillarum and form interactive networks with mRNAs and miRNAs that likely play important roles in the immune defense against pathogen infection.