RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo.

Circular RNAs (circRNAs) are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Here, we show that unmodified exogenous circRNA is able to bypass cellular RNA sensors and thereby avoid provoking an immune response in RIG-I and Toll-like receptor (TLR) competent cells and in mice. The immunogenicity and protein expression stability of circRNA preparations are found to be dependent on purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses. Unmodified circRNA is less immunogenic than unmodified linear mRNA in vitro, in part due to the evasion of TLR sensing. Finally, we provide the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and we show that circRNA translation is extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs.

Activation of host nucleic acid sensors by Mycobacterium: good for us or good for them?

Although the importance of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) sensors in controlling viral infection is well established, their role in promoting an effective immune response to pathogens other than viruses is less clear. This is particularly true for infections with mycobacteria, as studies point to both protective and detrimental roles for activation of nucleic acid sensors in controlling a mycobacterial infection. Some of the contradiction likely stems from the use of different model systems and different mycobacterial species/strains as well as from which nucleic acid sensors were studied and what downstream effectors were evaluated. In this review, we will describe the different nucleic acid sensors that have been studied in the context of mycobacterial infections, and how the different studies compare. We conclude with a section on how nucleic acid sensor agonists have been used therapeutically and what further information is needed to enhance their potential as therapeutic agents.

Epigenetic regulation of RNA sensors: Sentinels of immune response.

Living host system possess mechanisms like innate immune system to combat against inflammation, stress singling, and cancer. These mechanisms are initiated by PAMP and DAMP mediated recognition by PRR. PRR is consist of variety of nucleic acid sensors like-RNA sensors. They play crucial role in identifying exogenous and endogenous RNA molecules, which subsequently mediate pro/inflammatory cytokine, IFN and ISGs response in traumatized or tumorigenic conditions. The sensors can sensitize wide range of nucleic acid particle in term of size and structure, while each category sensors belongs subclasses with differentially expressed in cell and distinguished functioning mechanisms. They are also able to make comparison between self and non-self-nucleic acid molecules through specific mechanisms. Besides exhibiting anti-inflammatory and anti-tumorigenic responses, RNA sensors cover the broad spectrum of response mechanisms. Transcriptionally RNA sensors undergo with tight epigenetic regulations. In this review study, we will be going to discuss about the details of RNA sensors, their functional mechanisms and epi-transactional regulations.

The RLR intrinsic antiviral system is expressed in neural retina and restricts lentiviral transduction of human Mueller cells.

The retinoic acid-inducible gene I (RIG)-I-like receptor (RLR) family of RNA sensor proteins plays a key role in the innate immune response to viral nucleic acids, including viral gene delivery vectors, but little is known about the expression of RLR proteins in the retina. The purpose of this study was to characterize cell-specific expression patterns of RLR proteins in non-human primate (NHP) neural retina tissue and to examine if RLR pathway signaling restricts viral gene delivery transduction. Since RLR protein signaling converges at the mitochondrial antiviral signaling protein (MAVS), experiments were performed to determine if knockdown of MAVS affected FIVGFP transduction efficiency in the human Mueller cell line MIO-M1. Immunoblotting confirmed expression of RIG-I, melanoma differentiation-associated protein 5 (MDA5), laboratory of genetics and physiology 2 (LGP2), and MAVS proteins in MIO-M1 cells and NHP retina tissue. Double label immunofluorescence (IF) studies revealed RIG-I, LGP2, and MAVS were expressed in Mueller microglial cells in the NHP retina. In addition, LGP2 and MDA5 proteins were detected in cone and retinal ganglion cells (RGC). MDA5 was also present in a subset of calretinin positive amacrine cells, and in nuclei within the inner nuclear layer (INL). Knockdown of MAVS significantly increased the transduction efficiency of the lentiviral vector FIVGFP in MIO-M1 cells, compared to control cells. FIVGFP or AAVGFP challenge did not alter expression of the LGP2, MAVS, MDA5 or RIG-I genes in MIO-M1 cells or NHP retina tissue compared to media treated controls. Our data demonstrate that innate immune response proteins involved in viral RNA sensing, including MDA5, RIG-I, LGP2, and MAVS, are expressed in several cell types within the NHP neural retina. In addition, the MAVS protein restricts non-human lentiviral transduction efficiency in MIO-M1 cells.

Electron-Rich EDOT Linkers in Tetracationic bis-Triarylborane Chromophores: Influence on Water Stability, Biomacromolecule Sensing, and Photoinduced Cytotoxicity.

Three novel tetracationic bis-triarylboranes with 3,4-ethylenedioxythiophene (EDOT) linkers, and their neutral precursors, showed significant red-shifted absorption and emission compared to their thiophene-containing analogues, with one of the EDOT-derivatives emitting in the NIR region. Only the EDOT-linked trixylylborane tetracation was stable in aqueous solution, indicating that direct attachment of a thiophene or even 3-methylthiophene to the boron atom is insufficient to provide hydrolytic stability in aqueous solution. Further comparative analysis of the EDOT-linked trixylylborane tetracation and its bis-thiophene analogue revealed efficient photo-induced singlet oxygen production, with the consequent biological implications. Thus, both analogues bind strongly to ds-DNA and BSA, very efficiently enter living human cells, accumulate in several different cytoplasmic organelles with no toxic effect but, under intense visible light irradiation, they exhibit almost instantaneous and very strong cytotoxic effects, presumably attributed to singlet oxygen production. Thus, both compounds are intriguing theranostic agents, whose intracellular and probably intra-tissue location can be monitored by strong fluorescence, allowing switching on of the strong bioactivity by well-focused visible light.

Poly(I:C) transfection induces a pro-inflammatory cascade in murine mammary carcinoma and fibrosarcoma cells.

Germline-encoded pattern recognition receptors [PRRs] in mammalian cells function in the detection of molecular patterns associated with pathogen invasion or cellular damage. A PRR subset is activated by the atypical presence and location of double-stranded RNA [dsRNA] or its synthetic analogue polyinosinic-polycytidylic acid [poly(I:C)], triggering pro-inflammatory signalling and death in many cell types. Poly(I:C) has been tested as a sole or combination cancer therapy in preclinical studies and clinical trials. The purpose of this study was to evaluate the effects of poly(I:C) transfection via electroporation on cell lines from a cancer of epithelial origin, 4T1 mammary carcinoma, and a cancer of mesenchymal origin, WEHI 164 fibrosarcoma. The effects of the poly(I:C) delivery on cell metabolism implicate the induction of cell death. A pro-inflammatory response was demonstrated by mRNA upregulation and the secretion of Type I interferon and several cytokines and chemokines. The mRNAs of dsRNA sensor DExD/H-box helicase 58/retinoic acid-inducible gene I protein [Ddx58/RIG-I] and sensor/co-sensor DEAH-box helicase 9 [Dhx9] were not regulated, but the mRNAs of RNA sensors toll-like receptor 3 [TLR3], interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5 [Ifih1/MDA5] and Z-DNA binding protein 1 [Zbp1] and co-sensors DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 [Ddx60] and interferon-inducible protein 204 [Ifi204] were upregulated in both cell lines. The mRNAs encoding signalling pathways components were present or upregulated in both cell types. These data demonstrate that RNA sensing effects can be amplified by electroporation delivery, potentially expanding the practicality of this immunotherapeutic approach.

Roles of RNA Sensors in Host Innate Response to Influenza Virus and Coronavirus Infections.

Influenza virus and coronavirus are two important respiratory viruses, which often cause serious respiratory diseases in humans and animals after infection. In recent years, highly pathogenic avian influenza virus (HPAIV) and SARS-CoV-2 have become major pathogens causing respiratory diseases in humans. Thus, an in-depth understanding of the relationship between viral infection and host innate immunity is particularly important to the stipulation of effective control strategies. As the first line of defense against pathogens infection, innate immunity not only acts as a natural physiological barrier, but also eliminates pathogens through the production of interferon (IFN), the formation of inflammasomes, and the production of pro-inflammatory cytokines. In this process, the recognition of viral pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) is the initiation and the most important part of the innate immune response. In this review, we summarize the roles of RNA sensors in the host innate immune response to influenza virus and coronavirus infections in different species, with a particular focus on innate immune recognition of viral nucleic acids in host cells, which will help to develop an effective strategy for the control of respiratory infectious diseases.

The Second Class of Tetrahydrofolate (THF-II) Riboswitches Recognizes the Tetrahydrofolic Acid Ligand via Local Conformation Changes.

Riboswitches are regulatory noncoding RNAs found in bacteria, fungi and plants, that modulate gene expressions through structural changes in response to ligand binding. Understanding how ligands interact with riboswitches in solution can shed light on the molecular mechanisms of this ancient regulators. Previous studies showed that riboswitches undergo global conformation changes in response to ligand binding to relay information. Here, we report conformation switching models of the recently discovered tetrahydrofolic acid-responsive second class of tetrahydrofolate (THF-II) riboswitches in response to ligand binding. Using a combination of selective 2'-hydroxyl acylation, analyzed by primer extension (SHAPE) assay, 3D modeling and small-angle X-ray scattering (SAXS), we found that the ligand specifically recognizes and reshapes the THF-II riboswitch loop regions, but does not affect the stability of the P3 helix. Our results show that the THF-II riboswitch undergoes only local conformation changes in response to ligand binding, rearranging the Loop1-P3-Loop2 region and rotating Loop1 from a ~120° angle to a ~75° angle. This distinct conformation changes suggest a unique regulatory mechanism of the THF-II riboswitch, previously unseen in other riboswitches. Our findings may contribute to the fields of RNA sensors and drug design.

Bis(phenylethynyl)arene Linkers in Tetracationic Bis-triarylborane Chromophores Control Fluorimetric and Raman Sensing of Various DNAs and RNAs.

We report four new luminescent tetracationic bis-triarylborane DNA and RNA sensors that show high binding affinities, in several cases even in the nanomolar range. Three of the compounds contain substituted, highly emissive and structurally flexible bis(2,6-dimethylphenyl-4-ethynyl)arene linkers (3: arene=5,5'-2,2'-bithiophene; 4: arene=1,4-benzene; 5: arene=9,10-anthracene) between the two boryl moieties and serve as efficient dual Raman and fluorescence chromophores. The shorter analogue 6 employs 9,10-anthracene as the linker and demonstrates the importance of an adequate linker length with a certain level of flexibility by exhibiting generally lower binding affinities than 3-5. Pronounced aggregation-deaggregation processes are observed in fluorimetric titration experiments with DNA for compounds 3 and 5. Molecular modelling of complexes of 5 with AT-DNA, suggest the minor groove as the dominant binding site for monomeric 5, but demonstrate that dimers of 5 can also be accommodated. Strong SERS responses for 3-5 versus a very weak response for 6, particularly the strong signals from anthracene itself observed for 5 but not for 6, demonstrate the importance of triple bonds for strong Raman activity in molecules of this compound class. The energy of the characteristic stretching vibration of the C≡C bonds is significantly dependent on the aromatic moiety between the triple bonds. The insertion of aromatic moieties between two C≡C bonds thus offers an alternative design for dual Raman and fluorescence chromophores, applicable in multiplex biological Raman imaging.

RNA Signaling in Pulmonary Arterial Hypertension-A Double-Stranded Sword.

Recognition of and response to pathogens and tissue injury is driven by the innate immune system via activation of pattern recognition receptors. One of the many patterns recognized is RNA and, while several receptors bind RNA, Toll-like receptor 3 (TLR3) is well placed for initial recognition of RNA molecules due to its localization within the endosome. There is a growing body of work describing a role for TLR3 in maintenance of vascular homeostasis. For example, TLR3 deficiency has been shown to play repair and remodeling roles in the systemic vasculature and in lung parenchyma. A hallmark of pulmonary arterial hypertension (PAH) is pulmonary vascular remodeling, yet drivers and triggers of this remodeling remain incompletely understood. Based on its role in the systemic vasculature, our group discovered reduced endothelial TLR3 expression in PAH and revealed a protective role for a TLR3 agonist in rodent models of pulmonary hypertension. This review will provide an overview of RNA signaling in the vasculature and how it relates to PAH pathobiology, including whether targeting double-stranded RNA signaling is a potential treatment option for PAH.

Posttranslational Modification Control of Inflammatory Signaling.

Inflammation is usually the defensive reaction of the immune system to the invasion of pathogen and the exogenous objects. The activation of inflammation helps our body to eliminate pathogenic microbe, virus, and parasite harming our health, while under many circumstances inflammation is the direct cause of the pathological damage in tissues and dysfunction of organs. The posttranslational modification (PTM) of the inflammatory pathways, such as TLR pathways, RLR pathways, NLR pathway, intracellular DNA sensors, intracellular RNA sensors, and inflammasomes, is crucial in the regulation of these signaling trails. Ubiquitination, phosphorylation, polyubiquitination, methylation, and acetylation are the main forms of the PTM, and they respectively play different roles in signaling regulation. The effects of the PTM range from the production of pro-inflammatory factors and the interaction between adaptors and receptors to cell translocation in response to the infectious or other dangerous factors. In this chapter, we will have an overview of the different ways of the posttranslational modifications in different inflammatory signaling pathways and their essential roles in regulation of inflammation.

Double-stranded RNA analog and type I interferon regulate expression of Trem paired receptors in murine myeloid cells.

BACKGROUND: Triggering receptors expressed on myeloid cells (Trem) proteins are a family of cell surface receptors used to control innate immune responses such as proinflammatory cytokine production in mice. Trem genes belong to a rapidly expanding family of receptors that include activating and inhibitory paired-isoforms. RESULTS: By comparative genomic analysis, we found that Trem4, Trem5 and Trem-like transcript-6 (Treml6) genes typically paired receptors. These paired Trem genes were murine-specific and originated from an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing gene. Treml6 encoded ITIM, whereas Trem4 and Trem5 lacked the ITIM but possessed positively-charged residues to associate with DNAX activating protein of 12 kDa (DAP12). DAP12 was directly associated with Trem4 and Trem5, and DAP12 coupling was mandatory for their expression on the cell surface. In bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs), and splenic DC subsets, polyinosinic-polycytidylic acid (polyI:C) followed by type I interferon (IFN) production induced Trem4 and Treml6 whereas polyI:C or other TLR agonists failed to induce the expression of Trem5. PolyI:C induced Treml6 and Trem4 more efficiently in BMDMs than BMDCs. Treml6 was more potentially up-regulated in conventional DC (cDCs) and plasmacytoid DC (pDCs) than Trem4 in mice upon in vivo stimulation with polyI:C. DISCUSSION: Treml6-dependent inhibitory signal would be dominant in viral infection compared to resting state. Though no direct ligands of these Trem receptors have been determined, the results infer that a set of Trem receptors are up-regulated in response to viral RNA to regulate myeloid cell activation through modulation of DAP12-associated Trem4 and ITIM-containing Treml6.

Perspective: The RNA exosome, cytokine gene regulation and links to autoimmunity.

The RNA exosome is a highly conserved exoribonuclease complex that is involved in RNA processing, quality control and turnover regulation. The exosome plays pleiotropic functions by recruiting different cofactors that regulate its target specificity. Recently, the exosome has been implicated in the regulation of immune processes including cytokine production and negative regulation of innate sensing of nucleic acids. Careful regulation of such mechanisms is critical to avoid a breakdown of self-tolerance and the pathogenesis of autoimmune disorders. This perspective briefly introduces the exosome, its its normal function in RNA biology and summarizes regulatory roles of the RNA exosome in immunity. Finally we discuss how dysregulation of exosome function can lead to autoimmune disease.

Infection of human islets of Langerhans with two strains of Coxsackie B virus serotype 1: assessment of virus replication, degree of cell death and induction of genes involved in the innate immunity pathway.

Type 1 diabetes mellitus is believed to be triggered, in part, by one or more environmental factors and human enteroviruses (HEVs) are among the candidates. Therefore, this study has examined whether two strains of HEV may differentially affect the induction of genes involved in pathways leading to the synthesis of islet hormones, chemokines and cytokines in isolated, highly purified, human islets. Isolated, purified human pancreatic islets were infected with strains of Coxsackievirus B1.Viral replication and the degree of CPE/islet dissociation were monitored. The expression of insulin, glucagon, CXCL10, TLR3, IF1H1, CCL5, OAS-1, IFNß, and DDX58 was analyzed. Both strains replicated in islets but only one of strain caused rapid islet dissociation/CPE. Expression of the insulin gene was reduced during infection of islets with either viral strain but the gene encoding glucagon was unaffected. All genes analyzed which are involved in viral sensing and the development of innate immunity were induced by Coxsackie B viruses, with the notable exception of TLR3. There was no qualitative difference in the expression pattern between each strain but the magnitude of the response varied between donors. The lack of virus induced expression of TLR3, together with the differential regulation of IF1H1, OAS1 and IFNß, (each of which has polymorphic variants influence the predisposition to type 1 diabetes), that might result in defective clearance of virus from islet cells. The reduced expression of the insulin gene and the unaffected expression of the gene encoding glucagon by Coxsackie B1 infection is consistent with the preferential ß-cell tropism of the virus.

RNA-Based Sensor Systems for Affordable Diagnostics in the Age of Pandemics.

In the era of the COVID-19 pandemic, the significance of point-of-care (POC) diagnostic tools has become increasingly vital, driven by the need for quick and precise virus identification. RNA-based sensors, particularly toehold sensors, have emerged as promising candidates for POC detection systems due to their selectivity and sensitivity. Toehold sensors operate by employing an RNA switch that changes the conformation when it binds to a target RNA molecule, resulting in a detectable signal. This review focuses on the development and deployment of RNA-based sensors for POC viral RNA detection with a particular emphasis on toehold sensors. The benefits and limits of toehold sensors are explored, and obstacles and future directions for improving their performance within POC detection systems are presented. The use of RNA-based sensors as a technology for rapid and sensitive detection of viral RNA holds great potential for effectively managing (dealing/coping) with present and future pandemics in resource-constrained settings.

Optimization of RNA Pepper Sensors for the Detection of Arbitrary RNA Targets.

The development of fluorescent light-up RNA aptamers (FLAPs) has paved the way for the creation of sensors to track RNA in live cells. A major challenge with FLAP sensors is their brightness and limited signal-to-background ratio both in vivo and in vitro. To address this, we develop sensors using the Pepper aptamer, which exhibits superior brightness and photostability when compared to other FLAPs. The sensors are designed to fold into a low fluorescence conformation and to switch to a high fluorescence conformation through toehold or loop-mediated interactions with their RNA target. Our sensors detect RNA targets as short as 20 nucleotides in length with a wide dynamic range over 300-fold in vitro, and we describe strategies for optimizing the sensor's performance for any given RNA target. To demonstrate the versatility of our design approach, we generated Pepper sensors for a range of specific, biologically relevant RNA sequences. Our design and optimization strategies are portable to other FLAPs and offer a promising foundation for future development of RNA sensors with high specificity and sensitivity for detecting RNA biomarkers with multiple applications.

Innate immune responses to RNA: sensing and signaling.

Nucleic acids are among the most essential PAMPs (pathogen-associated molecular patterns). Animals have evolved numerous sensors to recognize nucleic acids and trigger immune signaling against pathogen replication, cellular stress and cancer. Many sensor proteins (e.g., cGAS, AIM2, and TLR9) recognize the molecular signature of infection or stress and are responsible for the innate immune response to DNA. Remarkably, recent evidence demonstrates that cGAS-like receptors acquire the ability to sense RNA in some forms of life. Compared with the nucleic-acid sensing by cGAS, innate immune responses to RNA are based on various RNA sensors, including RIG-I, MDA5, ADAR1, TLR3/7/8, OAS1, PKR, NLRP1/6, and ZBP1, via a broad-spectrum signaling axis. Importantly, new advances have brought to light the potential clinical application of targeting these signaling pathways. Here, we highlight the latest discoveries in the field. We also summarize the activation and regulatory mechanisms of RNA-sensing signaling. In addition, we discuss how RNA sensing is tightly controlled in cells and why the disruption of immune homeostasis is linked to disease.

RNAs as Sensors of Oxidative Stress in Bacteria.

Oxidative stress is an important and pervasive physical stress encountered by all kingdoms of life, including bacteria. In this review, we briefly describe the nature of oxidative stress, highlight well-characterized protein-based sensors (transcription factors) of reactive oxygen species that serve as standards for molecular sensors in oxidative stress, and describe molecular studies that have explored the potential of direct RNA sensitivity to oxidative stress. Finally, we describe the gaps in knowledge of RNA sensors-particularly regarding the chemical modification of RNA nucleobases. RNA sensors are poised to emerge as an essential layer of understanding and regulating dynamic biological pathways in oxidative stress responses in bacteria and, thus, also represent an important frontier of synthetic biology.

Expression of down-regulated ERV LTR elements associates with immune activation in human small-cell lung cancers.

Small-cell lung cancer (SCLC) is an aggressive cancer characterized by immunosuppressive features leading to poor responses to current immunotherapies. Activation of transposable elements (TE) can trigger an innate immune response, which can synergize with immunotherapeutic protocols in patients. However, TE activity in relation to immune gene response is not fully known in human SCLC. Here, we compared TE expression in 104 human SCLC and 24 normal tissues and established their involvement in innate immune responses. We observed that different intergenic TEs, mainly endogenous retroviral (ERV) families, are deregulated in SCLC. Similarly to other cancers, we detected a subset of LTRs that correlate with innate immune gene signatures and cytosolic RNA sensors, such as RIG-I. These LTRs are downregulated in SCLC tumors vs. normal tissues, and are mainly located at transcriptional repressed regions, marked with H3K4me2 in different cell lines. Analyses of different genomic datasets show that chromatin repression is likely due to de-methylase LSD1 activity. Moreover, high expression levels of ERV LTRs predict a better survival upon chemotherapy of SCLC patients. The findings reveal a specific pattern of TE-mediated activation of innate immune genes in SCLC, which can be exploited to establish more effective immunotherapeutic combinations.

Exploring the Molecular Basis of Vesicular Stomatitis Virus Pathogenesis in Swine: Insights from Expression Profiling of Primary Macrophages Infected with M51R Mutant Virus.

Vesicular stomatitis virus (VSV) is an emergent virus affecting livestock in the US. Previously, using a recombinant VSV carrying the M51R mutation in the matrix protein (rNJ0612NME6-M51R), we evaluated the pathogenesis of this virus in pigs. Our results indicated that rNJ0612NME6-M51R represented an attenuated phenotype in in-vivo and in ex-vivo in pig macrophages, resembling certain clinical features observed in field VSV isolates. In order to gain more insight into the molecular basis leading to the attenuation of rNJ0612NME6-M51R in pigs, we conducted a microarray analysis to assess the gene expression profiles of primary porcine macrophages infected with rNJ0612NME6-M51R compared to its parental virus (rNJ0612NME6). Our results showed an overall higher gene expression in macrophages infected with rNJ0612NME6-M51R. Specifically, we observed that the pathways related with immune cytokine signaling and interferon (IFN)-related responses (including activation, signaling, induction, and antiviral mechanisms) were the ones comprising most of the relevant genes identified during this study. Collectively, the results presented herein highlight the relevance of type I interferon during the pathogenesis of VSV in pigs. The information generated from this study may represent a framework for future studies intended to understand the molecular bases of the pathogenesis of field strains in livestock.