Grainyhead-like-2, an epithelial master programmer, promotes interferon induction and suppresses breast cancer recurrence.

Type-I and -III interferons play a central role in immune rejection of pathogens and tumors, thus promoting immunogenicity and suppressing tumor recurrence. Double strand RNA is an important ligand that stimulates tumor immunity via interferon responses. Differentiation of embryonic stem cells to pluripotent epithelial cells activates the interferon response during development, raising the question of whether epithelial vs. mesenchymal gene signatures in cancer potentially regulate the interferon pathway as well. Here, using genomics and signaling approaches, we show that Grainyhead-like-2 (GRHL2), a master programmer of epithelial cell identity, promotes type-I and -III interferon responses to double-strand RNA. GRHL2 enhanced the activation of IRF3 and relA/NF-kB and the expression of IRF1; a functional GRHL2 binding site in the IFNL1 promoter was also identified. Moreover, time to recurrence in breast cancer correlated positively with GRHL2 protein expression, indicating that GRHL2 is a tumor recurrence suppressor, consistent with its enhancement of interferon responses. These observations demonstrate that epithelial cell identity supports interferon responses in the context of cancer.

Caenorhabditis elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA.

Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

Control of Sclerotinia sclerotiorum via an RNA interference (RNAi)-mediated targeting of SsPac1 and SsSmk1.

MAIN CONCLUSION: The study evaluates the potential of Spray-Induced Gene Silencing and Host-Induced Gene Silencing for sustainable crop protection against the broad-spectrum necrotrophic fungus Sclerotinia sclerotiorum. Sclerotinia sclerotiorum (Lib.) de Bary, an aggressive ascomycete fungus causes white rot or cottony rot on a broad range of crops including Brassica juncea. The lack of sustainable control measures has necessitated biotechnological interventions such as RNA interference (RNAi) for effective pathogen control. Here we adopted two RNAi-based strategies-Spray-Induced Gene Silencing (SIGS) and Host-Induced Gene Silencing (HIGS) to control S. sclerotiorum. SIGS was successful in controlling white rot on Nicotiana benthamiana and B. juncea by targeting SsPac1, a pH-responsive transcription factor and SsSmk1, a MAP kinase involved in fungal development and pathogenesis. Topical application of dsRNA targeting SsPac1 and SsSmk1 delayed infection initiation and progression on B. juncea. Further, altered hyphal morphology and reduced radial growth were also observed following dsRNA application. We also explored the impact of stable dsRNA expression in A. thaliana against S. sclerotiorum. In this report, we highlight the utility of RNAi as a biofungicide and a tool for preliminary functional genomics.

MiR-21 Regulates Growth and Migration of Cervical Cancer Cells by RECK Signaling Pathway.

Expression of miR-21 has been found to be altered in almost all types of cancers, and it has been classified as an oncogenic microRNA. In addition, the expression of tumor suppressor gene RECK is associated with miR-21 overexpression in high-grade cervical lesions. In the present study, we analyze the role of miR-21 in RECK gene regulation in cervical cancer cells. To identify the downstream cellular target genes of upstream miR-21, we silenced endogenous miR-21 expression using siRNAs. We analyzed the expression of miR-21 and RECK, as well as functional effects on cell proliferation and migration. We found that in cervical cancer cells, there was an inverse correlation between miR-21 expression and RECK mRNA and protein expression. SiRNAs to miR-21 increased luciferase reporter activity in construct plasmids containing the RECK-3'-UTR microRNA response elements MRE21-1, MRE21-2, and MRE21-3. The role of miR-21 in cell proliferation was also analyzed, and cancer cells transfected with siRNAs exhibited a markedly reduced cell proliferation and migration. Our findings indicate that miR-21 post-transcriptionally down-regulates the expression of RECK to promote cell proliferation and cell migration inhibition in cervical cancer cell survival. Therefore, miR-21 and RECK may be potential therapeutic targets in gene therapy for cervical cancer.

RNA sequences that direct selective ADAR editing from a SELEX library bearing 8-azanebularine.

Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs' ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.

The dsRNA isolated from Escherichia coli infected with the MS2 bacteriophage induces the production of interferons.

Viral infections pose a significant threat to public health, and the production of interferons represents one of the most critical antiviral innate immune responses of the host. Consequently, the screening and identification of compounds or reagents that induce interferon production are of paramount importance. This study commenced with the cultivation of host bacterium 15,597, followed by the infection of Escherichia coli with the MS2 bacteriophage. Utilizing the J2 capture technique, a class of dsRNA mixtures (MS2+15,597) was isolated from the E. coli infected with the MS2 bacteriophage. Subsequent investigations were conducted on the immunostimulatory activity of the MS2+15,597 mixture. The results indicated that the dsRNA mixtures (MS2+15,597) extracted from E. coli infected with the MS2 bacteriophage possess the capability to activate innate immunity, thereby inducing the production of interferon-ß. These dsRNA mixtures can activate the RIG-I and TLR3 pattern recognition receptors, stimulating the expression of interferon stimulatory factors 3/7, which in turn triggers the NF-κB signaling pathway, culminating in the cellular production of interferon-ß to achieve antiviral effects. This study offers novel insights and strategies for the development of broad-spectrum antiviral drugs, potentially providing new modalities for future antiviral therapies.

Limited dsRNA editing impedes leukemia stem cells.

Understanding the mechanisms underlying the generation and maintenance of leukemia stem cells (LSCs) is crucial for the development of effective therapies against T cell acute lymphoblastic leukemia (T-ALL). In a recent study, Rivera et al. discovered that elevated adenosine deaminase acting on RNA (ADAR)-1-mediated RNA editing is a distinguishing feature of T-ALL relapse, and that ADAR1 suppresses apoptosis triggered by the double-stranded (ds)RNA-sensing pathway.

RNA is a pro-apoptotic target of cisplatin in cancer cell lines and C. elegans.

Cisplatin not only targets DNA but also RNA. However, it is largely unknown whether platinated RNA (Pt-RNA) causes apoptosis and thus contributes to the cytotoxic effects of cisplatin. Consequently, cellular RNA was isolated from HepG2 and LS180 cells, exposed to cisplatin, and the resulting Pt-RNA (20 ng Pt/µg RNA) was transfected into these cancer cell lines or used to treat an apoptosis reporter Caenorhabditis elegans (C. elegans) strain (MD701, expressing CED-1::GFP). Cellular and molecular effects of Pt-RNA were evaluated by luminogenic caspase 3/7 assays, PCR array analysis, and fluorescence microscopy-based quantification of apoptosis in C. elegans gonads. Assuming RNA cross-linking (pseudo double-stranded RNA), the contribution of the Toll-like receptor 3 (TLR3, a sensor of double-stranded RNA) to apoptosis induction in cancer cell lines was investigated by pharmacological TLR3 inhibition and overexpression. In contrast to controls, Pt-RNA significantly enhanced apoptosis in C. elegans (2-fold) and in the cancer cell lines (2-fold to 4-fold). TLR3 overexpression significantly enhanced the pro-apoptotic effects of Pt-RNA in HepG2 cells. TLR3 inhibition reduced the pro-apoptotic effects of Pt-RNA and cisplatin, but not of paclitaxel (off-target control). Gene expression analysis showed that Pt-RNA (but not RNA) significantly enhanced the mRNA levels of nuclear factor kappa B subunit 2 and interleukin-8 in HepG2 cells, suggesting that Pt-RNA is a damage-associated molecular pattern that additionally causes pro-inflammatory responses. Together, this data suggests that not only DNA but also cellular RNA is a functionally relevant target of cisplatin, leading to pro-apoptotic and immunogenic effects.

Interior modification of Macrobrachium rosenbergii nodavirus-like particle enhances encapsulation of VP37-dsRNA against shrimp white spot syndrome infection.

BACKGROUND: Application of a virus-like particle (VLP) as a nanocontainer to encapsulate double stranded (ds)RNA to control viral infection in shrimp aquaculture has been extensively reported. In this study, we aimed at improving VLP's encapsulation efficiency which should lead to a superior fighting weapon with disastrous viruses. RESULTS: We constructed 2 variants of chimeric Macrobrachium rosenbergii nodavirus (MrNV)-like particles (V1- and V2-MrN-VLPs) and tested their efficiency to encapsulate VP37 double stranded RNA as well as WSSV protection in P. vannamei. Two types of short peptides, RNA-binding domain (RBD) and deca-arginine (10R) were successfully engineered into the interior surface of VLP, the site where the contact with VP37-dsRNA occurs. TEM and dynamic light scattering (DLS) analyses revealed that the chimeric VLPs remained their assembling property to be an icosahedral symmetric particle with a diameter of about 30 nm, similar to the original MrN-VLP particle. The superior encapsulation efficiency of VP37-dsRNA into V2-MrN-VLP was achieved, which was slightly better than that of V1-MrN-VLP but far better (1.4-fold) than its parental V0-MrN-VLP which the mole ratio of 7.5-10.5 for all VLP variants. The protection effect against challenging WSSV (as gauged from the level of VP37 gene and the remaining viral copy number in shrimp) was significantly improved in both V1- and V2-MrN-VLP compared with an original V0-MrN-VLP template. CONCLUSION: MrN-VLP (V0-) were re-engineered interiorly with RBD (V1-) and 10R (V2-) peptides which had an improved VP37-dsRNA encapsulation capability. The protection effect against WSSV infection through shrimp administration with dsRNA + V1-/V2-MrN VLPs was experimentally evident.

Unraveling the Role of RNase L Knockout in Alleviating Immune Response Activation in Mice Bone Marrow after Irradiation.

Ionizing radiation (IR) induces severe hematopoietic injury by causing DNA and RNA damage as well as activating the immune responses, necessitating the development of effective therapeutic strategies. Ribonuclease L (RNase L) as an innate immune response pathway is triggered by exogenous and endogenous abnormal dsRNA under viral infection and dyshomeostasis, thereby activating the immune responses. Thus, we investigated the effect of RNase L on irradiation-induced bone marrow damage using RNase L knockout (RNase L-/-) mice. Phenotypic analysis revealed that RNase L knockout mitigates irradiation-induced injury in the bone marrow. Further investigation into the mechanism of RNase L by RNA-seq, qRT-PCR, and CBA analysis demonstrated that RNase L deficiency counteracts the upregulation of genes related to immune responses induced by irradiation, including cytokines and interferon-stimulated genes. Moreover, RNase L deficiency inhibits the increased levels of immunoglobulins in serum induced by irradiation. These findings indicate that RNase L plays a role in the immune response induced by irradiation in the bone marrow. This study further enhances our understanding of the biological functions of RNase L in the immune response induced by irradiation and offers a novel approach for managing irradiation-induced bone marrow injury through the regulation of RNase L activation.

Induction of Viral Mimicry Upon Loss of DHX9 and ADAR1 in Breast Cancer Cells.

Detection of viral double-stranded RNA (dsRNA) is an important component of innate immunity. However, many endogenous RNAs containing double-stranded regions can be misrecognized and activate innate immunity. The IFN-inducible ADAR1-p150 suppresses dsRNA sensing, an essential function for adenosine deaminase acting on RNA 1 (ADAR1) in many cancers, including breast. Although ADAR1-p150 has been well established in this role, the functions of the constitutively expressed ADAR1-p110 isoform are less understood. We used proximity labeling to identify putative ADAR1-p110-interacting proteins in breast cancer cell lines. Of the proteins identified, the RNA helicase DHX9 was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of the dsRNA sensor PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Together, these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways. SIGNIFICANCE: These findings implicate DHX9 as a suppressor of dsRNA sensing. In some cell lines, loss of DHX9 alone is sufficient to cause activation of dsRNA sensing pathways, while in other cell lines DHX9 functions redundantly with ADAR1 to suppress pathway activation.

Multifaceted roles of RNA editing enzyme ADAR1 in innate immunity.

Innate immunity must be tightly regulated to enable sensitive pathogen detection while averting autoimmunity triggered by pathogen-like host molecules. A hallmark of viral infection, double-stranded RNAs (dsRNAs) are also abundantly encoded in mammalian genomes, necessitating surveillance mechanisms to distinguish "self" from "nonself." ADAR1, an RNA editing enzyme, has emerged as an essential safeguard against dsRNA-induced autoimmunity. By converting adenosines to inosines (A-to-I) in long dsRNAs, ADAR1 covalently marks endogenous dsRNAs, thereby blocking the activation of the cytoplasmic dsRNA sensor MDA5. Moreover, beyond its editing function, ADAR1 binding to dsRNA impedes the activation of innate immune sensors PKR and ZBP1. Recent landmark studies underscore the utility of silencing ADAR1 for cancer immunotherapy, by exploiting the ADAR1-dependence developed by certain tumors to unleash an antitumor immune response. In this perspective, we summarize the genetic and mechanistic evidence for ADAR1's multipronged role in suppressing dsRNA-mediated autoimmunity and explore the evolving roles of ADAR1 as an immuno-oncology target.

Adenosine deaminases that act on RNA, then and now.

In this article, I recount my memories of key experiments that led to my entry into the RNA editing/modification field. I highlight initial observations made by the pioneers in the ADAR field, and how they fit into our current understanding of this family of enzymes. I discuss early mysteries that have now been solved, as well as those that still linger. Finally, I discuss important, outstanding questions and acknowledge my hope for the future of the RNA editing/modification field.

Initiation of a ZAKα-dependent ribotoxic stress response by the innate immunity endoribonuclease RNase L.

RNase L is an endoribonuclease of higher vertebrates that functions in antiviral innate immunity. Interferons induce oligoadenylate synthetase enzymes that sense double-stranded RNA of viral origin leading to the synthesis of 2',5'-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L remodels the host cell transcriptome. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A is directly introduced into cells. Here, we report that RNase L activation by 2-5A causes a ribotoxic stress response involving the MAP kinase kinase kinase (MAP3K) ZAKα, MAP2Ks, and the stress-activated protein kinases JNK and p38α. RNase L activation profoundly alters the transcriptome by widespread depletion of mRNAs associated with different cellular functions but also by JNK/p38α-stimulated induction of inflammatory genes. These results show that the 2-5A/RNase L system triggers a protein kinase cascade leading to proinflammatory signaling and apoptosis.

Contrasting functions of ATP hydrolysis by MDA5 and LGP2 in viral RNA sensing.

Cytosolic long dsRNA, among the most potent proinflammatory signals, is recognized by melanoma differentiation-associated protein 5 (MDA5). MDA5 binds dsRNA cooperatively forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-ß responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. Consecutive rounds of ATP hydrolysis amplify the filament footprint, displacing tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling identifies an internally bound MDA5-LGP2-RNA complex, with the LGP2 C-terminal tail forming the key contacts with MDA5. These contacts are specifically required for NTP-dependent internal RNA binding. We conclude that NTPase-dependent binding of LGP2 to internal dsRNA sites complements NTPase-independent binding to dsRNA ends, via distinct binding modes, to increase the number and signaling output of MDA5-dsRNA complexes.

Blockade of pan-viral propagation by inhibition of host cell PNPT1.

For successful viral propagation within infected cells, the virus needs to overcome the cellular integrated stress response (ISR), triggered during viral infection, which, in turn, inhibits general protein translation. This paper reports a tactic employed by viruses to suppress the ISR by upregulating host cell polyribonucleotide nucleotidyltransferase 1 (PNPT1). The propagation of adenovirus, murine cytomegalovirus and hepatovirus within their respective host cells induces PNPT1 expression. Notably, when PNPT1 is knocked down, the propagation of all three viruses is prevented. Mechanistically, the inhibition of PNPT1 facilitates the relocation of mitochondrial double-stranded RNAs (mt-dsRNAs) to the cytoplasm, where they activate RNA-activated protein kinase (PKR). This activation leads to eukaryotic initiation factor 2α (eIF2α) phosphorylation, resulting in the suppression of translation. Furthermore, by scrutinizing the PNPT1 recognition element and screening 17,728 drugs and bioactive compounds approved by the US Food and Drug Administration, lanatoside C was identified as a potent PNPT1 inhibitor. This compound impedes the propagation of adenovirus, murine cytomegalovirus and hepatovirus, and suppresses production of the severe acute respiratory syndrome coronavirus-2 spike protein. These discoveries shed light on a novel strategy to impede pan-viral propagation by activating the host cell mt-dsRNA-PKR-eIF2α signalling axis.

Cholesterol Conjugates of Small Interfering RNA: Linkers and Patterns of Modification.

Cholesterol siRNA conjugates attract attention because they allow the delivery of siRNA into cells without the use of transfection agents. In this study, we compared the efficacy and duration of silencing induced by cholesterol conjugates of selectively and totally modified siRNAs and their heteroduplexes of the same sequence and explored the impact of linker length between the 3' end of the sense strand of siRNA and cholesterol on the silencing activity of "light" and "heavy" modified siRNAs. All 3'-cholesterol conjugates were equally active under transfection, but the conjugate with a C3 linker was less active than those with longer linkers (C8 and C15) in a carrier-free mode. At the same time, they were significantly inferior in activity to the 5'-cholesterol conjugate. Shortening the sense strand carrying cholesterol by two nucleotides from the 3'-end did not have a significant effect on the activity of the conjugate. Replacing the antisense strand or both strands with fully modified ones had a significant effect on silencing as well as improving the duration in transfection-mediated and carrier-free modes. A significant 78% suppression of MDR1 gene expression in KB-8-5 xenograft tumors developed in mice promises an advantage from the use of fully modified siRNA cholesterol conjugates in combination chemotherapy.

DsRNA-based pesticides: Considerations for efficiency and risk assessment.

In view of the ongoing climate change and the ever-growing world population, novel agricultural solutions are required to ensure sustainable food supply. Microbials, natural substances, semiochemicals and double stranded RNAs (dsRNAs) are all considered potential low risk pesticides. DsRNAs function at the molecular level, targeting specific regions of specific genes of specific organisms, provided that they share a minimal sequence complementarity of approximately 20 nucleotides. Thus, dsRNAs may offer a great alternative to conventional chemicals in environmentally friendly pest control strategies. Any low-risk pesticide needs to be efficient and exhibit low toxicological potential and low environmental persistence. Having said that, in the current review, the mode of dsRNA action is explored and the parameters that need to be taken into consideration for the development of efficient dsRNA-based pesticides are highlighted. Moreover, since dsRNAs mode of action differs from those of synthetic pesticides, custom-made risk assessment schemes may be required and thus, critical issues related to the risk assessment of dsRNA pesticides are discussed here.

Satellite double-stranded RNA induces mesenchymal transition in pancreatic cancer by regulating alternative splicing.

Human satellite II (HSATII), composed of tandem repeats in pericentromeric regions, is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of repetitive elements in cancer tissues can facilitate incidental dsRNA formation; however, it remains controversial whether dsRNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the double-stranded formation of HSATII RNA and explored its molecular function. The overexpression of double-stranded HSATII (dsHSATII) RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, spermatid perinuclear RNA-binding protein (STRBP), which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. The mesenchymal transition of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with epithelial-mesenchymal transition (EMT). We also confirmed that isoform switching of CLSTN1, driven by dsHSATII overexpression or STRBP depletion, induced EMT-like morphological changes. These findings reveal a novel tumor-promoting function of dsHSATII RNA, inducing EMT-like changes and cell invasiveness, thus enhancing our understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors.

Significant Variations in Double-Stranded RNA Levels in Cultured Skin Cells.

Endogenous double-stranded RNA has emerged as a potent stimulator of innate immunity. Under physiological conditions, endogenous dsRNA is maintained in the cell nucleus or the mitochondria; however, if protective mechanisms are breached, it leaches into the cytoplasm and triggers immune signaling pathways. Ectopic activation of innate immune pathways is associated with various diseases and senescence and can trigger apoptosis. Hereby, the level of cytoplasmic dsRNA is crucial. We have enriched dsRNA from two melanoma cell lines and primary dermal fibroblasts, including a competing probe, and analyzed the dsRNA transcriptome using RNA sequencing. There was a striking difference in read counts between the cell lines and the primary cells, and the effect was confirmed by northern blotting and immunocytochemistry. Both mitochondria (10-20%) and nuclear transcription (80-90%) contributed significantly to the dsRNA transcriptome. The mitochondrial contribution was lower in the cancer cells compared to fibroblasts. The expression of different transposable element families was comparable, suggesting a general up-regulation of transposable element expression rather than stimulation of a specific sub-family. Sequencing of the input control revealed minor differences in dsRNA processing pathways with an upregulation of oligoadenylate synthase and RNP125 that negatively regulates the dsRNA sensors RIG1 and MDA5. Moreover, RT-qPCR, Western blotting, and immunocytochemistry confirmed the relatively minor adaptations to the hugely different dsRNA levels. As a consequence, these transformed cell lines are potentially less tolerant to interventions that increase the formation of endogenous dsRNA.