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1.
Parasit Vectors ; 17(1): 255, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38863029

RESUMO

BACKGROUND: RNA interference (RNAi) is a target-specific gene silencing method that can be used to determine gene functions and investigate host-pathogen interactions, as well as facilitating the development of ecofriendly pesticides. Commercially available transfection reagents (TRs) can improve the efficacy of RNAi. However, we currently lack a product and protocol for the transfection of insect cell lines with long double-stranded RNA (dsRNA). METHODS: We used agarose gel electrophoresis to determine the capacity of eight TRs to form complexes with long dsRNA. A CellTiter-Glo assay was then used to assess the cytotoxicity of the resulting lipoplexes. We also measured the cellular uptake of dsRNA by fluorescence microscopy using the fluorophore Cy3 as a label. Finally, we analyzed the TRs based on their transfection efficacy and compared the RNAi responses of Aedes albopictus C6/36 and U4.4 cells by knocking down an mCherry reporter Semliki Forest virus in both cell lines. RESULTS: The TRs from Biontex (K4, Metafectene Pro, and Metafectene SI+) showed the best complexing capacity and the lowest dsRNA:TR ratio needed for complete complex formation. Only HiPerFect was unable to complex the dsRNA completely, even at a ratio of 1:9. Most of the complexes containing mCherry-dsRNA were nontoxic at 2 ng/µL, but Lipofectamine 2000 was toxic at 1 ng/µL in U4.4 cells and at 2 ng/µL in C6/36 cells. The transfection of U4.4 cells with mCherry-dsRNA/TR complexes achieved significant knockdown of the virus reporter. Comparison of the RNAi response in C6/36 and U4.4 cells suggested that C6/36 cells lack the antiviral RNAi response because there was no significant knockdown of the virus reporter in any of the treatments. CONCLUSIONS: C6/36 cells have an impaired RNAi response as previously reported. This investigation provides valuable information for future RNAi experiments by showing how to mitigate the adverse effects attributed to TRs. This will facilitate the judicious selection of TRs and transfection conditions conducive to RNAi research in mosquitoes.


Assuntos
Aedes , Interferência de RNA , RNA de Cadeia Dupla , Transfecção , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Animais , Linhagem Celular , Aedes/genética , Inativação Gênica , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/efeitos dos fármacos
2.
Proc Natl Acad Sci U S A ; 121(25): e2322765121, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38865263

RESUMO

Antiviral RNA interference (RNAi) is conserved from yeasts to mammals. Dicer recognizes and cleaves virus-derived double-stranded RNA (dsRNA) and/or structured single-stranded RNA (ssRNA) into small-interfering RNAs, which guide effector Argonaute to homologous viral RNAs for digestion and inhibit virus replication. Thus, Argonaute is believed to be essential for antiviral RNAi. Here, we show Argonaute-independent, Dicer-dependent antiviral defense against dsRNA viruses using Cryphonectria parasitica (chestnut blight fungus), which is a model filamentous ascomycetous fungus and hosts a variety of viruses. The fungus has two dicer-like genes (dcl1 and dcl2) and four argonaute-like genes (agl1 to agl4). We prepared a suite of single to quadruple agl knockout mutants with or without dcl disruption. We tested these mutants for antiviral activities against diverse dsRNA viruses and ssRNA viruses. Although both DCL2 and AGL2 worked as antiviral players against some RNA viruses, DCL2 without argonaute was sufficient to block the replication of other RNA viruses. Overall, these results indicate the existence of a Dicer-alone defense and different degrees of susceptibility to it among RNA viruses. We discuss what determines the great difference in susceptibility to the Dicer-only defense.


Assuntos
Vírus de RNA , Ribonuclease III , Ribonuclease III/metabolismo , Ribonuclease III/genética , Vírus de RNA/imunologia , Vírus de RNA/genética , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Ascomicetos/virologia , Interferência de RNA , Replicação Viral/genética , RNA Viral/metabolismo , RNA Viral/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , RNA de Cadeia Dupla/metabolismo
3.
Int J Mol Sci ; 25(10)2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38791257

RESUMO

In this study, we report the complexities and challenges associated with achieving robust RNA interference (RNAi)-mediated gene knockdown in the mosquitoes Aedes aegypti and Aedes albopictus, a pivotal approach for genetic analysis and vector control. Despite RNAi's potential for species-specific gene targeting, our independent efforts to establish oral delivery of RNAi for identifying genes critical for mosquito development and fitness encountered significant challenges, failing to reproduce previously reported potent RNAi effects. We independently evaluated a range of RNAi-inducing molecules (siRNAs, shRNAs, and dsRNAs) and administration methods (oral delivery, immersion, and microinjection) in three different laboratories. We also tested various mosquito strains and utilized microorganisms for RNA delivery. Our results reveal a pronounced inconsistency in RNAi efficacy, characterized by minimal effects on larval survival and gene expression levels in most instances despite strong published effects for the tested targets. One or multiple factors, including RNase activity in the gut, the cellular internalization and processing of RNA molecules, and the systemic dissemination of the RNAi signal, could be involved in this variability, all of which are barely understood in mosquitoes. The challenges identified in this study highlight the necessity for additional research into the underlying mechanisms of mosquito RNAi to develop more robust RNAi-based methodologies. Our findings emphasize the intricacies of RNAi application in mosquitoes, which present a substantial barrier to its utilization in genetic control strategies.


Assuntos
Aedes , Interferência de RNA , Animais , Aedes/genética , RNA Interferente Pequeno/genética , Mosquitos Vetores/genética , Larva/genética , RNA de Cadeia Dupla/genética , Inativação Gênica , Técnicas de Silenciamento de Genes/métodos
4.
Arch Insect Biochem Physiol ; 116(1): e22118, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38713637

RESUMO

We detected enzymatic activity that generates 20-nucleotide (nt) RNA from double-stranded RNAs (dsRNAs) in crude extracts prepared from various silkworm (Bombyx mori) organs. The result using knocked-down cultured cells indicated that this dicing activity originated from B. mori Dicer-2 (BmDcr2). Biochemical analyses revealed that BmDcr2 preferentially cleaves 5'-phosphorylated dsRNAs at the 20-nt site-counted from the 5'-phosphorylated end-and required ATP and magnesium ions for the dicing reaction. This is the first report of the biochemical characterization of Dicer-2 in lepidopteran insects. This enzymatic property of BmDcr2 in vitro is consistent with the in vivo small interfering RNA profile in virus-infected silkworm cells.


Assuntos
Bombyx , RNA de Cadeia Dupla , Ribonuclease III , Animais , Bombyx/genética , Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Larva/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Magnésio/metabolismo , Ribonuclease III/metabolismo , Ribonuclease III/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo
5.
Mol Immunol ; 170: 156-169, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38692097

RESUMO

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.


Assuntos
Neoplasias da Mama , Proteínas de Ligação a DNA , Fatores de Transcrição , Humanos , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Feminino , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Recidiva Local de Neoplasia/imunologia , Interferons/metabolismo , Interferons/imunologia , Interferons/genética , Linhagem Celular Tumoral , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Animais , RNA de Cadeia Dupla/imunologia , Fator de Transcrição RelA/metabolismo , Camundongos , Regulação Neoplásica da Expressão Gênica , Transdução de Sinais/imunologia , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/imunologia
6.
Sci Signal ; 17(837): eadi9844, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38771918

RESUMO

Oligoadenylate synthetase 3 (OAS3) and ribonuclease L (RNase L) are components of a pathway that combats viral infection in mammals. Upon detection of viral double-stranded RNA (dsRNA), OAS3 synthesizes 2'-5'-oligo(A), which activates the RNase domain of RNase L by promoting the homodimerization and oligomerization of RNase L monomers. Activated RNase L rapidly degrades all cellular mRNAs, shutting off several cellular processes. We sought to understand the molecular mechanisms underlying the rapid activation of RNase L in response to viral infection. Through superresolution microscopy and live-cell imaging, we showed that OAS3 and RNase L concentrated into higher-order cytoplasmic complexes known as dsRNA-induced foci (dRIF) in response to dsRNA or infection with dengue virus, Zika virus, or West Nile virus. The concentration of OAS3 and RNase L at dRIF corresponded with the activation of RNase L-mediated RNA decay. We showed that dimerized/oligomerized RNase L concentrated in a liquid-like shell surrounding a core OAS3-dRIF structure and dynamically exchanged with the cytosol. These data establish that the condensation of dsRNA, OAS3, and RNase L into dRIF is a molecular switch that promotes the rapid activation of RNase L upon detection of dsRNA in mammalian cells.


Assuntos
2',5'-Oligoadenilato Sintetase , Endorribonucleases , RNA de Cadeia Dupla , Zika virus , Endorribonucleases/metabolismo , Endorribonucleases/genética , Endorribonucleases/química , Humanos , 2',5'-Oligoadenilato Sintetase/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/química , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , Zika virus/metabolismo , Animais , Vírus da Dengue/metabolismo , RNA Viral/metabolismo , RNA Viral/genética , Estabilidade de RNA , Vírus do Nilo Ocidental/metabolismo , Vírus do Nilo Ocidental/genética , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologia , Ativação Enzimática , Células HeLa , Células HEK293
7.
J Agric Food Chem ; 72(22): 12508-12515, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38788129

RESUMO

Nanotechnology-based RNA interference (RNAi) offers a promising approach to pest control. However, current methods for producing RNAi nanopesticides are mainly implemented in a batch-to-batch manner, lacking consistent quality control. Herein, we present a microfluidic-based nanoplatform for RNA nanopesticide preparation using lipid nanoparticles (LNPs) as nanocarriers, taking advantage of the enhanced mass transfer and continuous processing capabilities of microfluidic technology. The dsRNA@LNPs were rapidly formed within seconds, which showed uniform size distribution, improved leaf wettability, and excellent dispersion properties. The delivery efficiency of dsRNA@LNPs was evaluated by targeting the chitin synthetase B (CHSB) gene ofSpodoptera exigua. The dsRNA@LNPs can effectively resist nuclease-rich midgut fluid degradation. Importantly, dsCHSB@LNPs exhibited increased mortality rates, significant reduction of larvae growth, and enhanced gene suppression efficiency. Therefore, a continuous nanoplatform for RNAi nanopesticide preparation is demonstrated by utilizing microfluidic technology, representing a new route to produce RNAi nanopesticides with enhanced quality control and might accelerate their practical applications.


Assuntos
Larva , Interferência de RNA , RNA de Cadeia Dupla , Spodoptera , Animais , Spodoptera/genética , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Larva/crescimento & desenvolvimento , Larva/genética , Nanopartículas/química , Microfluídica/instrumentação , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/química , Controle de Insetos/métodos
8.
Biochem Biophys Res Commun ; 719: 150103, 2024 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-38761636

RESUMO

The RNA-binding protein PKR serves as a crucial antiviral innate immune factor that globally suppresses translation by sensing viral double-stranded RNA (dsRNA) and by phosphorylating the translation initiation factor eIF2α. Recent findings have unveiled that single-stranded RNAs (ssRNAs), including in vitro transcribed (IVT) mRNA, can also bind to and activate PKR. However, the precise mechanism underlying PKR activation by ssRNAs, remains incompletely understood. Here, we developed a NanoLuc Binary Technology (NanoBiT)-based in vitro PKR dimerization assay to assess the impact of ssRNAs on PKR dimerization. Our findings demonstrate that, akin to double-stranded polyinosinic:polycytidylic acid (polyIC), an encephalomyocarditis virus (EMCV) RNA, as well as NanoLuc luciferase (Nluc) mRNA, can induce PKR dimerization. Conversely, homopolymeric RNA lacking secondary structure fails to promote PKR dimerization, underscoring the significance of secondary structure in this process. Furthermore, adenovirus VA RNA 1, another ssRNA, impedes PKR dimerization by competing with Nluc mRNA. Additionally, we observed structured ssRNAs capable of forming G-quadruplexes induce PKR dimerization. Collectively, our results indicate that ssRNAs have the ability to either induce or inhibit PKR dimerization, thus representing potential targets for the development of antiviral and anti-inflammatory agents.


Assuntos
Vírus da Encefalomiocardite , Multimerização Proteica , RNA de Cadeia Dupla , RNA Viral , eIF-2 Quinase , eIF-2 Quinase/metabolismo , eIF-2 Quinase/química , Humanos , RNA Viral/metabolismo , RNA Viral/genética , RNA Viral/química , Vírus da Encefalomiocardite/genética , RNA de Cadeia Dupla/metabolismo , RNA de Cadeia Dupla/química , Poli I-C/farmacologia , Conformação de Ácido Nucleico
9.
Nat Commun ; 15(1): 4644, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38821943

RESUMO

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.


Assuntos
Retículo Endoplasmático , Organelas , RNA Viral , SARS-CoV-2 , Replicação Viral , SARS-CoV-2/fisiologia , SARS-CoV-2/ultraestrutura , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , RNA Viral/metabolismo , RNA Viral/genética , Replicação Viral/fisiologia , Humanos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Retículo Endoplasmático/ultraestrutura , Organelas/virologia , Organelas/metabolismo , Organelas/ultraestrutura , Chlorocebus aethiops , Células Vero , Animais , COVID-19/virologia , COVID-19/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , Microscopia de Fluorescência , Compartimentos de Replicação Viral/metabolismo , RNA de Cadeia Dupla/metabolismo
10.
Mol Cell ; 84(11): 2087-2103.e8, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38815579

RESUMO

RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo M , Íntrons , Elementos Nucleotídeos Longos e Dispersos , Splicing de RNA , RNA de Cadeia Dupla , Humanos , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , Interferons/metabolismo , Interferons/genética , Animais , Células HEK293 , Camundongos , Transcriptoma , Éxons , Sítios de Splice de RNA , Elementos Alu/genética
11.
J Agric Food Chem ; 72(20): 11381-11391, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38728113

RESUMO

RNA interference (RNAi)-based biopesticides offer an attractive avenue for pest control. Previous studies revealed high RNAi sensitivity in Holotrichia parallela larvae, showcasing its potential for grub control. In this study, we aimed to develop an environmentally friendly RNAi method for H. parallela larvae. The double-stranded RNA (dsRNA) of the V-ATPase-a gene (HpVAA) was loaded onto layered double hydroxide (LDH). The dsRNA/LDH nanocomplex exhibited increased environmental stability, and we investigated the absorption rate and permeability of dsRNA-nanoparticle complexes and explored the RNAi controlling effect. Silencing the HpVAA gene was found to darken the epidermis of H. parallela larvae, with growth cessation or death or mortality, disrupting the epidermis and midgut structure. Quantitative reverse transcription-polymerase chain reaction and confocal microscopy confirmed the effective absorption of the dsRNA/LDH nanocomplex by peanut plants, with distribution in roots, stems, and leaves. Nanomaterial-mediated RNAi silenced the target genes, leading to the death of pests. Therefore, these findings indicate the successful application of the nanomaterial-mediated RNAi system for underground pests, thus establishing a theoretical foundation for developing a green, safe, and efficient pest control strategy.


Assuntos
Larva , Interferência de RNA , RNA de Cadeia Dupla , Animais , Larva/crescimento & desenvolvimento , Larva/genética , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Hidróxidos/química , Hidróxidos/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , Arachis/genética , Arachis/química , Arachis/crescimento & desenvolvimento , Arachis/metabolismo , Controle Biológico de Vetores , Besouros/genética , Besouros/crescimento & desenvolvimento , Química Verde , Agentes de Controle Biológico/química , Agentes de Controle Biológico/metabolismo , Nanopartículas/química
12.
Biotechnol J ; 19(5): e2400024, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38797726

RESUMO

The development of RNA interference (RNAi) is crucial for studying plant gene function. Its use, is limited to a few plants with well-established transgenic techniques. Spray-induced gene silencing (SIGS) introduces exogenous double-stranded RNA (dsRNA) into plants by spraying, injection, or irrigation, triggering the RNAi pathway to instantly silence target genes. As is a transient RNAi technology that does not rely on transgenic methods, SIGS has significant potential for studying gene function in plants lacking advanced transgenic technology. In this study, to enhance their stability and delivery efficiency, siRNAs were used as structural motifs to construct RNA nanoparticles (NPs) of four shapes: triangle, square, pentagon, and hexagon. These NPs, when synthesized by Escherichia coli, showed that triangular and square shapes accumulated more efficiently than pentagon and hexagon shapes. Bioassays revealed that RNA squares had the highest RNAi efficiency, followed by RNA triangles, with GFP-dsRNA showing the lowest efficiency at 4 and 7 days post-spray. We further explored the use of RNA squares in inducing transient RNAi in plants that are difficult to transform genetically. The results indicated that Panax notoginseng-derived MYB2 (PnMYB2) and Camellia oleifera-derived GUT (CoGUT) were significantly suppressed in P. notoginseng and C. oleifera, respectively, following the application of PnMYB2- and CoGUT-specific RNA squares. These findings suggest that RNA squares are highly effective in SIGS and can be utilized for gene function research in plants.


Assuntos
Plantas Geneticamente Modificadas , Interferência de RNA , Plantas Geneticamente Modificadas/genética , RNA Interferente Pequeno/genética , Nanopartículas/química , RNA de Cadeia Dupla/genética , Escherichia coli/genética , Nicotiana/genética
13.
Nat Commun ; 15(1): 4127, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38750080

RESUMO

Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.


Assuntos
DNA Helicases , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Transdução de Sinais , Grânulos de Estresse , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Grânulos de Estresse/metabolismo , RNA Helicases/metabolismo , DNA Helicases/metabolismo , Proteína DEAD-box 58/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Imunidade Inata , RNA de Cadeia Dupla/metabolismo , Células HEK293 , Células HeLa , Grânulos Citoplasmáticos/metabolismo , Infecções por Vírus de RNA/virologia , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/imunologia , Receptores Imunológicos/metabolismo
14.
Methods Mol Biol ; 2808: 71-88, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38743363

RESUMO

Copy-back defective interfering RNAs are major contaminants of viral stock preparations of morbilliviruses and other negative strand RNA viruses. They are hybrid molecules of positive sense antigenome and negative sense genome. They possess perfectly complementary ends allowing the formation of extremely stable double-stranded RNA panhandle structures. The presence of the 3'-terminal promoter allows replication of these molecules by the viral polymerase. They thereby negatively interfere with replication of standard genomes. In addition, the double-stranded RNA stem structures are highly immunostimulatory and activate antiviral cell-intrinsic innate immune responses. Thus, copy-back defective interfering RNAs severely affect the virulence and pathogenesis of morbillivirus stocks. We describe two biochemical methods to analyze copy-back defective interfering RNAs in virus-infected samples, or purified viral RNA. First, we present our Northern blotting protocol that allows accurate size determination of defective interfering RNA molecules and estimation of the relative contamination level of virus preparations. Second, we describe a PCR approach to amplify defective interfering RNAs specifically, which allows detailed sequence analysis.


Assuntos
Morbillivirus , RNA Viral , RNA Viral/genética , Morbillivirus/genética , Animais , Northern Blotting , Replicação Viral/genética , Reação em Cadeia da Polimerase/métodos , RNA Interferente Pequeno/genética , Genoma Viral , RNA de Cadeia Dupla/genética , Humanos
15.
J Agric Food Chem ; 72(19): 10936-10943, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38691835

RESUMO

RNAi plays a crucial role in insect gene function research and pest control field. Nonetheless, the variable efficiency of RNAi across diverse insects and off-target effects also limited its further application. In this study, we cloned six essential housekeeping genes from Solenopsis invicta and conducted RNAi experiments by orally administering dsRNA. Then, we found that mixing with liposomes significantly enhanced the RNAi efficiency by targeting for SiV-ATPaseE. Additionally, we observed a certain lethal effect of this dsRNA on queens by our established RNAi system. Furthermore, no strict sequence-related off-target effects were detected. Finally, the RNAi effect of large-scale bacteria expressing dsRNA was successfully confirmed for controlling S. invicta. In summary, this study established an RNAi system for S. invicta and provided a research template for the future development of nucleic acid drugs based on RNAi.


Assuntos
Formigas , Proteínas de Insetos , Interferência de RNA , Animais , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Formigas/genética , Controle de Insetos/métodos , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Controle Biológico de Vetores/métodos , Feminino , Formigas Lava-Pés
16.
Planta ; 259(6): 153, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38744752

RESUMO

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.


Assuntos
Ascomicetos , Nicotiana , Doenças das Plantas , Interferência de RNA , Ascomicetos/fisiologia , Ascomicetos/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Nicotiana/genética , Nicotiana/microbiologia , Mostardeira/genética , Mostardeira/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , RNA de Cadeia Dupla/genética
17.
Elife ; 132024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38747717

RESUMO

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.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , RNA de Cadeia Dupla , Ribonuclease III , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/metabolismo , Ribonuclease III/química , Ribonuclease III/genética , Microscopia Crioeletrônica , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , RNA Helicases/metabolismo , RNA Helicases/genética , RNA Helicases/química , Ligação Proteica , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteína DEAD-box 58/metabolismo , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/química
18.
Methods Mol Biol ; 2775: 91-106, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38758313

RESUMO

RNA interference (RNAi) is a molecular biology technique for silencing specific eukaryotic genes without altering the DNA sequence in the genome. The silencing effect occurs because of decreased levels of mRNA that then result in decreased protein levels for the gene. The specificity of the silencing is dependent upon the presence of sequence-specific double-stranded RNA (dsRNA) that activates the cellular RNAi machinery. This chapter describes the process of silencing a specific target gene in Cryptococcus using a dual promoter vector. The plasmid, pIBB103, was designed with two convergent GAL7 promoters flanking a ura5 fragment that acts as a reporter for efficient RNAi. The target gene fragment is inserted between the promoters to be transcribed from both directions leading to the production of dsRNA in cells that activate the RNAi pathway.


Assuntos
Cryptococcus , Regiões Promotoras Genéticas , Interferência de RNA , Cryptococcus/genética , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Vetores Genéticos/genética , Plasmídeos/genética , Inativação Gênica
19.
J Immunother Cancer ; 12(5)2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38749537

RESUMO

BACKGROUND: Cancer-intrinsic type I interferon (IFN-I) production triggered by radiotherapy (RT) is mainly dependent on cytosolic double-stranded DNA (dsDNA)-mediated cGAS/STING signaling and increases cancer immunogenicity and enhances the antitumor immune response to increase therapeutic efficacy. However, cGAS/STING deficiency in colorectal cancer (CRC) may suppress the RT-induced antitumor immunity. Therefore, we aimed to evaluate the importance of the dsRNA-mediated antitumor immune response induced by RT in patients with CRC. METHODS: Cytosolic dsRNA level and its sensors were evaluated via cell-based assays (co-culture assay, confocal microscopy, pharmacological inhibition and immunofluorescent staining) and in vivo experiments. Biopsies and surgical tissues from patients with CRC who received preoperative chemoradiotherapy (neoCRT) were collected for multiplex cytokine assays, immunohistochemical analysis and SNP genotyping. We also generated a cancer-specific adenovirus-associated virus (AAV)-IFNß1 construct to evaluate its therapeutic efficacy in combination with RT, and the immune profiles were analyzed by flow cytometry and RNA-seq. RESULTS: Our studies revealed that RT stimulates the autonomous release of dsRNA from cancer cells to activate TLR3-mediated IFN-I signatures to facilitate antitumor immune responses. Patients harboring a dysfunctional TLR3 variant had reduced serum levels of IFN-I-related cytokines and intratumoral CD8+ immune cells and shorter disease-free survival following neoCRT treatment. The engineered cancer-targeted construct AAV-IFNß1 significantly improved the response to RT, leading to systematic eradication of distant tumors and prolonged survival in defective TLR3 preclinical models. CONCLUSION: Our results support that increasing cancer-intrinsic IFNß1 expression is an immunotherapeutic strategy that enhances the RT-induced antitumor immune response in locally patients with advanced CRC with dysfunctional TLR3.


Assuntos
Neoplasias Colorretais , Interferon Tipo I , Interferon beta , RNA de Cadeia Dupla , Humanos , Neoplasias Colorretais/radioterapia , Neoplasias Colorretais/imunologia , Interferon beta/metabolismo , Camundongos , Animais , Interferon Tipo I/metabolismo , Transdução de Sinais , Feminino , Masculino
20.
Curr Opin Genet Dev ; 86: 102195, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38643591

RESUMO

Adenosine-to-inosine RNA editing, catalyzed by the enzymes ADAR1 and ADAR2, stands as a pervasive RNA modification. A primary function of ADAR1-mediated RNA editing lies in labeling endogenous double-stranded RNAs (dsRNAs) as 'self', thereby averting their potential to activate innate immune responses. Recent findings have highlighted additional roles of ADAR1, independent of RNA editing, that are crucial for immune control. Here, we focus on recent progress in understanding ADAR1's RNA editing-dependent and -independent roles in immune control. We describe how ADAR1 regulates various dsRNA innate immune receptors through distinct mechanisms. Furthermore, we discuss the implications of ADAR1 and RNA editing in diseases, including autoimmune diseases and cancers.


Assuntos
Adenosina Desaminase , Imunidade Inata , Edição de RNA , Proteínas de Ligação a RNA , Humanos , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Imunidade Inata/genética , RNA de Cadeia Dupla/genética , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia
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