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1.
Mol Plant Pathol ; 25(3): e13441, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38462774

RESUMO

RNA interference, or RNA silencing, is an important defence mechanism against viroid infection in plants. Plants encode multiple DICER-LIKE (DCL) proteins that are key components of the RNA silencing pathway. However, the roles of different DCLs in defence responses against viroid infection remain unclear. Here, we determined the function of tomato DCL2b (SlDCL2b) in defence responses against potato spindle tuber viroid (PSTVd) infection using SlDCL2b loss-of-function tomato mutant plants. Compared with wild-type plants, mutant plants were more susceptible to PSTVd infection, developing more severe symptoms earlier and accumulating higher levels of PSTVd RNAs. Moreover, we verified the feedback mechanism for the regulation of SlDCL2b expression by miR6026. Functional blocking of tomato miR6026, by expressing its target mimics, can enhance resistance to PSTVd infection in tomato plants. These findings deepen the current understanding of RNAi-based resistance against viroid infection and provide a potentially new strategy for viroid control.


Assuntos
Solanum lycopersicum , Solanum tuberosum , Viroides , Viroides/genética , Solanum lycopersicum/genética , Solanum tuberosum/genética , Interferência de RNA , RNA Viral/metabolismo
2.
Cells ; 13(5)2024 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-38474355

RESUMO

While glucose is the primary fuel for fetal growth, the placenta utilizes the majority of glucose taken up from the maternal circulation. Of the facilitative glucose transporters in the placenta, SLC2A8 (GLUT8) is thought to primarily function as an intracellular glucose transporter; however, its function in trophoblast cells has not been determined. To gain insight into the function of SLC2A8 in the placenta, lentiviral-mediated RNA interference (RNAi) was performed in the human first-trimester trophoblast cell line ACH-3P. Non-targeting sequence controls (NTS RNAi; n = 4) and SLC2A8 RNAi (n = 4) infected ACH-3P cells were compared. A 79% reduction in SLC2A8 mRNA concentration was associated with an 11% reduction (p ≤ 0.05) in ACH-3P glucose uptake. NTS RNAi and SLC2A8 RNAi ACH-3P mRNA were subjected to RNAseq, identifying 1525 transcripts that were differentially expressed (|log2FC| > 1 and adjusted p-value < 0.05), with 273 transcripts derived from protein-coding genes, and the change in 10 of these mRNAs was validated by real-time qPCR. Additionally, there were 147 differentially expressed long non-coding RNAs. Functional analyses revealed differentially expressed genes involved in various metabolic pathways associated with cellular respiration, oxidative phosphorylation, and ATP synthesis. Collectively, these data indicate that SLC2A8 deficiency may impact placental uptake of glucose, but that its likely primary function in trophoblast cells is to support cellular respiration. Since the placenta oxidizes the majority of the glucose it takes up to support its own metabolic needs, impairment of SLC2A8 function could set the stage for functional placental insufficiency.


Assuntos
Placenta , Transcriptoma , Humanos , Gravidez , Feminino , Placenta/metabolismo , Interferência de RNA , Trofoblastos/metabolismo , Glucose/metabolismo , RNA Mensageiro/metabolismo
3.
Pestic Biochem Physiol ; 199: 105773, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38458680

RESUMO

Chemical signals play a central role in mediating insect feeding and reproductive behavior, and serve as the primary drivers of the insect-plant interactions. The detection of chemical signals, particularly host plant volatiles, relies heavily on the insect's complex olfactory system. The Bemisia tabaci cryptic species complex is a group of globally important whitefly pests of agricultural and ornamental crops that have a wide range of host plants, but the molecular mechanism of their host plant recognition is not yet clear. In this study, the odorant coreceptor gene of the Whitefly MEAM1 cryptic species (BtOrco) was cloned. The coding sequence of BtOrco was 1413 bp in length, with seven transmembrane structural domains, and it was expressed primarily in the heads of both male and female adult whiteflies, rather than in other tissues. Knockdown of BtOrco using transgenic plant-mediated RNAi technology significantly inhibited the foraging behavior of whiteflies. This inhibition was manifested as a reduced percentage of whiteflies responding to the host plant and a prolonged foraging period. Moreover, there was a substantial suppression of egg-laying activity among adult female whiteflies. These results indicate that BtOrco has the potential to be used as a target for the design of novel active compounds for the development of environmentally friendly whitefly control strategies.


Assuntos
Hemípteros , Animais , Feminino , Hemípteros/genética , Oviposição , Plantas Geneticamente Modificadas , Interferência de RNA
4.
Prog Mol Biol Transl Sci ; 204: 1-43, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38458734

RESUMO

Atherosclerosis represents a pathological state that affects the arterial system of the organism. This chronic, progressive condition is typified by the accumulation of atheroma within arterial walls. Modulation of RNA molecules through RNA-based therapies has expanded the range of therapeutic options available for neurodegenerative diseases, infectious diseases, cancer, and, more recently, cardiovascular disease (CVD). Presently, microRNAs and small interfering RNAs (siRNAs) are the most widely employed therapeutic strategies for targeting RNA molecules, and for regulating gene expression and protein production. Nevertheless, for these agents to be developed into effective medications, various obstacles must be overcome, including inadequate binding affinity, instability, challenges of delivering to the tissues, immunogenicity, and off-target toxicity. In this comprehensive review, we discuss in detail the current state of RNA interference (RNAi)-based therapies.


Assuntos
Aterosclerose , MicroRNAs , Neoplasias , Humanos , Interferência de RNA , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Interferente Pequeno/uso terapêutico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Neoplasias/terapia , Aterosclerose/terapia , Aterosclerose/tratamento farmacológico
5.
Prog Mol Biol Transl Sci ; 204: 249-294, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38458740

RESUMO

Over the past decades, drug discovery utilizing small pharmacological compounds, fragment-based therapeutics, and antibody therapy have significantly advanced treatment options for many human diseases. However, a major bottleneck has been that>70% of human proteins/genomic regions are 'undruggable' by the above-mentioned approaches. Many of these proteins constitute essential drug targets against complex multifactorial diseases like cancer, immunological disorders, and neurological diseases. Therefore, alternative approaches are required to target these proteins or genomic regions in human cells. RNA therapeutics is a promising approach for many of the traditionally 'undruggable' targets by utilizing methods such as antisense oligonucleotides, RNA interference, CRISPR/Cas-based genome editing, aptamers, and the development of mRNA therapeutics. In the following chapter, we will put emphasis on recent advancements utilizing these approaches against challenging drug targets, such as intranuclear proteins, intrinsically disordered proteins, untranslated genomic regions, and targets expressed in inaccessible tissues.


Assuntos
Edição de Genes , RNA , Humanos , Interferência de RNA , RNA Mensageiro , Proteínas
6.
Prog Mol Biol Transl Sci ; 204: 45-67, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38458743

RESUMO

Unhealthy lifestyles have given rise to a growing epidemic of metabolic liver diseases, including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). NAFLD often occurs as a consequence of obesity, and currently, there is no FDA-approved drug for its treatment. However, therapeutic oligonucleotides, such as RNA interference (RNAi), represent a promising class of pharmacotherapy that can target previously untreatable conditions. The potential significance of RNAi in maintaining physiological homeostasis, understanding pathogenesis, and improving metabolic liver diseases, including NAFLD, is discussed in this article. We explore why NAFLD/NASH is an ideal target for therapeutic oligonucleotides and provide insights into the delivery platforms of RNAi and its therapeutic role in addressing NAFLD/NASH.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/terapia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Interferência de RNA , Cirrose Hepática , Oligonucleotídeos/uso terapêutico
7.
BMC Genom Data ; 25(1): 31, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38491426

RESUMO

BACKGROUND: Dates contain various minerals that are essential for good health. The major RNA interference (RNAi) gene families play a vital role in plant growth and development by controlling the expression of protein-coding genes against different biotic and abiotic stresses. However, these gene families for date palm are not yet studied. Therefore, this study has explored major RNAi genes and their characteristics in date palm. RESULTS: We have identified 4 PdDCLs, 7 PdAGOs, and 3 PdRDRs as RNAi proteins from the date palm genome by using AtRNAi genes as query sequences in BLASTp search. Domain analysis of predicted RNAi genes has revealed the Helicase_C, Dicer_dimer, PAZ, RNase III, and Piwi domains that are associated with the gene silencing mechanisms. Most PdRNAi proteins have been found in the nucleus and cytosol associated with the gene silencing actions. The gene ontology (GO) enrichment analysis has revealed some important GO terms including RNA interference, dsRNA fragmentation, and ribonuclease_III activity that are related to the protein-coding gene silencing mechanisms. Gene regulatory network (GRN) analysis has identified PAZ and SNF2 as the transcriptional regulators of PdRNAi genes. Top-ranked 10 microRNAs including Pda-miR156b, Pda-miR396a, Pda-miR166a, Pda-miR167d, and Pda-miR529a have been identified as the key post-transcriptional regulators of PdRNAi genes that are associated with different biotic/abiotic stresses. The cis-acting regulatory element analysis of PdRNAi genes has detected some vital cis-acting elements including ABRE, MBS, MYB, MYC, Box-4, G-box, I-box, and STRE that are linked with different abiotic stresses. CONCLUSION: The results of this study might be valuable resources for the improvement of different characteristics in date palm by further studies in wet-lab.


Assuntos
MicroRNAs , Phoeniceae , Phoeniceae/genética , Interferência de RNA , Genoma , Sequências Reguladoras de Ácido Nucleico
8.
Proc Natl Acad Sci U S A ; 121(11): e2307802121, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38437557

RESUMO

RNA interference (RNAi) therapeutics are an emerging class of medicines that selectively target mRNA transcripts to silence protein production and combat disease. Despite the recent progress, a generalizable approach for monitoring the efficacy of RNAi therapeutics without invasive biopsy remains a challenge. Here, we describe the development of a self-reporting, theranostic nanoparticle that delivers siRNA to silence a protein that drives cancer progression while also monitoring the functional activity of its downstream targets. Our therapeutic target is the transcription factor SMARCE1, which was previously identified as a key driver of invasion in early-stage breast cancer. Using a doxycycline-inducible shRNA knockdown in OVCAR8 ovarian cancer cells both in vitro and in vivo, we demonstrate that SMARCE1 is a master regulator of genes encoding proinvasive proteases in a model of human ovarian cancer. We additionally map the peptide cleavage profiles of SMARCE1-regulated proteases so as to design a readout for downstream enzymatic activity. To demonstrate the therapeutic and diagnostic potential of our approach, we engineered self-assembled layer-by-layer nanoparticles that can encapsulate nucleic acid cargo and be decorated with peptide substrates that release a urinary reporter upon exposure to SMARCE1-related proteases. In an orthotopic ovarian cancer xenograft model, theranostic nanoparticles were able to knockdown SMARCE1 which was in turn reported through a reduction in protease-activated urinary reporters. These LBL nanoparticles both silence gene products by delivering siRNA and noninvasively report on downstream target activity by delivering synthetic biomarkers to sites of disease, enabling dose-finding studies as well as longitudinal assessments of efficacy.


Assuntos
Neoplasias Ovarianas , Peptídeos , Humanos , Feminino , Interferência de RNA , Peptídeos/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Peptídeo Hidrolases , RNA Interferente Pequeno/genética , Endopeptidases , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA
9.
J Am Heart Assoc ; 13(6): e032031, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38456415

RESUMO

Lowering low-density lipoprotein cholesterol (LDL-C) is a cornerstone of reducing risk for atherosclerotic cardiovascular disease. Despite the approval of nonstatin therapies for LDL-C lowering over the past 2 decades, these medications are underused, and most patients are still not at guideline-recommended LDL-C goals. Barriers include poor adherence, clinical inertia, concern for side effects, cost, and complex prior authorization processes. With atherosclerotic cardiovascular disease-related mortality increasing globally, there remains a need for additional therapeutic options for lowering LDL-C as part of an atherosclerotic cardiovascular disease prevention strategy. Following the identification of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a promising therapeutic target, inclisiran was developed using the natural process of RNA interference for robust, sustained prevention of hepatic PCSK9 synthesis. Twice-yearly maintenance subcutaneous inclisiran (following initial loading doses at Day 1 and Day 90) reduces circulating LDL-C levels by ≈50% versus placebo when added to maximally tolerated statins. Long-term safety and tolerability of inclisiran have been assessed, with studies underway to evaluate the effects of inclisiran on cardiovascular outcomes and to provide additional safety and effectiveness data. In 2021, <20 years after the discovery of PCSK9, inclisiran became the first RNA interference therapeutic approved in the United States for LDL-C lowering in patients with established atherosclerotic cardiovascular disease or familial hypercholesterolemia and has since been approved for use in patients with primary hyperlipidemia. This article reviews the journey of inclisiran from bench to bedside, including early development, the clinical trial program, key characteristics of inclisiran, and practical points for its use in the clinic.


Assuntos
Anticolesterolemiantes , Doenças Cardiovasculares , Humanos , LDL-Colesterol , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Interferência de RNA , Inibidores de PCSK9 , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/prevenção & controle , Colesterol , RNA Interferente Pequeno/efeitos adversos , Anticolesterolemiantes/efeitos adversos
10.
Bioconjug Chem ; 35(3): 381-388, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38446033

RESUMO

Long noncoding RNA (lncRNA) differentiation antagonizing noncoding RNA (DANCR) is overexpressed in human triple-negative breast cancer (TNBC) and promotes cell migration and proliferation. TNBC is limited in treatment options relative to hormone-receptor-positive breast cancer and is commonly treated with chemotherapy, which is often compromised by acquired resistance. DANCR has been implicated in the development of chemoresistance across multiple cancer types. Here, we applied magnetic resonance molecular imaging (MRMI) with a targeted contrast agent, MT218, specific to extradomain-B fibronectin (EDB-FN), a marker for epithelial-to-mesenchymal transition, to assess the therapeutic efficacy of the combination of paclitaxel and ZD2-PEG-ECO/siDANCR nanoparticles (ZD2-siDANCR-ELNP) to treat TNBC. The treatment of orthotopic MDA-MB-231 TNBC in mice with paclitaxel significantly suppressed tumor growth but with a significant increase of EDB-FN in the tumor, as revealed by MRMI and immunohistochemistry. Combining ZD2-siDANCR-ELNP with paclitaxel further reduced tumor sizes, along with reduced EDB-FN expression. Interestingly, MT218-MRMI revealed a lower reduction of tumor signal enhancement with the combination treatment than that with the siDANCR treatment alone, which was supported by higher cell density in the tumors treated with the combination therapy, as shown by histochemical analysis. MT218-MRMI clearly revealed the changes of the tumor microenvironment in response to various therapies and is effective to noninvasively assess the response of TNBC tumors to the therapies. Regulating oncogenic lncRNA DANCR is an effective strategy for improving the outcomes of chemotherapy in TNBC.


Assuntos
RNA Longo não Codificante , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , RNA Longo não Codificante/genética , Interferência de RNA , Linhagem Celular Tumoral , Paclitaxel/uso terapêutico , Espectroscopia de Ressonância Magnética , Imagem Molecular/métodos , Proliferação de Células , Microambiente Tumoral
11.
PLoS One ; 19(3): e0298766, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38498505

RESUMO

PVD neuron of C. elegans has become an attractive model for the study of dendrite development and regeneration due to its elaborate and stereotype dendrite morphology. RNA interference (RNAi) by feeding E. coli expressing dsRNA has been the basis of several genome wide screens performed using C. elegans. However, the feeding method often fails when it comes to knocking down genes in nervous system. In order to optimize the RNAi conditions for PVD neuron, we fed the worm strains with E. coli HT115 bacteria expressing dsRNA against mec-3, hpo-30, and tiam-1, whose loss of function are known to show dendrite morphology defects in PVD neuron. We found that RNAi of these genes in the available sensitive backgrounds including the one expresses sid-1 under unc-119 promoter, although resulted in reduction of dendrite branching, the phenotypes were significantly modest compared to the respective loss of function mutants. In order to enhance RNAi in PVD neurons, we generated a strain that expressed sid-1 under the promoter mec-3, which exhibits strong expression in PVD. When Pmec-3::sid-1 is expressed in either nre-1(-)lin-15b(-) or lin-15b(-) backgrounds, the higher order branching phenotype after RNAi of mec-3, hpo-30, and tiam-1 was significantly enhanced as compared to the genetic background alone. Moreover, knockdown of genes playing role in dendrite regeneration in the nre-1(-)lin-15b(-), Pmec-3-sid-1[+] background resulted in significant reduction in dendrite regeneration following laser injury. The extent of dendrite regrowth due to the RNAi of aff-1 or ced-10 in our optimized strain was comparable to that of aff-1 and ced-10 mutants. Essentially, our strain expressing sid-1 in PVD neuron, provides an RNAi optimized platform for high throughput screening of genes involved in PVD development, maintenance and regeneration.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Interferência de RNA , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Escherichia coli/metabolismo , Neurônios/metabolismo
12.
Planta ; 259(4): 79, 2024 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-38431538

RESUMO

MAIN CONCLUSION: Tomato transgenics expressing dsRNA against FoFLPs act as biofungicides and result in enhanced disease resistance upon Fol infection, by downregulating the endogenous gene expression levels of FoFLPs within Fol. Fusarium oxysporum f. sp. lycopersici (Fol) hijacks plant immunity by colonizing within the host and further instigating secondary infection causing vascular wilt disease in tomato that leads to significant yield loss. Here, RNA interference (RNAi) technology was used to determine its potential in enduring resistance against Fusarium wilt in tomato. To gain resistance against Fol infection, host-induced gene silencing (HIGS) of Fol-specific genes encoding for fasciclin-like proteins (FoFLPs) was done by generating tomato transgenics harbouring FoFLP1, FoFLP4 and FoFLP5 RNAi constructs confirmed by southern hybridizations. These tomato transgenics were screened for stable siRNA production in T0 and T1 lines using northern hybridizations. This confirmed stable dsRNAhp expression in tomato transgenics and suggested durable trait heritability in the subsequent progenies. FoFLP-specific siRNAs producing T1 tomato progenies were further selected to ascertain its disease resistance ability using seedling infection assays. We observed a significant reduction in FoFLP1, FoFLP4 and FoFLP5 transcript levels in Fol, upon infecting their respective RNAi tomato transgenic lines. Moreover, tomato transgenic lines, expressing intended siRNA molecules in the T1 generation, exhibit delayed disease onset with improved resistance. Furthermore, reduced fungal colonization was observed in the roots of Fol-infected T1 tomato progenies, without altering the plant photosynthetic efficiency of transgenic plants. These results substantiate the cross-kingdom dsRNA or siRNA delivery from transgenic tomato to Fol, leading to enhanced resistance against Fusarium wilt disease. The results also demonstrated that HIGS is a successful approach in rendering resistance to Fol infection in tomato plants.


Assuntos
Fusarium , Solanum lycopersicum , Interferência de RNA , Solanum lycopersicum/genética , Fusarium/fisiologia , Resistência à Doença/genética , RNA Interferente Pequeno , Doenças das Plantas/genética , Doenças das Plantas/microbiologia
13.
Genomics ; 116(2): 110815, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38431033

RESUMO

Small interfering RNAs (siRNAs) exemplify the promise of genetic medicine in the discovery of novel therapeutic modalities. Their ability to selectively suppress gene expression makes them ideal candidates for the development of oligonucleotide pharmaceuticals. Recent advancements in machine learning (ML) have facilitated the design of unmodified siRNA and efficacy prediction. However, a model trained to predict the silencing activity of siRNAs with diverse chemical modification patterns is yet to be published despite the importance of such modifications in designing siRNAs with the potential to reach the level of clinical use. This study presents the first application of ML to efficiently classify chemically modified siRNAs on the basis of sequence and chemical modification patterns alone. Three algorithms were evaluated at three classification thresholds and compared according to sensitivity, specificity, consistency of feature weights with empirical knowledge, and performance using an external validation dataset. Finally, possible directions for future research were proposed.


Assuntos
Algoritmos , Oligonucleotídeos , RNA Interferente Pequeno/genética , Aprendizado de Máquina , Interferência de RNA
14.
Wiley Interdiscip Rev RNA ; 15(2): e1832, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38448799

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs that play a fundamental role in enabling miRNA-mediated target repression, a post-transcriptional gene regulatory mechanism preserved across metazoans. Loss of certain animal miRNA genes can lead to developmental abnormalities, disease, and various degrees of embryonic lethality. These short RNAs normally guide Argonaute (AGO) proteins to target RNAs, which are in turn translationally repressed and destabilized, silencing the target to fine-tune gene expression and maintain cellular homeostasis. Delineating miRNA-mediated target decay has been thoroughly examined in thousands of studies, yet despite these exhaustive studies, comparatively less is known about how and why miRNAs are directed for decay. Several key observations over the years have noted instances of rapid miRNA turnover, suggesting endogenous means for animals to induce miRNA degradation. Recently, it was revealed that certain targets, so-called target-directed miRNA degradation (TDMD) triggers, can "trigger" miRNA decay through inducing proteolysis of AGO and thereby the bound miRNA. This process is mediated in animals via the ZSWIM8 ubiquitin ligase complex, which is recruited to AGO during engagement with triggers. Since its discovery, several studies have identified that ZSWIM8 and TDMD are indispensable for proper animal development. Given the rapid expansion of this field of study, here, we summarize the key findings that have led to and followed the discovery of ZSWIM8-dependent TDMD. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA in Disease and Development > RNA in Development.


Assuntos
MicroRNAs , Riboswitch , Animais , MicroRNAs/genética , Interferência de RNA , Proteínas Argonautas/genética
15.
J Agric Food Chem ; 72(8): 3973-3983, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38361393

RESUMO

The lepidopteran-specific RNAi efficiency-related nuclease (REase) has been shown to contribute to double-strand RNA (dsRNA) degradation in several lepidopteran insects. However, little is known about its regulatory mechanism. In this study, we identified and characterized SfREase in Spodoptera frugiperda. The exposure of the third-instar larvae to dsEGFP and high temperature led to the upregulation of SfREase, whereas starvation treatment resulted in the downregulation of SfREase. Further experiments revealed that dsRNA degraded more slowly in the hemolymph or midgut fluid extracted from dsSfREase-injected or dsSfREase-ingested larvae compared with those from dsEGFP-treated larvae, and the recombinant SfREase degraded dsRNA in a concentration-dependent manner. Additionally, the knockdown of SfREase improved RNAi efficiency. Finally, both RNAi and dual-luciferase reporter assay in Sf9 cells revealed that SfREase is negatively regulated by FOXO. These data provide insights into the function and regulatory mechanism of REase and have applied implications for the development of an RNAi-based control strategy of S. frugiperda.


Assuntos
Insetos , RNA de Cadeia Dupla , Animais , Interferência de RNA , Spodoptera , Insetos/genética , Larva/genética , Larva/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo
16.
Nat Commun ; 15(1): 957, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38302462

RESUMO

RNA interference (RNAi) is a conserved gene silencing process that exists in diverse organisms to protect genome integrity and regulate gene expression. In C. elegans, the majority of RNAi pathway proteins localize to perinuclear, phase-separated germ granules, which are comprised of sub-domains referred to as P granules, Mutator foci, Z granules, and SIMR foci. However, the protein components and function of the newly discovered SIMR foci are unknown. Here we demonstrate that HRDE-2 localizes to SIMR foci and interacts with the germline nuclear Argonaute HRDE-1 in its small RNA unbound state. In the absence of HRDE-2, HRDE-1 exclusively loads CSR-class 22G-RNAs rather than WAGO-class 22G-RNAs, resulting in inappropriate H3K9me3 deposition on CSR-target genes. Thus, our study demonstrates that the recruitment of unloaded HRDE-1 to germ granules, mediated by HRDE-2, is critical to ensure that the correct small RNAs are used to guide nuclear RNA silencing in the C. elegans germline.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
17.
Sci Rep ; 14(1): 3545, 2024 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-38347054

RESUMO

RNA interference (RNAi) is an effective approach to suppress gene expression and monitor gene regulation. Despite its wide application, its use is limited in certain taxonomic groups, including cnidarians. Myxozoans are a unique group of cnidarian parasites that diverged from their free-living ancestors about 600 million years ago, with several species causing acute disease in farmed and wild fish populations. In this pioneering study we successfully applied RNAi in blood stages of the myxozoan Sphaerospora molnari, combining a dsRNA soaking approach, real-time PCR, confocal microscopy, and Western blotting. For proof of concept, we knocked down two unusual actins, one of which is known to play a critical role in S. molnari cell motility. We observed intracellular uptake of dsRNA after 30 min and accumulation in all cells of the typical myxozoan cell-in-cell structure. We successfully knocked down actin in S. molnari in vitro, with transient inhibition for 48 h. We observed the disruption of the cytoskeletal network within the primary cell and loss of the characteristic rotational cell motility. This RNAi workflow could significantly advance functional research within the Myxozoa, offering new prospects for investigating therapeutic targets and facilitating drug discovery against economically important fish parasites.


Assuntos
Cnidários , Doenças dos Peixes , Myxozoa , Parasitos , Animais , Cnidários/genética , Interferência de RNA , Myxozoa/genética , Movimento Celular , Peixes , Actinas/genética , Doenças dos Peixes/genética , Filogenia
18.
J Am Chem Soc ; 146(10): 6665-6674, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38412223

RESUMO

RNA-cleaving ribozymes are promising candidates as general tools of RNA interference (RNAi) in gene manipulation. However, compared with other RNA systems, such as siRNA and CRISPR technologies, the ribozyme tools are still far from broad applications on RNAi due to their poor performance in the cellular context. In this work, we report an efficient RNAi tool based on chemically modified hammerhead ribozyme (HHR). By the introduction of an intramolecular linkage into the minimal HHR to reconstruct the distal interaction within the tertiary ribozyme structure, this cross-linked HHR exhibits efficient RNA substrate cleavage activities with almost no sequence constraint. Cellular experiments suggest that both exogenous and endogenous RNA expression can be dramatically knocked down by this HHR tool with levels comparable to those of siRNA. Unlike the widely applied protein-recruiting RNA systems (siRNA and CRISPR), this ribozyme tool functions solely on RNA itself with great simplicity, which may provide a new approach for gene manipulation in both fundamental and translational studies.


Assuntos
RNA Catalítico , RNA Catalítico/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Processamento de Proteína Pós-Traducional , Conformação de Ácido Nucleico
19.
Acta Trop ; 252: 107149, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38360259

RESUMO

The enzyme NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochromes P450 activity. Gene expression analysis of cytochromes P450 and CPR in deltamethrin-resistant and susceptible populations revealed that P450s genes are involved in the development of insecticide resistance in Triatoma infestans. To clarify the role of cytochromes P450 in insecticide resistance, it was proposed to investigate the effect of CPR gene silencing by RNA interference (RNAi) in a pyrethroid resistant population of T. infestans. Silencing of the CPR gene showed a significant increase in susceptibility to deltamethrin in the population analysed. This result support the hypothesis that the metabolic process of detoxification mediated by cytochromes P450 contributes to the decreased deltamethrin susceptibility observed in the resistant strain of T. infestans.


Assuntos
Doença de Chagas , Inseticidas , Piretrinas , Triatoma , Animais , Inseticidas/farmacologia , Interferência de RNA , Piretrinas/farmacologia , Doença de Chagas/genética , Nitrilas/farmacologia , Resistência a Inseticidas/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia
20.
Mol Biol Rep ; 51(1): 355, 2024 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-38400844

RESUMO

Nanoparticle-based delivery systems have emerged as powerful tools in the field of pest management, offering precise and effective means of delivering double-stranded RNA (dsRNA), a potent agent for pest control through RNA interference (RNAi). This comprehensive review aims to evaluate and compare various types of nanoparticles for their suitability in dsRNA delivery for pest management applications. The review begins by examining the unique properties and advantages of different nanoparticle materials, including clay, chitosan, liposomes, carbon, gold and silica. Each material's ability to protect dsRNA from degradation and its potential for targeted delivery to pests are assessed. Furthermore, this review delves into the surface modification strategies employed to enhance dsRNA delivery efficiency. Functionalization with oligonucleotides, lipids, polymers, proteins and peptides is discussed in detail, highlighting their role in improving stability, cellular uptake, and specificity of dsRNA delivery.This review also provides valuable guidance on choosing the most suitable nanoparticle-based system for delivering dsRNA effectively and sustainably in pest management. Moreover, it identifies existing knowledge gaps and proposes potential research directions aimed at enhancing pest control strategies through the utilization of nanoparticles and dsRNA.


Assuntos
Nanopartículas , RNA de Cadeia Dupla , Animais , Insetos/genética , Interferência de RNA , Lipossomos/metabolismo , Controle de Pragas
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