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NIKOLOV, SVETOSLAV, and VALKO PETROV. "TIME DELAY MODEL OF RNA SILENCING." Journal of Mechanics in Medicine and Biology 07, no. 03 (2007): 297–314. http://dx.doi.org/10.1142/s0219519407002315.
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RNA silencing (also known as RNA interference) suppresses the expression of genes posttranscriptionally. We propose a time delay model of RNA silencing through a system consisting of double-stranded RNA (dsRNA), RNA-induced silencing complex (RISC), messenger RNA (mRNA), and RISC–mRNA complex. The time delay model is based on the consideration that the regeneration (or degradation) of the RISC–mRNA complex needs a finite time τ. The model equations are analyzed using nonlinear dynamics methods, in particular the Hopf bifurcation theorem, and they are solved numerically. From the accomplished analytical and numerical calculations, it becomes clear that time delay τ is a key factor in the behavior of the model. In this case, it has a destabilizing effect on the silencing process.
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Prakash, Prerana, and M. G. Hariprasad. "SIRNA: ITS TRANSLATIONFROM RESEARCH TO THERAPEUTIC APPLICATIONS." International Journal of Advanced Research 13, no. 05 (2025): 538–43. https://doi.org/10.21474/ijar01/20930.
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Small interfering RNA (siRNA) is a short, double-stranded RNA molecule that has emerged as a pivotal tool for gene silencing through the RNA interference (RNAi) pathway. It is a powerful post-transcriptional gene-silencing molecule central to RNA interference (RNAi) mechanisms1. Typically 21–25 nucleotides in length, it guides the RNA-induced silencing complex (RISC) to a target messenger RNA (mRNA), enabling sequence-specific degradation and effectively silencing gene expression2. This process is highly specific and forms the foundation for siRNA’s role in research and therapeutic interventions3. By guiding the RNA-induced silencing complex (RISC) to target mRNA, siRNA enables post-transcriptional gene regulation with unparalleled precision. This targeted action holds immense promise in treating genetic, infectious, and degenerative diseases4. Therapeutically, siRNA offers unique advantages over traditional treatments, including the ability to target undruggable proteins, rapid development timelines, and reduced systemic toxicity. These advantages have catalyzed interest in siRNA for precision medicine and rare genetic disorders5
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Pantaleo, Vitantonio, György Szittya, and József Burgyán. "Molecular Bases of Viral RNA Targeting by Viral Small Interfering RNA-Programmed RISC." Journal of Virology 81, no. 8 (2007): 3797–806. http://dx.doi.org/10.1128/jvi.02383-06.
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ABSTRACT RNA silencing is conserved in a broad range of eukaryotes and operates in the development and maintenance of genome integrity in many organisms. Plants have adapted this system for antiviral defense, and plant viruses have in turn developed mechanisms to suppress RNA silencing. RNA silencing-related RNA inactivation is likely based on target RNA cleavage or translational arrest. Although it is widely assumed that virus-induced gene silencing (VIGS) promotes the endonucleolytic cleavage of the viral RNA genome, this popular assumption has never been tested experimentally. Here we analyzed the viral RNA targeting by VIGS in tombusvirus-infected plants, and we show evidence that antiviral response of VIGS is based on viral RNA cleavage by RNA-induced silencing effector complex (RISC) programmed by virus-specific small interfering RNAs (siRNAs). In addition, we found that the RISC-mediated cleavages do not occur randomly on the viral genome. Indeed, sequence analysis of cloned cleavage products identified hot spots for target RNA cleavage, and the regions of specific RISC-mediated cleavages are asymmetrically distributed along the positive- and negative-sense viral RNA strands. In addition, we identified viral siRNAs containing high-molecular-mass protein complexes purified from the recovery leaves of the silencing suppressor mutant virus-infected plants. Strikingly, these large nucleoproteins cofractionated with microRNA-containing complexes, suggesting that these nucleoproteins are silencing related effector complexes.
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Nitin, B. Ghiware Pawan Wankhade Sangameshwar B. Kanthale Ajay D. Kshirsagar Haidarali M. Shaikh*. "REGULATION OF MICRO-RNA IN CANCER." INDO AMERICAN JOURNAL OF PHARMACEUTICAL SCIENCES 05, no. 01 (2018): 502–9. https://doi.org/10.5281/zenodo.1161661.
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Cancer is a dreadful disease of mankind, the treatment for cancer is not revealed as per expectation. The illuminating way come out with understanding and grab the molecular alteration in cell. Therefore, miRNAs is a novel notation for procurement of cancer. MicroRNAs are small, highly conserved non-coding RNA molecules involved in the regulation of gene expression. MicroRNAs are transcribed by RNA polymerases II and III which forms precursors that undergoes series of cleavage to form mature microRNA. There are two types of biogenesis pathways, one nuclear and one cytoplasmic. However there are some alternative biogenesis pathways exist that differ from conventional pathway in the number of cleavage events and enzymes responsible. The mechanism of sorting of microRNA precursors to the different pathways is unclear but it can be determined by the site of origin, its sequence and thermodynamic stability. The regulatory functions of microRNAs are able through the RNA-induced silencing complex (RISC). The regulation level of miRNAs in cell i.e. up regulation and down regulation, leads to cancer. In this review, highlighted the role of miRNAs in physiological way and explain the molecular mechanism involved in development of cancer. Key words:MicroRNA, Cancer, RNA-induced silencing complex (RISC), RNA polymerases II and III.
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Martínez-Turiño, Sandra, and Carmen Hernández. "Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors." Journal of General Virology 90, no. 2 (2009): 519–25. http://dx.doi.org/10.1099/vir.0.006098-0.
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Viral-derived double-stranded RNAs (dsRNAs) activate RNA silencing, generating small interfering RNAs (siRNAs) which are incorporated into an RNA-induced silencing complex (RISC) that promotes homology-dependent degradation of cognate RNAs. To counteract this, plant viruses express RNA silencing suppressors. Here, we show that the coat protein (CP) of Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, is able to efficiently inhibit RNA silencing. Interestingly, PFBV CP blocked both sense RNA- and dsRNA-triggered RNA silencing and did not preclude generation of siRNAs, which is in contrast with the abilities that have been reported for other carmoviral CPs. We have also found that PFBV CP can bind siRNAs and that this ability correlates with silencing suppression activity and enhancement of potato virus X pathogenicity. Collectively, the results indicate that PFBV CP inhibits RNA silencing by sequestering siRNAs and preventing their incorporation into a RISC, thus behaving similarly to unrelated viral suppressors but dissimilarly to orthologous ones.
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Deshpande, Sonal, and Neetu Singh. "Probing the nanoparticle–AGO2 interaction for enhanced gene knockdown." Soft Matter 14, no. 20 (2018): 4169–77. http://dx.doi.org/10.1039/c8sm00534f.
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RNA interference is a promising technology for treatment of various diseases. Here, we systematically probe the effect of steric hindrance of nanoparticles on the RNA induced silencing complex (RISC) interaction, by modulating two parameters, the nanoparticle size and hardness.
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Itaya, Asuka, Xuehua Zhong, Ralf Bundschuh, et al. "A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation." Journal of Virology 81, no. 6 (2007): 2980–94. http://dx.doi.org/10.1128/jvi.02339-06.
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ABSTRACT RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.
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Pashupathi, M. and Rashmi Mishra. "RNA Interference in Veterinary Parasitology." Vet Farm Frontier 02, no. 04 (2025): 155–56. https://doi.org/10.5281/zenodo.15401258.
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<strong>Introduction</strong> RNA interference (RNAi) is a naturally occurring mechanism of gene silencing that regulates gene expression post-transcriptionally. This process utilizes small double-stranded RNA molecules—particularly small interfering RNAs (siRNAs) and microRNAs (miRNAs)—to bind complementary sequences on target messenger RNAs (mRNAs), ultimately preventing their translation into proteins. Initially observed in plants and later in nematodes, the discovery of RNAi fundamentally changed our understanding of gene regulation and introduced a powerful tool for genetic research and therapy. <strong>Discovery and Mechanism of Action</strong> The landmark discovery of RNA interference was made in the nematode <em>Caenorhabditis elegans</em> by scientists Andrew Fire and Craig Mello in 1998. Their research demonstrated that the injection of double-stranded RNA (dsRNA) could lead to the silencing of genes with matching sequences. This discovery earned them the Nobel Prize in Physiology or Medicine in 2006. The process of RNAi begins with the introduction or endogenous production of dsRNA, which is cleaved by the RNase III enzyme Dicer into short fragments known as siRNAs. These siRNAs are then loaded into the RNA-induced silencing complex (RISC), guiding the complex to the target mRNA through sequence complementarity. Once bound, the RISC complex cleaves the mRNA, leading to its degradation and silencing of gene expression.
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Yoo, Byoung Kwon, Prasanna K. Santhekadur, Rachel Gredler, et al. "Increased RNA-induced silencing complex (RISC) activity contributes to hepatocellular carcinoma." Hepatology 53, no. 5 (2011): 1538–48. http://dx.doi.org/10.1002/hep.24216.
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Piao, Xianghua, Xue Zhang, Ligang Wu, and Joel G. Belasco. "CCR4-NOT Deadenylates mRNA Associated with RNA-Induced Silencing Complexes in Human Cells." Molecular and Cellular Biology 30, no. 6 (2010): 1486–94. http://dx.doi.org/10.1128/mcb.01481-09.
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ABSTRACT MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of “off-target” mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal.
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Grasso, Giuseppa, and Rosemary Kiernan. "The Polyvalent Role of NF90 in RNA Biology." International Journal of Molecular Sciences 23, no. 21 (2022): 13584. http://dx.doi.org/10.3390/ijms232113584.
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Double-stranded RNA-binding proteins (dsRBPs) are major players in the regulation of gene expression patterns. Among them, Nuclear Factor 90 (NF90) has a plethora of well-known functions in viral infection, transcription, and translation as well as RNA stability and degradation. In addition, NF90 has been identified as a regulator of microRNA (miRNA) maturation by competing with Microprocessor for the binding of pri-miRNAs in the nucleus. NF90 was recently shown to control the biogenesis of a subset of human miRNAs, which ultimately influences, not only the abundance, but also the expression of the host gene and the fate of the mRNA target repertoire. Moreover, recent evidence suggests that NF90 is also involved in RNA-Induced Silencing Complex (RISC)-mediated silencing by binding to target mRNAs and controlling their translation and degradation. Here, we review the many, and growing, functions of NF90 in RNA biology, with a focus on the miRNA pathway and RISC-mediated gene silencing.
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Iwakawa, Hiro-oki, and Yukihide Tomari. "Life of RISC: Formation, action, and degradation of RNA-induced silencing complex." Molecular Cell 82, no. 1 (2022): 30–43. http://dx.doi.org/10.1016/j.molcel.2021.11.026.
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Cambronne, X. A., R. Shen, P. L. Auer, and R. H. Goodman. "Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach." Proceedings of the National Academy of Sciences 109, no. 50 (2012): 20473–78. http://dx.doi.org/10.1073/pnas.1218887109.
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Vigh, Maria L., Simon Bressendorff, Axel Thieffry, Laura Arribas-Hernández, and Peter Brodersen. "Nuclear and cytoplasmic RNA exosomes and PELOTA1 prevent miRNA-induced secondary siRNA production in Arabidopsis." Nucleic Acids Research 50, no. 3 (2022): 1396–415. http://dx.doi.org/10.1093/nar/gkab1289.
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Abstract Amplification of short interfering RNA (siRNAs) via RNA-dependent RNA polymerases (RdRPs) is of fundamental importance in RNA silencing. Plant microRNA (miRNA) action generally does not involve engagement of RdRPs, in part thanks to a poorly understood activity of the cytoplasmic exosome adaptor SKI2. Here, we show that inactivation of the exosome subunit RRP45B and SKI2 results in similar patterns of miRNA-induced siRNA production. Furthermore, loss of the nuclear exosome adaptor HEN2 leads to secondary siRNA production from miRNA targets largely distinct from those producing siRNAs in ski2. Importantly, mutation of the Release Factor paralogue PELOTA1 required for subunit dissociation of stalled ribosomes causes siRNA production from miRNA targets overlapping with, but distinct from, those affected in ski2 and rrp45b mutants. We also show that in exosome mutants, miRNA targets can be sorted into producers and non-producers of illicit secondary siRNAs based on trigger miRNA levels and miRNA:target affinity rather than on presence of 5′-cleavage fragments. We propose that stalled RNA-Induced Silencing Complex (RISC) and ribosomes, but not mRNA cleavage fragments released from RISC, trigger siRNA production, and that the exosome limits siRNA amplification by reducing RISC dwell time on miRNA target mRNAs while PELOTA1 does so by reducing ribosome stalling.
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Csorba, Tibor, Aurelie Bovi, Tamás Dalmay, and József Burgyán. "The p122 Subunit of Tobacco Mosaic Virus Replicase Is a Potent Silencing Suppressor and Compromises both Small Interfering RNA- and MicroRNA-Mediated Pathways." Journal of Virology 81, no. 21 (2007): 11768–80. http://dx.doi.org/10.1128/jvi.01230-07.
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ABSTRACT One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3′ overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised.
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Suzuki, Junya, and Sadaki Yokota. "PRKRA Localizes to Nuage Structures and the Ectoplasmic Specialization and Tubulobulbar Complexes in Rat and Mouse Testis." Journal of Histology 2014 (February 25, 2014): 1–9. http://dx.doi.org/10.1155/2014/634290.
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The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including PRKRA, TRBP, and Dicer. RISC localizes to P-bodies. The nuage of the spermatogenic cells has function similar to the P-bodies. We study whether PRKRA localizes to nuage of spermatogenic cells of rat and mouse. PRKRA localized to four types of nuage structures, including aggregates of 60–90 nm particles, irregularly-shaped perinuclear granules, and intermitochondrial cement of pachytene spermatocytes, and chromatoid bodies of round spermatids. In addition, PRKRA is associated with dense material surrounding tubulobulbar complexes and with the ectoplasmic specialization. The results suggest that PRKRA functions in the nuage as an element of RNA silencing system and plays unknown role in the ectoplasmic specialization and at the tubulobulbar complexes of Sertoli cells attaching the head of late spermatids.
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Duan, Cheng-Guo, Chun-Han Wang, Rong-Xiang Fang, and Hui-Shan Guo. "Artificial MicroRNAs Highly Accessible to Targets Confer Efficient Virus Resistance in Plants." Journal of Virology 82, no. 22 (2008): 11084–95. http://dx.doi.org/10.1128/jvi.01377-08.
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ABSTRACT Short-hairpin RNAs based on microRNA (miRNA) precursors to express the artificial miRNAs (amiRNAs) can specifically induce gene silencing and confer virus resistance in plants. The efficacy of RNA silencing depends not only on the nature of amiRNAs but also on the local structures of the target mRNAs. However, the lack of tools to accurately and reliably predict secondary structures within long RNAs makes it very hard to predict the secondary structures of a viral genome RNA in the natural infection conditions in vivo. In this study, we used an experimental approach to dissect how the endogenous silencing machinery acts on the 3′ untranslated region (UTR) of the Cucumber mosaic virus (CMV) genome. Transiently expressed 3′UTR RNAs were degraded by site-specific cleavage. By comparing the natural cleavage hotspots within the 3′UTR of the CMV-infected wild-type Arabidopsis to those of the triple dcl2/3/4 mutant, we acquired true small RNA programmed RNA-induced silencing complex (siRISC)-mediated cleavage sites to design valid amiRNAs. We showed that the tRNA-like structure within the 3′UTR impeded target site access and restricted amiRNA-RISC-mediated cleavage of the target viral RNA. Moreover, target recognition in the less-structured area also influenced siRISC catalysis, thereby conferring different degrees of resistance to CMV infection. Transgenic plants expressing the designed amiRNAs that target the putative RISC accessible target sites conferred high resistance to the CMV challenge from both CMV subgroup strains. Our work suggests that the experimental approach is credible for studying the course of RISC target recognition to engineer effective gene silencing and virus resistance in plants by amiRNAs.
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Akbar, Sehrish, Yao Wei, and Mu-Qing Zhang. "RNA Interference: Promising Approach to Combat Plant Viruses." International Journal of Molecular Sciences 23, no. 10 (2022): 5312. http://dx.doi.org/10.3390/ijms23105312.
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Plant viruses are devastating plant pathogens that severely affect crop yield and quality. Plants have developed multiple lines of defense systems to combat viral infection. Gene silencing/RNA interference is the key defense system in plants that inhibits the virulence and multiplication of pathogens. The general mechanism of RNAi involves (i) the transcription and cleavage of dsRNA into small RNA molecules, such as microRNA (miRNA), or small interfering RNA (siRNA), (ii) the loading of siRNA/miRNA into an RNA Induced Silencing Complex (RISC), (iii) complementary base pairing between siRNA/miRNA with a targeted gene, and (iv) the cleavage or repression of a target gene with an Argonaute (AGO) protein. This natural RNAi pathway could introduce transgenes targeting various viral genes to induce gene silencing. Different RNAi pathways are reported for the artificial silencing of viral genes. These include Host-Induced Gene Silencing (HIGS), Virus-Induced Gene Silencing (VIGS), and Spray-Induced Gene Silencing (SIGS). There are significant limitations in HIGS and VIGS technology, such as lengthy and time-consuming processes, off-target effects, and public concerns regarding genetically modified (GM) transgenic plants. Here, we provide in-depth knowledge regarding SIGS, which efficiently provides RNAi resistance development against targeted genes without the need for GM transgenic plants. We give an overview of the defense system of plants against viral infection, including a detailed mechanism of RNAi, small RNA molecules and their types, and various kinds of RNAi pathways. This review will describe how RNA interference provides the antiviral defense, recent improvements, and their limitations.
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Sjögren, Lars, Maïna Floris, Andrea Barghetti, Franziska Völlmy, Rune Linding, and Peter Brodersen. "Farnesylated heat shock protein 40 is a component of membrane-bound RISC in Arabidopsis." Journal of Biological Chemistry 293, no. 43 (2018): 16608–22. http://dx.doi.org/10.1074/jbc.ra118.003887.
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ARGONAUTE1 (AGO1) binds directly to small regulatory RNA and is a key effector protein of post-transcriptional gene silencing mediated by microRNA (miRNA) and small interfering RNA (siRNA) in Arabidopsis. The formation of an RNA-induced silencing complex (RISC) of AGO1 and small RNA requires the function of the heat shock protein 70/90 chaperone system. Some functions of AGO1 occur in association with endomembranes, in particular the rough endoplasmic reticulum (RER), but proteins interacting with AGO1 in membrane fractions remain unidentified. In this study, we show that the farnesylated heat shock protein 40 homologs, J2 and J3, associate with AGO1 in membrane fractions in a manner that involves protein farnesylation. We also show that three changes in AGO1 function are detectable in mutants in protein farnesylation and J2/J3. First, perturbations of the HSP40/70/90 pathway by mutation of J3, HSP90, and farnesyl transferase affect the amounts of AGO1 associated with membranes. Second, miRNA association with membrane-bound polysomes is increased in farnesyl transferase and farnesylation-deficient J2/J3 mutants. Third, silencing by noncell autonomously acting short interfering RNAs is impaired. These observations highlight the involvement of farnesylated J2/J3 in small RNA-mediated gene regulation, and suggest that the importance of chaperone-AGO1 interaction is not limited to the RISC assembly process.
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Goodier, John L., Lili Zhang, Melissa R. Vetter, and Haig H. Kazazian. "LINE-1 ORF1 Protein Localizes in Stress Granules with Other RNA-Binding Proteins, Including Components of RNA Interference RNA-Induced Silencing Complex." Molecular and Cellular Biology 27, no. 18 (2007): 6469–83. http://dx.doi.org/10.1128/mcb.00332-07.
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ABSTRACT LINE-1 retrotransposons constitute one-fifth of human DNA and have helped shape our genome. A full-length L1 encodes a 40-kDa RNA-binding protein (ORF1p) and a 150-kDa protein (ORF2p) with endonuclease and reverse transcriptase activities. ORF1p is distinctive in forming large cytoplasmic foci, which we identified as cytoplasmic stress granules. A phylogenetically conserved central region of the protein is critical for wild-type localization and retrotransposition. Yeast two-hybrid screens revealed several RNA-binding proteins that coimmunoprecipitate with ORF1p and colocalize with ORF1p in foci. Two of these proteins, YB-1 and hnRNPA1, were previously reported in stress granules. We identified additional proteins associated with stress granules, including DNA-binding protein A, 9G8, and plasminogen activator inhibitor RNA-binding protein 1 (PAI-RBP1). PAI-RBP1 is a homolog of VIG, a part of the Drosophila melanogaster RNA-induced silencing complex (RISC). Other RISC components, including Ago2 and FMRP, also colocalize with PAI-RBP1 and ORF1p. We suggest that targeting ORF1p, and possibly the L1 RNP, to stress granules is a mechanism for controlling retrotransposition and its associated genetic and cellular damage.
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Liang, Chao, Xiaoliu Wang, Hualong He, Chi Xu, and Jie Cui. "Beyond Loading: Functions of Plant ARGONAUTE Proteins." International Journal of Molecular Sciences 24, no. 22 (2023): 16054. http://dx.doi.org/10.3390/ijms242216054.
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ARGONAUTE (AGO) proteins are key components of the RNA-induced silencing complex (RISC) that mediates gene silencing in eukaryotes. Small-RNA (sRNA) cargoes are selectively loaded into different members of the AGO protein family and then target complementary sequences to in-duce transcriptional repression, mRNA cleavage, or translation inhibition. Previous reviews have mainly focused on the traditional roles of AGOs in specific biological processes or on the molecular mechanisms of sRNA sorting. In this review, we summarize the biological significance of canonical sRNA loading, including the balance among distinct sRNA pathways, cross-regulation of different RISC activities during plant development and defense, and, especially, the emerging roles of AGOs in sRNA movement. We also discuss recent advances in novel non-canonical functions of plant AGOs. Perspectives for future functional studies of this evolutionarily conserved eukaryotic protein family will facilitate a more comprehensive understanding of the multi-faceted AGO proteins.
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Xu, Ning, Bo Segerman, Xiaofu Zhou, and Göran Akusjärvi. "Adenovirus Virus-Associated RNAII-Derived Small RNAs Are Efficiently Incorporated into the RNA-Induced Silencing Complex and Associate with Polyribosomes." Journal of Virology 81, no. 19 (2007): 10540–49. http://dx.doi.org/10.1128/jvi.00885-07.
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ABSTRACT Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3′ strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes.
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Evgenia, Bloch M., and Donald R. Branch. "Argonaute 2 Silencing of Band 3 in Early Reticulocytes." Blood 128, no. 22 (2016): 1273. http://dx.doi.org/10.1182/blood.v128.22.1273.1273.
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Abstract Post-transcriptional gene silencing by RNA-induced silencing complex (RISC) in erythropoiesis is a rapidly evolving field of study. Disruption of microRNA (miRNA) synthesis has been shown to hinder erythropoiesis and has been linked to disease. The presence of miRNA in circulating RBCs has recently been reported; however, clear understanding of their role and effectors has yet to be determined. In this study, freshly drawn RBC were density separated on discontinuous percoll gradients. The top two fractions, fraction 1 (F1) representative of the top layer containing the youngest, reticulocyte-like cells, and a second fraction with greater density and non-reticulocyte properties (F2), were isolated. Cell populations were analyzed to compare RBC protein levels using western blotting (WB). qRT-PCR was used to evaluate band 3 RNA. Argonaute (AGO2) RNA immunoprecipitation (RIP), was used to ascertain RISC mediated transcriptional repression of band 3 using qRT-PCR. Band 3 protein expression was elevated in the denser F2 compared to F1, in freshly drawn normal RBCs. RNA levels of band 3 complemented protein levels, as increased band 3 RNA was observed in F2. RIP experiments revealed elevated post-transcriptional control of band 3 through increased association of its RNA with AGO2 in F1 compared to F2 thereby, correlating with greatly reduced, sometimes undetectable protein levels of band 3 in F1. We confirm the presence of AGO2 in circulating RBCs. We report, for the first time, the post-transcriptional control of band 3, an integral RBC transmembrane protein, in circulating reticulocyte-like cells. These results suggest a possible subpopulation of reticulocytes, a pro-reticulocyte, with ongoing control of band 3 expression following enucleation, and a potential role of miRNA-RISC in band 3 pathology. Disclosures No relevant conflicts of interest to declare.
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Agrawal, Neema, P. V. N. Dasaradhi, Asif Mohmmed, Pawan Malhotra, Raj K. Bhatnagar, and Sunil K. Mukherjee. "RNA Interference: Biology, Mechanism, and Applications." Microbiology and Molecular Biology Reviews 67, no. 4 (2003): 657–85. http://dx.doi.org/10.1128/mmbr.67.4.657-685.2003.
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SUMMARY Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes.
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Nowak, Iwona, and Aishe A. Sarshad. "Argonaute Proteins Take Center Stage in Cancers." Cancers 13, no. 4 (2021): 788. http://dx.doi.org/10.3390/cancers13040788.
Full textAbstract:
Argonaute proteins (AGOs) play crucial roles in RNA-induced silencing complex (RISC) formation and activity. AGOs loaded with small RNA molecules (miRNA or siRNA) either catalyze endoribonucleolytic cleavage of target RNAs or recruit factors responsible for translational silencing and target destabilization. miRNAs are well characterized and broadly studied in tumorigenesis; nevertheless, the functions of the AGOs in cancers have lagged behind. Here, we discuss the current state of knowledge on the role of AGOs in tumorigenesis, highlighting canonical and non-canonical functions of AGOs in cancer cells, as well as the biomarker potential of AGO expression in different of tumor types. Furthermore, we point to the possible application of the AGOs in development of novel therapeutic approaches.
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Rusinowska, Barbara. "Using RNAi in the treatment of cardiovascular diseases - therapeutics based on siRNA overview." Journal of Education, Health and Sport 12, no. 7 (2022): 890–96. http://dx.doi.org/10.12775/jehs.2022.12.07.089.
Full textAbstract:
RNA interference (RNAi) discovered in the 1990s by Fire and Mello plays a role in silencing gene function. One type of RNAi is siRNA, which is a double-stranded molecule of 20-25 base pairs. This molecule is made by cleaving double-stranded RNA by the enzyme Dicer. siRNA binds to the protein complex with ribonuclease activity - RISC (RNA-induced silencing complex). The resulting complex binds to the mRNA and cuts it into parts, which blocks the formation of the protein encoded by the mRNA. This property has been exploited in the production of siRNA-based drugs. However, the instability of siRNA molecules turned out to be difficult - the challenge was to properly modify the structure of siRNA in order to increase the stability and half-life and select the appropriate method of delivering the molecule to the body. Many siRNA-based drugs have already been developed, but most are in clinical trials. In this review, we present the role of RNA interference in therapeutics production and use of siRNA in cardiovascular diseases treatment.
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Sullivan, Christopher S., and Don Ganem. "A Virus-Encoded Inhibitor That Blocks RNA Interference in Mammalian Cells." Journal of Virology 79, no. 12 (2005): 7371–79. http://dx.doi.org/10.1128/jvi.79.12.7371-7379.2005.
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ABSTRACT Nodamura virus (NoV) is a small RNA virus that is infectious for insect and mammalian hosts. We have developed a highly sensitive assay of RNA interference (RNAi) in mammalian cells that shows that the NoV B2 protein functions as an inhibitor of RNAi triggered by either short hairpin RNAs or small interfering RNAs. In the cell, NoV B2 binds to pre-Dicer substrate RNA and RNA-induced silencing complex (RISC)-processed RNAs and inhibits the Dicer cleavage reaction and, potentially, one or more post-Dicer activities. In vitro, NoV B2 inhibits Dicer-mediated RNA cleavage in the absence of any other host factors and specifically binds double-stranded RNAs corresponding in structure to Dicer substrates and products. Its abilities to bind to Dicer precursor and post-Dicer RISC-processed RNAs suggest a mechanism of inhibition that is unique among known viral inhibitors of RNAi.
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Andersson, M. Gunnar, P. C. Joost Haasnoot, Ning Xu, Saideh Berenjian, Ben Berkhout, and Göran Akusjärvi. "Suppression of RNA Interference by Adenovirus Virus-Associated RNA." Journal of Virology 79, no. 15 (2005): 9556–65. http://dx.doi.org/10.1128/jvi.79.15.9556-9565.2005.
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ABSTRACT We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3′ strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.
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Pare, Justin M., Paul LaPointe, and Tom C. Hobman. "Hsp90 cochaperones p23 and FKBP4 physically interact with hAgo2 and activate RNA interference–mediated silencing in mammalian cells." Molecular Biology of the Cell 24, no. 15 (2013): 2303–10. http://dx.doi.org/10.1091/mbc.e12-12-0892.
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Argonaute proteins and small RNAs together form the RNA-induced silencing complex (RISC), the central effector of RNA interference (RNAi). The molecular chaperone Hsp90 is required for the critical step of loading small RNAs onto Argonaute proteins. Here we show that the Hsp90 cochaperones Cdc37, Aha1, FKBP4, and p23 are required for efficient RNAi. Whereas FKBP4 and p23 form a stable complex with hAgo2, the function of Cdc37 in RNAi appears to be indirect and may indicate that two or more Hsp90 complexes are involved. Our data also suggest that p23 and FKBP4 interact with hAgo2 before small RNA loading and that RISC loading takes place in the cytoplasm rather than in association with RNA granules. Given the requirement for p23 and FKBP4 for efficient RNAi and that these cochaperones bind to hAgo2, we predict that loading of hAgo2 is analogous to Hsp90-mediated steroid hormone receptor activation. To this end, we outline a model in which FKBP4, p23, and Aha1 cooperatively regulate the progression of hAgo2 through the chaperone cycle. Finally, we propose that hAgo2 and RNAi can serve as a robust model system for continued investigation into the Hsp90 chaperone cycle.
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Barbato, Christian, Paola Frisone, Laura Braccini, et al. "Silencing of Ago-2 Interacting Protein SERBP1 Relieves KCC2 Repression by miR-92 in Neurons." Cells 11, no. 6 (2022): 1052. http://dx.doi.org/10.3390/cells11061052.
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RNA-binding proteins (RBPs) play important roles in modulating miRNA-mediated mRNA target repression. Argonaute2 (Ago2) is an essential component of the RNA-induced silencing complex (RISC) that plays a central role in silencing mechanisms via small non-coding RNA molecules known as siRNAs and miRNAs. Small RNAs loaded into Argonaute proteins catalyze endoribonucleolytic cleavage of target RNAs or recruit factors responsible for translational silencing and mRNA target destabilization. In previous studies we have shown that KCC2, a neuronal Cl (−) extruding K (+) Cl (−) co-transporter 2, is regulated by miR-92 in neuronal cells. Searching for Ago2 partners by immunoprecipitation and LC-MS/MS analysis, we isolated among other proteins the Serpine mRNA binding protein 1 (SERBP1) from SH-SY5Y neuroblastoma cells. Exploring the role of SERBP1 in miRNA-mediated gene silencing in SH-SY5Y cells and primary hippocampal neurons, we demonstrated that SERBP1 silencing regulates KCC2 expression through the 3′ untranslated region (UTR). In addition, we found that SERBP1 as well as Ago2/miR-92 complex bind to KCC2 3′UTR. Finally, we demonstrated the attenuation of miR-92-mediated repression of KCC2 3′UTR by SERBP1 silencing. These findings advance our knowledge regarding the miR-92-mediated modulation of KCC2 translation in neuronal cells and highlight SERBP1 as a key component of this gene regulation.
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Lou, Qiang, Yanzhong Hu, Yanfang Ma, and Zheng Dong. "RNA interference may suppress stress granule formation by preventing argonaute 2 recruitment." American Journal of Physiology-Cell Physiology 316, no. 1 (2019): C81—C91. http://dx.doi.org/10.1152/ajpcell.00251.2018.
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RNA-induced silencing complex (RISC) is formed during RNA interference (RNAi), whereas stress granules (SG) are assembled in response to cellular stress. Here, we demonstrate an interesting connection between RISC and SG that may involve argonaute 2 (Ago2), a core component of RISC. We analyzed SG induction by arsenite, the commonly used SG inducer. SG formation was suppressed in heat shock transcription factor 1 (Hsf1) or hypoxia-inducible factor-1α (Hif1α) shRNA-transfected cells but not in Hsf1 or Hif1α-knockout cells, suggesting that RNAi per se (rather than gene deficiency) may account for the suppressive effect on SG. In support, the suppressive effect of RNAi on SG formation was reversed by the RISC-loading inhibitor aurintricarboxylic acid. In non-RNAi cells, arsenite induced the accumulation of Ago2 in SGs as shown by its colocalization and coimmunoprecipitation with SG proteins, but Ago2 was not recruited to SG in the cells with RNAi. Consistently, arsenite induced the dissociation of Ago2 from RISC proteins in non-RNAi cells but not in RNAi cells. CRISPR-Cas9-medicated ablation of Ago2 attenuated SG formation during arsenite treatment, suggesting a critical role of Ago2 in SG assembly. Together, these results indicate that RISC and SG may compete for some key components, such as Ago2. In response to cellular stress, Ago2 is recruited for SG assembly; however, during RNAi, Ago2 is held in RISC, becoming unavailable for SG formation.
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Ji, Jingmin, Andrea Glaser, Marion Wernli, Jan Martin Berke, Darius Moradpour, and Peter Erb. "Suppression of short interfering RNA-mediated gene silencing by the structural proteins of hepatitis C virus." Journal of General Virology 89, no. 11 (2008): 2761–66. http://dx.doi.org/10.1099/vir.0.2008/002923-0.
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Viruses have evolved strategies to overcome the antiviral effects of the host at different levels. Besides specific defence mechanisms, the host responds to viral infection via the interferon pathway and also by RNA interference (RNAi). However, several viruses have been identified that suppress RNAi. We addressed the question of whether hepatitis C virus (HCV) suppresses RNAi, using cell lines constitutively expressing green fluorescent protein (GFP) and inducibly expressing HCV proteins. It was found that short interfering RNA-mediated GFP gene silencing was inhibited when the entire HCV polyprotein was expressed. Further studies showed that HCV structural proteins, and in particular envelope protein 2 (E2), were responsible for this inhibition. Co-precipitation assays demonstrated that E2 bound to Argonaute-2 (Ago-2), a member of the RNA-induced silencing complex, RISC. Thus, HCV E2 that interacts with Ago-2 is able to suppress RNAi.
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Pandolfini, Luca, Ettore Luzi, Dario Bressan, et al. "RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells." Genome Biology 17, no. 1 (2016): 94. https://doi.org/10.1186/s13059-016-0952-x.
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<strong>Background: </strong>Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. <strong>Results: </strong>We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. <strong>Conclusions: </strong>We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.
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Wilson, Briana, Zhangli Su, Pankaj Kumar, and Anindya Dutta. "XRN2 suppresses aberrant entry of tRNA trailers into argonaute in humans and Arabidopsis." PLOS Genetics 19, no. 5 (2023): e1010755. http://dx.doi.org/10.1371/journal.pgen.1010755.
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MicroRNAs (miRNAs) are a well-characterized class of small RNAs (sRNAs) that regulate gene expression post-transcriptionally. miRNAs function within a complex milieu of other sRNAs of similar size and abundance, with the best characterized being tRNA fragments or tRFs. The mechanism by which the RNA-induced silencing complex (RISC) selects for specific sRNAs over others is not entirely understood in human cells. Several highly expressed tRNA trailers (tRF-1s) are strikingly similar to microRNAs in length but are generally excluded from the microRNA effector pathway. This exclusion provides a paradigm for identifying mechanisms of RISC selectivity. Here, we show that 5’ to 3’ exoribonuclease XRN2 contributes to human RISC selectivity. Although highly abundant, tRF-1s are highly unstable and degraded by XRN2 which blocks tRF-1 accumulation in RISC. We also find that XRN mediated degradation of tRF-1s and subsequent exclusion from RISC is conserved in plants. Our findings reveal a conserved mechanism that prevents aberrant entry of a class of highly produced sRNAs into Ago2.
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Kaya, Çağlar, and Tolga Sarıyer. "Gene Silencing RNAi Technology: Uses in Plants." JOURNAL OF GLOBAL CLIMATE CHANGE 1, no. 1 (2022): 7–14. http://dx.doi.org/10.56768/jytp.1.1.02.
Full textAbstract:
Ensuring sustainable food production in national and global area depends on the determination of plant species and varieties that can survive under the influence of various stress factors that may occur due to global climate changes and other factors that adversely limit growth and development, and depends on the protection and development of existing ones. It is important to develop new plant varieties that are resistant to abiotic stress factors that have occurred as a result of global climate changes. At this point, modern biotechnological methods have been widely needed in plant breeding in recent years. One of these techniques is RNAi technology. The mechanism of RNA interference (RNAi) is defined as post-transcriptional gene silencing or regulation of gene expression, resulting in the degradation of mRNA chain, which is the complement of double-stranded RNA (dsRNA) entering the cell. RNA interference begins when double-stranded RNA is cut into small inhibitory RNAs (siRNA) by an RNase III enzyme called as Dicer. These siRNAs then bind to the RNA-inducing silencing complex (RISC) which is a multiprotein-RNA nuclease complex. RISC uses siRNAs to find complementary mRNA and cuts the target mRNA endonucleolytically. The resulting decrease in specific mRNA leads to a decrease in available protein(s). Post transcriptional gene silencing, RNA interference and other forms of RNA silencing have been observed particularly in plants. In recent years, RNAi studies, which are among the leading topics in the global area, have shown that non-coding RNAs in plants play a role in the control of tissue differentiation and development, signal transmission, interaction with phytohormones, abiotic (drought, salinity, etc.) and environmental factors such as biotic stress. In this review paper, the basics of RNAi mechanism and the usage of RNAi in plants are explained.
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Peng, Shaohong, Pei Guo, Xiao Lin, et al. "CAG RNAs induce DNA damage and apoptosis by silencing NUDT16 expression in polyglutamine degeneration." Proceedings of the National Academy of Sciences 118, no. 19 (2021): e2022940118. http://dx.doi.org/10.1073/pnas.2022940118.
Full textAbstract:
DNA damage plays a central role in the cellular pathogenesis of polyglutamine (polyQ) diseases, including Huntington’s disease (HD). In this study, we showed that the expression of untranslatable expanded CAG RNA per se induced the cellular DNA damage response pathway. By means of RNA sequencing (RNA-seq), we found that expression of the Nudix hydrolase 16 (NUDT16) gene was down-regulated in mutant CAG RNA-expressing cells. The loss of NUDT16 function results in a misincorporation of damaging nucleotides into DNAs and leads to DNA damage. We showed that small CAG (sCAG) RNAs, species generated from expanded CAG transcripts, hybridize with CUG-containing NUDT16 mRNA and form a CAG-CUG RNA heteroduplex, resulting in gene silencing of NUDT16 and leading to the DNA damage and cellular apoptosis. These results were further validated using expanded CAG RNA-expressing mouse primary neurons and in vivo R6/2 HD transgenic mice. Moreover, we identified a bisamidinium compound, DB213, that interacts specifically with the major groove of the CAG RNA homoduplex and disfavors the CAG-CUG heteroduplex formation. This action subsequently mitigated RNA-induced silencing complex (RISC)-dependent NUDT16 silencing in both in vitro cell and in vivo mouse disease models. After DB213 treatment, DNA damage, apoptosis, and locomotor defects were rescued in HD mice. This work establishes NUDT16 deficiency by CAG repeat RNAs as a pathogenic mechanism of polyQ diseases and as a potential therapeutic direction for HD and other polyQ diseases.
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Kourtidis, Antonis, Brian Necela, Wan-Hsin Lin, et al. "Cadherin complexes recruit mRNAs and RISC to regulate epithelial cell signaling." Journal of Cell Biology 216, no. 10 (2017): 3073–85. http://dx.doi.org/10.1083/jcb.201612125.
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Cumulative evidence demonstrates that most RNAs exhibit specific subcellular distribution. However, the mechanisms regulating this phenomenon and its functional consequences are still under investigation. Here, we reveal that cadherin complexes at the apical zonula adherens (ZA) of epithelial adherens junctions recruit the core components of the RNA-induced silencing complex (RISC) Ago2, GW182, and PABPC1, as well as a set of 522 messenger RNAs (mRNAs) and 28 mature microRNAs (miRNAs or miRs), via PLEKHA7. Top canonical pathways represented by these mRNAs include Wnt/β-catenin, TGF-β, and stem cell signaling. We specifically demonstrate the presence and silencing of MYC, JUN, and SOX2 mRNAs by miR-24 and miR-200c at the ZA. PLEKHA7 knockdown dissociates RISC from the ZA, decreases loading of the ZA-associated mRNAs and miRNAs to Ago2, and results in a corresponding increase of MYC, JUN, and SOX2 protein expression. The present work reveals a mechanism that directly links junction integrity to the silencing of a set of mRNAs that critically affect epithelial homeostasis.
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Thibaut, Olivier, and Bragard Claude. "Innate Immunity Activation and RNAi Interplay in Citrus Exocortis Viroid—Tomato Pathosystem." Viruses 10, no. 11 (2018): 587. http://dx.doi.org/10.3390/v10110587.
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Although viroids are the smallest and simplest plant pathogens known, the molecular mechanisms underlying their pathogenesis remain unclear. To unravel these mechanisms, a dual approach was implemented consisting of in silico identification of potential tomato silencing targets of pospiviroids, and the experimental validation of these targets through the sequencing of small RNAs and RNA ends extracted from tomatoes infected with a severe isolate of Citrus exocortis viroid (CEVd). The generated RNA ends were also used to monitor the differentially-expressed genes. These analyses showed that when CEVd symptoms are well established: (i) CEVd are degraded by at least three Dicer-like (DCL) proteins and possibly by RNA-induced silencing complex (RISC), (ii) five different mRNAs are partially degraded through post-transcriptional gene silencing (PTGS), including argonaute 2a, which is further degraded in phasiRNAs, (iii) Dicer-like 2b and 2d are both upregulated and degraded in phasiRNAs, and (iv) CEVd infection induced a significant shift in gene expression allowing to explain the usual symptoms of pospiviroids on tomato and to demonstrate the constant activation of host innate immunity and systemic acquired resistance (SAR) by these pathogenic RNAs. Finally, based on in silico analysis, potential immunity receptor candidates of viroid-derived RNAs are suggested.
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Wang, Haoyou, Qiming Shen, Xin Zhang, et al. "The Long Non-Coding RNA XIST Controls Non-Small Cell Lung Cancer Proliferation and Invasion by Modulating miR-186-5p." Cellular Physiology and Biochemistry 41, no. 6 (2017): 2221–29. http://dx.doi.org/10.1159/000475637.
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Background/Aims: Long non-coding RNAs (lncRNAs) are key players in the development and progression of human cancers. The lncRNA XIST (X-inactive specific transcript) has been shown to be upregulated in human non-small cell lung cancer (NSCLC); however, its role and molecular mechanisms in NSCLC cell progression remain unclear. Methods: qRT-PCR was conducted to assess the expression of XIST and miR-186. Cell proliferation was detected using MTT assay. Cell invasion and migration were evaluated using transwell assay. Cell cycle distribution and apoptosis rates were analyzed by flow cytometry. Luciferase reporter assay was used to identify the direct regulation of XIST and miR-186. A RNA immunoprecipitation was used to analyze whether XIST was associated with the RNA-induced silencing complex (RISC). Results: We confirmed that XIST was upregulated in NSCLC cell lines and tissues. Functionally, XIST knockdown inhibited cancer cell proliferation and invasion, and induced apoptosis in vitro, and suppressed subcutaneous tumor growth in vivo. Mechanistic investigations revealed a reciprocal repressive interaction between XIST and miR-186-5p. Furthermore, we showed that miR-186-5p has a binding site for XIST. Our data also indicated that XIST and miR-186-5p are likely in the same RNA induced silencing complex. Conclusion: Together, our data revealed that XIST knockdown confers suppressive function in NSCLC and XIST may be a novel therapeutic marker in this disease.
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Giudicatti, Axel J., Ariel H. Tomassi, Pablo A. Manavella, and Agustin L. Arce. "Extensive Analysis of miRNA Trimming and Tailing Indicates that AGO1 Has a Complex Role in miRNA Turnover." Plants 10, no. 2 (2021): 267. http://dx.doi.org/10.3390/plants10020267.
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MicroRNAs are small regulatory RNAs involved in several processes in plants ranging from development and stress responses to defense against pathogens. In order to accomplish their molecular functions, miRNAs are methylated and loaded into one ARGONAUTE (AGO) protein, commonly known as AGO1, to stabilize and protect the molecule and to assemble a functional RNA-induced silencing complex (RISC). A specific machinery controls miRNA turnover to ensure the silencing release of targeted-genes in given circumstances. The trimming and tailing of miRNAs are fundamental modifications related to their turnover and, hence, to their action. In order to gain a better understanding of these modifications, we analyzed Arabidopsis thaliana small RNA sequencing data from a diversity of mutants, related to miRNA biogenesis, action, and turnover, and from different cellular fractions and immunoprecipitations. Besides confirming the effects of known players in these pathways, we found increased trimming and tailing in miRNA biogenesis mutants. More importantly, our analysis allowed us to reveal the importance of ARGONAUTE 1 (AGO1) loading, slicing activity, and cellular localization in trimming and tailing of miRNAs.
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Redfern, A. D., S. M. Colley, D. J. Beveridge, et al. "RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators." Proceedings of the National Academy of Sciences 110, no. 16 (2013): 6536–41. http://dx.doi.org/10.1073/pnas.1301620110.
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Chen, Chen. "Interaction Between Argonaute2 and RNA Molecules: AGO2 Molecular Structure and Different Regions in Nucleotide Chain." BIO Web of Conferences 59 (2023): 01001. http://dx.doi.org/10.1051/bioconf/20235901001.
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Argonaute2 (AGO2) is an important protein connecting the construction of RNA-induced silencing complex (RISC) and micro-RNA (miRNA) biogenesis. This paper explores the mechanism during the function through previous studies which reveal the crystal structure and affinity comparisons. It is concluded that the AGO2 have compartmentalized domains for certain functions, which have both individual and cooperative role in the entire process. The characters of AGO2 and miRNA suggest a model of regiondifferent nucleotide chains in miRNA, which means its epigenetic information is based on base sequence and its special information. According to these findings, further studies are advised to monitor the reactions in a dynamical method, which would be a new potential entry point for clinical utilization.
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Vasselon, Thierry, Manuella Bouttier, Anne Saumet, and Charles-Henri Lecellier. "RNAi and retroviruses: are they in RISC?" BioMolecular Concepts 4, no. 1 (2013): 43–52. http://dx.doi.org/10.1515/bmc-2012-0041.
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AbstractRNA interference (RNAi) is a potent cellular system against viruses in various organisms. Although common traits are observed in plants, insects, and nematodes, the situation observed in mammals appears more complex. In mammalian somatic cells, RNAi is implicated in endonucleolytic cleavage mediated by artificially delivered small interfering RNAs (siRNAs) as well as in translation repression mediated by microRNAs (miRNAs). Because siRNAs and miRNAs recognize viral mRNAs, RNAi inherently limits virus production and participates in antiviral defense. However, several observations made in the cases of hepatitis C virus and retroviruses (including the human immunodeficiency virus and the primate foamy virus) bring evidence that this relationship is much more complex and that certain components of the RNAi effector complex [called the RNA-induced silencing complex (RISC)], such as AGO2, are also required for viral replication. Here, we summarize recent discoveries that have revealed this dual implication in virus biology. We further discuss their potential implications for the functions of RNAi-related proteins, with special emphasis on retrotransposition and genome stability.
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Stavast, Christiaan J., and Stefan J. Erkeland. "The Non-Canonical Aspects of MicroRNAs: Many Roads to Gene Regulation." Cells 8, no. 11 (2019): 1465. http://dx.doi.org/10.3390/cells8111465.
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MicroRNAs (miRNAs) are critical regulators of gene expression. As miRNAs are frequently deregulated in many human diseases, including cancer and immunological disorders, it is important to understand their biological functions. Typically, miRNA-encoding genes are transcribed by RNA Polymerase II and generate primary transcripts that are processed by RNase III-endonucleases DROSHA and DICER into small RNAs of approximately 21 nucleotides. All miRNAs are loaded into Argonaute proteins in the RNA-induced silencing complex (RISC) and act as post-transcriptional regulators by binding to the 3′- untranslated region (UTR) of mRNAs. This seed-dependent miRNA binding inhibits the translation and/or promotes the degradation of mRNA targets. Surprisingly, recent data presents evidence for a target-mediated decay mechanism that controls the level of specific miRNAs. In addition, several non-canonical miRNA-containing genes have been recently described and unexpected functions of miRNAs have been identified. For instance, several miRNAs are located in the nucleus, where they are involved in the transcriptional activation or silencing of target genes. These epigenetic modifiers are recruited by RISC and guided by miRNAs to specific loci in the genome. Here, we will review non-canonical aspects of miRNA biology, including novel regulators of miRNA expression and functions of miRNAs in the nucleus.
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Abaturov, A. E., and V. L. Babуch. "MicroRNA biogenesis. Part 2. Formation of mature miRNAs. Maturation of non-canonical miRNAs." CHILD`S HEALTH 16, no. 3 (2021): 257–63. http://dx.doi.org/10.22141/2224-0551.16.3.2021.233912.
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The scientific review presents the biogenesis of miRNAs. To write the article, information was searched using databases Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka. The article shows the stages of formation of mature miRNAs. It is noted that duplex RNAs resulting from DICER-mediated cleavage interact with Argonaute (AGO) proteins to form an effector RNA-induced silencing complex (RISC). It is shown that the deficiency of AGO proteins leads to a significant decrease in the amount of miRs, and overexpression of AGO proteins is accompanied by an increase in the level of miRs. The main stages of assembling a fully functional RISC are presented. The first stage is the loading of duplex miRs on AGO proteins. The second stage is the promotion of duplex miRs. Human diseases associated with processing disorders in the cytoplasm of the cell are presented. Numerous alternative mechanisms involved in the formation of functionally active miRs are is characterized. There are three classes of mirtrons: typical mirtrons, 5’-tailed mirtrons and 3’-tailed mirtrons. Endogenous csRNAs resemble Drosha-independent synthetic csRNAs used to experimentally induce gene knockout. Chimeric hairpins of non-canonical miR genes are transcribed in tandem or as a part of another type of small RNA gene. Thus, the formation of mature miRs occurs due to the formation of the RISC complex. The core of the RISC complex consists of microRNA, AGO and protein with a trinucleotide repeat 6. Loading dsRNA on AGO proteins and subsequent promotion of duplex RNA are the main stages of assembly of a fully functional RISC. Disorders of processing in the cytoplasm of the cell are associated with the development of some human diseases. There are alternative mechanisms involved in the formation of functionally active miRs: mirtrons, endogenous short RNAs containing hairpins, chimeric hairpins.
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Chattopadhyay, Maitreyi, Vera A. Stupina, Feng Gao, et al. "Requirement for Host RNA-Silencing Components and the Virus-Silencing Suppressor when Second-Site Mutations Compensate for Structural Defects in the 3′ Untranslated Region." Journal of Virology 89, no. 22 (2015): 11603–18. http://dx.doi.org/10.1128/jvi.01566-15.
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ABSTRACTTurnip crinkle virus (TCV) contains a structured 3′ region with hairpins and pseudoknots that form a complex network of noncanonical RNA:RNA interactions supporting higher-order structure critical for translation and replication. We investigated several second-site mutations in the p38 coat protein open reading frame (ORF) that arose in response to a mutation in the asymmetric loop of a critical 3′ untranslated region (UTR) hairpin that disrupts local higher-order structure. All tested second-site mutations improved accumulation of TCV in conjunction with a partial reversion of the primary mutation (TCV-rev1) but had neutral or a negative effect on wild-type (wt) TCV or TCV with the primary mutation. SHAPE (selective 2′-hydroxylacylation analyzed byprimerextension) structure probing indicated that these second-site mutations reside in an RNA domain that includes most of p38 (domain 2), and evidence for RNA:RNA interactions between domain 2 and 3′UTR-containing domain 1 was found. However, second-site mutations were not compensatory in the absence of p38, which is also the TCV silencing suppressor, or indcl-2/dcl4orago1/ago2backgrounds. One second-site mutation reduced silencing suppressor activity of p38 by altering one of two GW motifs that are required for p38 binding to double-stranded RNAs (dsRNAs) and interaction with RNA-induced silencing complex (RISC)-associated AGO1/AGO2. Another second-site mutation substantially reduced accumulation of TCV-rev1 in the absence of p38 or DCL2/DCL4. We suggest that the second-site mutations in the p38 ORF exert positive effects through a similar downstream mechanism, either by enhancing accumulation of beneficial DCL-produced viral small RNAs that positively regulate the accumulation of TCV-rev1 or by affecting the susceptibility of TCV-rev1 to RISC loaded with viral small RNAs.IMPORTANCEGenomes of positive-strand RNA viruses fold into high-order RNA structures. Viruses with mutations in regions critical for translation and replication often acquire second-site mutations that exert a positive compensatory effect through reestablishment of canonical base pairing with the altered region. In this study, two distal second-site mutations that individually arose in response to a primary mutation in a critical 3′ UTR hairpin in the genomic RNA of turnip crinkle virus did not directly interact with the primary mutation. Although different second-site changes had different attributes, compensation was dependent on the production of the viral p38 silencing suppressor and on the presence of silencing-required DCL and AGO proteins. Our results provide an unexpected connection between a 3′ UTR primary-site mutation proposed to disrupt higher-order structure and the RNA-silencing machinery.
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Yamada, Ken, Samuel Hildebrand, Sarah M. Davis, et al. "Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction, and modulates siRNA activity and allele specificity." Nucleic Acids Research 49, no. 21 (2021): 12069–88. http://dx.doi.org/10.1093/nar/gkab1126.
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Abstract Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (iE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for iE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the iE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of iE-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington's disease as an example, iE-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the iE-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs.
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Li, Hongying, Zhengbo Wang, Yanwu Gao, et al. "Genome-Wide Identification of the Argonaute Protein Family and Its Expression Analysis under PEG6000, ABA and Heat Treatments in Populus alba × P. glandulosa." Forests 14, no. 5 (2023): 1015. http://dx.doi.org/10.3390/f14051015.
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The argonaute (AGO) protein, as an important member of the small RNA (sRNA) regulatory pathway gene-silencing complex (RNA-induced silencing complex, RISC), is a key protein that mediates gene silencing and plays a key role in the recruitment of sRNAs. In this study, bioinformatics was used to identify the AGO gene family in poplar and study its expression in various tissues and in response to abiotic stress treatments. A total of 15 PtAGO genes were identified in poplar, which were unevenly distributed in 9 chromosomes. Most proteins were predicted to be located in the nucleus and chloroplast. The PtAGOs had similar motif structures and conserved motifs, except for PtAGO3. All the PtAGO genes could be clustered into 3 groups, and Group II, including PtAGO2/3/7, had the smallest number of exons, while the others had more than 20 exons. Cis-regulatory elements involved in light response, growth and development, abiotic stress and hormone-induced responses were found in the promoters of PtAGO members. Further expression analysis found that the PtAGO genes had tissue-specific expression patterns. For example, PtAGO7 and PtAGO10b were mainly expressed in the xylem and might be involved in secondary xylem development. Furthermore, abiotic stress tests, including heat, ABA and PEG treatments, showed that most PtAGO genes could respond quickly to ABA treatment, and multiple PtAGO genes were constantly regulated under heat-shock stress. These results provide a basis for the elucidation mechanism of PtAGO genes and further molecular breeding in poplar.
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Lewkowicz, P., H. Cwikli ska, M. P. Mycko, et al. "Dysregulated RNA-Induced Silencing Complex (RISC) Assembly within CNS Corresponds with Abnormal miRNA Expression during Autoimmune Demyelination." Journal of Neuroscience 35, no. 19 (2015): 7521–37. http://dx.doi.org/10.1523/jneurosci.4794-14.2015.
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Milochau, Alexandra, Valérie Lagrée, Marie-Noëlle Benassy та ін. "Synaptotagmin 11 interacts with components of the RNA-induced silencing complex RISC in clonal pancreatic β-cells". FEBS Letters 588, № 14 (2014): 2217–22. http://dx.doi.org/10.1016/j.febslet.2014.05.031.
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