Journal articles on the topic 'RNAi pathway'
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1
Dzianott, Aleksandra, Joanna Sztuba-Solińska, and Jozef J. Bujarski. "Mutations in the Antiviral RNAi Defense Pathway Modify Brome mosaic virus RNA Recombinant Profiles." Molecular Plant-Microbe Interactions® 25, no. 1 (January 2012): 97–106. http://dx.doi.org/10.1094/mpmi-05-11-0137.
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RNA interference (RNAi) mechanism targets viral RNA for degradation. To test whether RNAi gene products contributed to viral RNA recombination, a series of Arabidopsis thaliana RNAi-defective mutants were infected with Brome mosaic virus (BMV) RNAs that have been engineered to support crossovers within the RNA3 segment. Single-cross RNA3-RNA1, RNA3-RNA2, and RNA3-RNA3 recombinants accumulated in both the wild-type (wt) and all knock-out lines at comparable frequencies. However, a reduced accumulation of novel 3′ mosaic RNA3 recombinants was observed in ago1, dcl2, dcl4, and rdr6 lines but not in wt Col-0 or the dcl3 line. A BMV replicase mutant accumulated a low level of RNA3-RNA1 single-cross recombinants in Col-0 plants while, in a dcl2 dcl4 double mutant, the formation of both RNA3-RNA1 and mosaic recombinants was at a low level. A control infection in the cpr5-2 mutant, a more susceptible BMV Arabidopsis host, generated similar-to-Col-0 profiles of both single-cross and mosaic recombinants, indicating that recombinant profiles were, to some extent, independent of a viral replication rate. Also, the relative growth experiments revealed similar selection pressure for recombinants among the host lines. Thus, the altered recombinant RNA profiles have originated at the level of recombinant formation rather than because of altered selection. In conclusion, the viral replicase and the host RNAi gene products contribute in distinct ways to BMV RNA recombination. Our studies reveal that the antiviral RNAi mechanisms are utilized by plant RNA viruses to increase their variability, reminiscent of phenomena previously demonstrated in fungi.
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Yang, Li, Yuan Tian, Yuan-Yuan Peng, Jinzhi Niu, and Jin-Jun Wang. "Expression Dynamics of Core RNAi Machinery Genes in Pea Aphids Upon Exposure to Artificially Synthesized dsRNA and miRNAs." Insects 11, no. 2 (January 21, 2020): 70. http://dx.doi.org/10.3390/insects11020070.
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The pea aphid is an important pest of vegetables and causes serious losses worldwide. RNA interference (RNAi) is an effective pest control tool, and three sub-pathways have been described: The miRNA pathway, siRNA pathway, and piRNA pathway. A large number of genes in miRNA pathway and piRNA pathway are found to be expanded. To study the roles of these genes, the expression of 25 core RNAi genes was screened in spatiotemporal samples, artificially synthesized dsRNA and miRNA treated samples. The 25 genes were all expressed during different development stages and in different tissues. In dsRNA-treated samples and miRNA-treated samples, the expressions of genes in these three pathways were induced, especially the expanded genes. This suggests a complex network of RNAi core genes in the three sub-pathways. Treatment of miRNA seems to induce gene expression in a dosage-dependent manner. These results increase our knowledge of the siRNA pathway and related factors from RNAi pathway in aphids and promote the use of RNAi for the control of aphid pests.
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Soleimani, Saeed, Zahra Valizadeh Arshad, Sharif Moradi, Ali Ahmadi, Seyed Javad Davarpanah, and Sadegh Azimzadeh Jamalkandi. "Small regulatory noncoding RNAs in Drosophila melanogaster: biogenesis and biological functions." Briefings in Functional Genomics 19, no. 4 (March 27, 2020): 309–23. http://dx.doi.org/10.1093/bfgp/elaa005.
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Abstract RNA interference (RNAi) is an important phenomenon that has diverse genetic regulatory functions at the pre- and posttranscriptional levels. The major trigger for the RNAi pathway is double-stranded RNA (dsRNA). dsRNA is processed to generate various types of major small noncoding RNAs (ncRNAs) that include microRNAs (miRNAs), small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) in Drosophila melanogaster (D. melanogaster). Functionally, these small ncRNAs play critical roles in virtually all biological systems and developmental pathways. Identification and processing of dsRNAs and activation of RNAi machinery are the three major academic interests that surround RNAi research. Mechanistically, some of the important biological functions of RNAi are achieved through: (i) supporting genomic stability via degradation of foreign viral genomes; (ii) suppressing the movement of transposable elements and, most importantly, (iii) post-transcriptional regulation of gene expression by miRNAs that contribute to regulation of epigenetic modifications such as heterochromatin formation and genome imprinting. Here, we review various routes of small ncRNA biogenesis, as well as different RNAi-mediated pathways in D. melanogaster with a particular focus on signaling pathways. In addition, a critical discussion of the most relevant and latest findings that concern the significant contribution of small ncRNAs to the regulation of D. melanogaster physiology and pathophysiology is presented.
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Lew, A. E., L. A. Jackson, and M. I. Bellgard. "Comparative genomic analysis of non-coding sequences and the application of RNA interference tools for bovine functional genomics." Australian Journal of Experimental Agriculture 45, no. 8 (2005): 995. http://dx.doi.org/10.1071/ea05057.
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Non-coding (nc) RNAs are important regulators of developmental genes, and essential for the modification of cellular DNA and chromatin through a process known as RNA interference (RNAi). The mediators of RNAi can be in the form of short double stranded (ds) RNAs, micro (mi) RNAs or small interfering (si) RNAs. miRNAs are involved in a translation repression pathway that inhibits protein translation in mRNA targets. Comparative genomic screens have revealed conserved regulatory non-coding sequences, which assist to predict the function of endogenous miRNAs. Only a few comparative studies include bovine genomic sequence, and RNAi has yet to be applied in bovine genome functional screens. siRNAs target homologous mRNAs for degradation, and thereby, silence specific genes. The use of synthetic siRNAs facilitates the elucidation of gene pathways by specific gene knockdown. A survey of the literature identifies a small number of reports using RNAi to examine immune pathways in bovine cell lines; however, they do not target genes involved in specific production traits. Applications of RNAi to elucidate bovine immune pathways for relevant bacterial and parasite diseases are yet to be reported. The inhibition of viral replication using RNAi has been demonstrated with bovine RNA viruses such as pestivirus and foot and mouth disease virus signifying the potential of RNAi as an antiviral therapeutic. RNAi approaches combined with genome data for protozoan parasites, insects and nematodes, will expedite the identification of novel targets for the treatment and prevention of economically important parasitic infections. This review will examine the approaches used in mammalian RNAi research, the current status of its applications to livestock systems and will discuss potential applications in beef cattle programs.
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Zhuang, Jimmy J., and Craig P. Hunter. "The Influence of Competition Among C. elegans Small RNA Pathways on Development." Genes 3, no. 4 (October 19, 2012): 671–85. http://dx.doi.org/10.3390/genes3040671.
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Small RNAs play a variety of regulatory roles, including highly conserved developmental functions. Caenorhabditis elegans not only possesses most known small RNA pathways, it is also an easy system to study their roles and interactions during development. It has been proposed that in C. elegans, some small RNA pathways compete for access to common limiting resources. The strongest evidence supporting this model is that disrupting the production or stability of endogenous short interfering RNAs (endo-siRNAs) enhances sensitivity to experimentally induced exogenous RNA interference (exo-RNAi). Here, we examine the relationship between the endo-siRNA and microRNA (miRNA) pathways, and find that, consistent with competition among these endogenous small RNA pathways, endo-siRNA pathway mutants may enhance miRNA efficacy. Furthermore, we show that exo-RNAi may also compete with both endo-siRNAs and miRNAs. Our data thus provide support that all known Dicer-dependent small RNA pathways may compete for limiting common resources. Finally, we observed that both endo-siRNA mutants and animals experiencing exo-RNAi have increased expression of miRNA-regulated stage-specific developmental genes. These observations suggest that perturbing the small RNA flux and/or the induction of exo-RNAi, even in wild-type animals, may impact development via effects on the endo-RNAi and microRNA pathways.
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Szachnowski, Ugo, Sara Andus, Dominika Foretek, Antonin Morillon, and Maxime Wery. "Endogenous RNAi pathway evolutionarily shapes the destiny of the antisense lncRNAs transcriptome." Life Science Alliance 2, no. 5 (August 28, 2019): e201900407. http://dx.doi.org/10.26508/lsa.201900407.
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Antisense long noncoding (aslnc)RNAs are extensively degraded by the nuclear exosome and the cytoplasmic exoribonuclease Xrn1 in the budding yeast Saccharomyces cerevisiae, lacking RNAi. Whether the ribonuclease III Dicer affects aslncRNAs in close RNAi-capable relatives remains unknown. Using genome-wide RNA profiling, here we show that aslncRNAs are primarily targeted by the exosome and Xrn1 in the RNAi-capable budding yeast Naumovozyma castellii, Dicer only affecting Xrn1-sensitive aslncRNAs levels in Xrn1-deficient cells. The dcr1 and xrn1 mutants display synergic growth defects, indicating that Dicer becomes critical in the absence of Xrn1. Small RNA sequencing showed that Dicer processes aslncRNAs into small RNAs, with a preference for Xrn1-sensitive aslncRNAs. Consistently, Dicer localizes into the cytoplasm. Finally, we observed an expansion of the exosome-sensitive antisense transcriptome in N. castellii compared with S. cerevisiae, suggesting that the presence of cytoplasmic RNAi has reinforced the nuclear RNA surveillance machinery to temper aslncRNAs expression. Our data provide fundamental insights into aslncRNAs metabolism and open perspectives into the possible evolutionary contribution of RNAi in shaping the aslncRNAs transcriptome.
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Mondal, Mosharrof, Judith K. Brown, and Alex Flynt. "Exploiting somatic piRNAs in Bemisia tabaci enables novel gene silencing through RNA feeding." Life Science Alliance 3, no. 10 (August 6, 2020): e202000731. http://dx.doi.org/10.26508/lsa.202000731.
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RNAi promises to reshape pest control by being nontoxic, biodegradable, and species specific. However, due to the plastic nature of RNAi, there is a significant variability in responses. In this study, we investigate small RNA pathways and processing of ingested RNAi trigger molecules in a hemipteran plant pest, the whitefly Bemisia tabaci. Unlike Drosophila, where the paradigm for insect RNAi technology was established, whitefly has abundant somatic piwi-associated RNAs (piRNAs). Long regarded as germline restricted, piRNAs are common in the soma of many invertebrates. We sought to exploit this for a novel gene silencing approach. The main principle of piRNA biogenesis is the recruitment of target RNA fragments into the pathway. As such, we designed synthetic RNAs to possess complementarity to the loci we annotated. Following feeding of these exogenous piRNA triggers knockdown as effective as conventional siRNA-only approaches was observed. These results demonstrate a new approach for RNAi technology that could be applicable to dsRNA-recalcitrant pest species and could be fundamental to realizing insecticidal RNAi against pests.
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Cánovas-Márquez, José Tomás, Sebastian Falk, Francisco E. Nicolás, Subramanian Padmanabhan, Rubén Zapata-Pérez, Álvaro Sánchez-Ferrer, Eusebio Navarro, and Victoriano Garre. "A ribonuclease III involved in virulence of Mucorales fungi has evolved to cut exclusively single-stranded RNA." Nucleic Acids Research 49, no. 9 (April 20, 2021): 5294–307. http://dx.doi.org/10.1093/nar/gkab238.
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Abstract Members of the ribonuclease III (RNase III) family regulate gene expression by processing double-stranded RNA (dsRNA). This family includes eukaryotic Dicer and Drosha enzymes that generate small dsRNAs in the RNA interference (RNAi) pathway. The fungus Mucor lusitanicus, which causes the deadly infection mucormycosis, has a complex RNAi system encompassing a non-canonical RNAi pathway (NCRIP) that regulates virulence by degrading specific mRNAs. In this pathway, Dicer function is replaced by R3B2, an atypical class I RNase III, and small single-stranded RNAs (ssRNAs) are produced instead of small dsRNA as Dicer-dependent RNAi pathways. Here, we show that R3B2 forms a homodimer that binds to ssRNA and dsRNA molecules, but exclusively cuts ssRNA, in contrast to all known RNase III. The dsRNA cleavage inability stems from its unusual RNase III domain (RIIID) because its replacement by a canonical RIIID allows dsRNA processing. A crystal structure of R3B2 RIIID resembles canonical RIIIDs, despite the low sequence conservation. However, the groove that accommodates dsRNA in canonical RNases III is narrower in the R3B2 homodimer, suggesting that this feature could be responsible for the cleavage specificity for ssRNA. Conservation of this activity in R3B2 proteins from other mucormycosis-causing Mucorales fungi indicates an early evolutionary acquisition.
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Holding, Cathy. "RNAi dissects signal pathway." Genome Biology 5 (2004): spotlight—20040624–01. http://dx.doi.org/10.1186/gb-spotlight-20040624-01.
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Lucentini, Jack. "Second RNAi pathway emerges." Genome Biology 5 (2004): spotlight—20040517–01. http://dx.doi.org/10.1186/gb-spotlight-20040817-01.
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Varley, Andrew J., and Jean-Paul Desaulniers. "Chemical strategies for strand selection in short-interfering RNAs." RSC Advances 11, no. 4 (2021): 2415–26. http://dx.doi.org/10.1039/d0ra07747j.
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Mao, Kai, Peter Breen, and Gary Ruvkun. "Mitochondrial dysfunction induces RNA interference in C. elegans through a pathway homologous to the mammalian RIG-I antiviral response." PLOS Biology 18, no. 12 (December 2, 2020): e3000996. http://dx.doi.org/10.1371/journal.pbio.3000996.
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RNA interference (RNAi) is an antiviral pathway common to many eukaryotes that detects and cleaves foreign nucleic acids. In mammals, mitochondrially localized proteins such as mitochondrial antiviral signaling (MAVS), retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) mediate antiviral responses. Here, we report that mitochondrial dysfunction in Caenorhabditis elegans activates RNAi-directed silencing via induction of a pathway homologous to the mammalian RIG-I helicase viral response pathway. The induction of RNAi also requires the conserved RNA decapping enzyme EOL-1/DXO. The transcriptional induction of eol-1 requires DRH-1 as well as the mitochondrial unfolded protein response (UPRmt). Upon mitochondrial dysfunction, EOL-1 is concentrated into foci that depend on the transcription of mitochondrial RNAs that may form double-stranded RNA (dsRNA), as has been observed in mammalian antiviral responses. Enhanced RNAi triggered by mitochondrial dysfunction is necessary for the increase in longevity that is induced by mitochondrial dysfunction.
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Nishide, Yudai, Daisuke Kageyama, Kakeru Yokoi, Akiya Jouraku, Hiromitsu Tanaka, Ryo Futahashi, and Takema Fukatsu. "Functional crosstalk across IMD and Toll pathways: insight into the evolution of incomplete immune cascades." Proceedings of the Royal Society B: Biological Sciences 286, no. 1897 (February 20, 2019): 20182207. http://dx.doi.org/10.1098/rspb.2018.2207.
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In insects, antimicrobial humoral immunity is governed by two distinct gene cascades, IMD pathway mainly targeting Gram-negative bacteria and Toll pathway preferentially targeting Gram-positive bacteria, which are widely conserved among diverse metazoans. However, recent genomic studies uncovered that IMD pathway is exceptionally absent in some hemipteran lineages like aphids and assassin bugs. How the apparently incomplete immune pathways have evolved with functionality is of interest. Here we report the discovery that, in the hemipteran stinkbug Plautia stali , both IMD and Toll pathways are present but their functional differentiation is blurred. Injection of Gram-negative bacteria and Gram-positive bacteria upregulated effector genes of both pathways. Notably, RNAi experiments unveiled significant functional permeation and crosstalk between IMD and Toll pathways: RNAi of IMD pathway genes suppressed upregulation of effector molecules of both pathways, where the suppression was more remarkable for IMD effectors; and RNAi of Toll pathway genes reduced upregulation of effector molecules of both pathways, where the suppression was more conspicuous for Toll effectors. These results suggest the possibility that, in hemipterans and other arthropods, IMD and Toll pathways are intertwined to target wider and overlapping arrays of microbes, which might have predisposed and facilitated the evolution of incomplete immune pathways.
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Ashe, Alyson, Peter Sarkies, Jérémie Le Pen, Mélanie Tanguy, and Eric A. Miska. "Antiviral RNA Interference against Orsay Virus Is neither Systemic nor Transgenerational in Caenorhabditis elegans." Journal of Virology 89, no. 23 (September 23, 2015): 12035–46. http://dx.doi.org/10.1128/jvi.03664-14.
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ABSTRACTAntiviral RNA-mediated silencing (RNA interference [RNAi]) acts as a powerful innate immunity defense in plants, invertebrates, and mammals. InCaenorhabditis elegans, RNAi is systemic; i.e., RNAi silencing signals can move between cells and tissues. Furthermore, RNAi effects can be inherited transgenerationally and may last for many generations. Neither the biological relevance of systemic RNAi nor transgenerational RNAi is currently understood. Here we examined the role of both pathways in the protection ofC. elegansfrom viral infection. We studied the Orsay virus, a positive-strand RNA virus related toNodaviridaeand the first and only virus known to infectC. elegans. Immunity to Orsay virus infection requires the RNAi pathway. Surprisingly, we found that genes required for systemic or transgenerational RNAi did not have a role in antiviral defense. Furthermore, we found that Orsay virus infection did not elicit a systemic RNAi response even when a target for RNAi was provided by using transgenes. Finally, we show that viral siRNAs, the effectors of RNAi, are not inherited to a level that provides any significant resistance to viral infection in the next generation. We conclude that systemic or transgenerational RNAi does not play a role in the defense against natural Orsay virus infection. Furthermore, our data suggest that there is a qualitative difference between experimental RNAi and antiviral RNAi. Our data are consistent with a model of systemic and transgenerational RNAi that requires a nuclear or germ line component that is lacking in almost all RNA virus infections.IMPORTANCESince its discovery inCaenorhabditis elegans, RNAi has proven a valuable scientific tool in many organisms. InC. elegans, exogenous RNAi spreads throughout the organism and can be passed between generations; however, there has been controversy as to the endogenous role(s) that the RNAi pathway plays. One endogenous role for which spreading both within the infected organism and between generations would be advantageous is a role in viral defense. In plants, antiviral RNAi is systemic and the spread of RNAi between cells provides protection against subsequent viral infection. Here we investigated this by using the only naturally occurring virus known to infectC. elegans, Orsay virus, and surprisingly found that, in contrast to the exogenous RNAi pathway, the antiviral RNAi response targeted against this virus does not spread systemically throughout the organism and cannot be passed between generations. These results suggest that there are differences between the two pathways that remain to be discovered.
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Foda, Bardees M., and Upinder Singh. "Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway." Journal of Biological Chemistry 290, no. 34 (July 6, 2015): 21114–30. http://dx.doi.org/10.1074/jbc.m115.647263.
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RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5′-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica.
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Ahmed, Fee Faysal, Md Imran Hossen, Md Abdur Rauf Sarkar, Jesmin Naher Konak, Fatema Tuz Zohra, Md Shoyeb, and Samiran Mondal. "Genome-wide identification of DCL, AGO and RDR gene families and their associated functional regulatory elements analyses in banana (Musa acuminata)." PLOS ONE 16, no. 9 (September 2, 2021): e0256873. http://dx.doi.org/10.1371/journal.pone.0256873.
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RNA silencing is mediated through RNA interference (RNAi) pathway gene families, i.e., Dicer-Like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) and their cis-acting regulatory elements. The RNAi pathway is also directly connected with the post-transcriptional gene silencing (PTGS) mechanism, and the pathway controls eukaryotic gene regulation during growth, development, and stress response. Nevertheless, genome-wide identification of RNAi pathway gene families such as DCL, AGO, and RDR and their regulatory network analyses related to transcription factors have not been studied in many fruit crop species, including banana (Musa acuminata). In this study, we studied in silico genome-wide identification and characterization of DCL, AGO, and RDR genes in bananas thoroughly via integrated bioinformatics approaches. A genome-wide analysis identified 3 MaDCL, 13 MaAGO, and 5 MaRDR candidate genes based on multiple sequence alignment and phylogenetic tree related to the RNAi pathway in banana genomes. These genes correspond to the Arabidopsis thaliana RNAi silencing genes. The analysis of the conserved domain, motif, and gene structure (exon-intron numbers) for MaDCL, MaAGO, and MaRDR genes showed higher homogeneity within the same gene family. The Gene Ontology (GO) enrichment analysis exhibited that the identified RNAi genes could be involved in RNA silencing and associated metabolic pathways. A number of important transcription factors (TFs), e.g., ERF, Dof, C2H2, TCP, GATA and MIKC_MADS families, were identified by network and sub-network analyses between TFs and candidate RNAi gene families. Furthermore, the cis-acting regulatory elements related to light-responsive (LR), stress-responsive (SR), hormone-responsive (HR), and other activities (OT) functions were identified in candidate MaDCL, MaAGO, and MaRDR genes. These genome-wide analyses of these RNAi gene families provide valuable information related to RNA silencing, which would shed light on further characterization of RNAi genes, their regulatory elements, and functional roles, which might be helpful for banana improvement in the breeding program.
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Olson, Ken E., and Carol D. Blair. "Arbovirus–mosquito interactions: RNAi pathway." Current Opinion in Virology 15 (December 2015): 119–26. http://dx.doi.org/10.1016/j.coviro.2015.10.001.
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Li, Chunhong, Sreelatha Sarangapani, Qian Wang, Kumar Nadimuthu, and Rajani Sarojam. "Metabolic Engineering of the Native Monoterpene Pathway in Spearmint for Production of Heterologous Monoterpenes Reveals Complex Metabolism and Pathway Interactions." International Journal of Molecular Sciences 21, no. 17 (August 26, 2020): 6164. http://dx.doi.org/10.3390/ijms21176164.
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Spearmint produces and stores large amounts of monoterpenes, mainly limonene and carvone, in glandular trichomes and is the major natural source of these compounds. Towards producing heterologous monoterpenes in spearmint, we first reduced the flux into the native limonene pathway by knocking down the expression of limonene synthase (MsLS) by RNAi method. The MsLS RNAi lines exhibited a huge reduction in the synthesis of limonene and carvone. Detailed GC-MS and LC-MS analysis revealed that MsLS RNAi plants also showed an increase in sesquiterpene, phytosterols, fatty acids, flavonoids, and phenolic metabolites, suggesting an interaction between the MEP, MVA shikimate and fatty acid pathways in spearmint. Three different heterologous monoterpene synthases namely, linalool synthase and myrcene synthase from Picea abies and geraniol synthase from Cananga odorata were cloned and introduced independently into the MsLS RNAi mutant background. The expression of these heterologous terpene synthases resulted mainly in production of monoterpene derivatives. Of all the introduced monoterpenes geraniol showed the maximum number of derivatives. Our results provide new insights into MEP pathway interactions and regulation and reveals the existence of mechanisms for complex metabolism of monoterpenes in spearmint.
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Rogers, Alicia K., and Carolyn M. Phillips. "RNAi pathways repress reprogramming of C. elegans germ cells during heat stress." Nucleic Acids Research 48, no. 8 (March 18, 2020): 4256–73. http://dx.doi.org/10.1093/nar/gkaa174.
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Abstract Repression of cellular reprogramming in germ cells is critical to maintaining cell fate and fertility. When germ cells mis-express somatic genes they can be directly converted into other cell types, resulting in loss of totipotency and reproductive potential. Identifying the molecular mechanisms that coordinate these cell fate decisions is an active area of investigation. Here we show that RNAi pathways play a key role in maintaining germline gene expression and totipotency after heat stress. By examining transcriptional changes that occur in mut-16 mutants, lacking a key protein in the RNAi pathway, at elevated temperature we found that genes normally expressed in the soma are mis-expressed in germ cells. Furthermore, these genes displayed increased chromatin accessibility in the germlines of mut-16 mutants at elevated temperature. These findings indicate that the RNAi pathway plays a key role in preventing aberrant expression of somatic genes in the germline during heat stress. This regulation occurs in part through the maintenance of germline chromatin, likely acting through the nuclear RNAi pathway. Identification of new pathways governing germ cell reprogramming is critical to understanding how cells maintain proper gene expression and may provide key insights into how cell identity is lost in some germ cell tumors.
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Taglini, Francesca, Elliott Chapman, Rob van Nues, Emmanuelle Theron, and Elizabeth H. Bayne. "Mkt1 is required for RNAi-mediated silencing and establishment of heterochromatin in fission yeast." Nucleic Acids Research 48, no. 3 (December 11, 2019): 1239–53. http://dx.doi.org/10.1093/nar/gkz1157.
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Abstract Constitutive domains of repressive heterochromatin are maintained within the fission yeast genome through self-reinforcing mechanisms involving histone methylation and small RNAs. Non-coding RNAs generated from heterochromatic regions are processed into small RNAs by the RNA interference pathway, and are subject to silencing through both transcriptional and post-transcriptional mechanisms. While the pathways involved in maintenance of the repressive heterochromatin state are reasonably well understood, less is known about the requirements for its establishment. Here, we describe a novel role for the post-transcriptional regulatory factor Mkt1 in establishment of heterochromatin at pericentromeres in fission yeast. Loss of Mkt1 does not affect maintenance of existing heterochromatin, but does affect its recovery following depletion, as well as de novo establishment of heterochromatin on a mini-chromosome. Pathway dissection revealed that Mkt1 is required for RNAi-mediated post-transcriptional silencing, downstream of small RNA production. Mkt1 physically associates with pericentromeric transcripts, and is additionally required for maintenance of silencing and heterochromatin at centromeres when transcriptional silencing is impaired. Our findings provide new insight into the mechanism of RNAi-mediated post-transcriptional silencing in fission yeast, and unveil an important role for post-transcriptional silencing in establishment of heterochromatin that is dispensable when full transcriptional silencing is imposed.
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Gamez, Stephanie, Satyam Srivastav, Omar S. Akbari, and Nelson C. Lau. "Diverse Defenses: A Perspective Comparing Dipteran Piwi-piRNA Pathways." Cells 9, no. 10 (September 27, 2020): 2180. http://dx.doi.org/10.3390/cells9102180.
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Animals face the dual threat of virus infections hijacking cellular function and transposons proliferating in germline genomes. For insects, the deeply conserved RNA interference (RNAi) pathways and other chromatin regulators provide an important line of defense against both viruses and transposons. For example, this innate immune system displays adaptiveness to new invasions by generating cognate small RNAs for targeting gene silencing measures against the viral and genomic intruders. However, within the Dipteran clade of insects, Drosophilid fruit flies and Culicids mosquitoes have evolved several unique mechanistic aspects of their RNAi defenses to combat invading transposons and viruses, with the Piwi-piRNA arm of the RNAi pathways showing the greatest degree of novel evolution. Whereas central features of Piwi-piRNA pathways are conserved between Drosophilids and Culicids, multiple lineage-specific innovations have arisen that may reflect distinct genome composition differences and specific ecological and physiological features dividing these two branches of Dipterans. This perspective review focuses on the most recent findings illuminating the Piwi/piRNA pathway distinctions between fruit flies and mosquitoes, and raises open questions that need to be addressed in order to ameliorate human diseases caused by pathogenic viruses that mosquitoes transmit as vectors.
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Wang, Huan, Anne Spang, Mark A. Sullivan, Jennifer Hryhorenko, and Fred K. Hagen. "The Terminal Phase of Cytokinesis in the Caenorhabditis elegans Early Embryo Requires Protein Glycosylation." Molecular Biology of the Cell 16, no. 9 (September 2005): 4202–13. http://dx.doi.org/10.1091/mbc.e05-05-0472.
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RNA interference (RNAi) was used to characterize the requirement of protein glycosylation for cell membrane stability during cytokinesis in the early embryo. This screen targeted 13 enzymes or components of polypeptide sugar transferases that initiate either N-glycosylation or three different pathways of O-glycosylation. RNAi of genes in the mucin-type and epidermal growth factor-fringe glycosylation pathways did not affect cytokinesis. However, embryos deficient in N-glycosylation exhibited a variable inability to complete cytokinesis. The most potent block in early embryonic cell division was obtained by RNAi of the polypeptide xylose transferase (ppXyl-T), which is required to initiate the proteoglycan modification pathway. Two generations of ppXyl-T RNAi-feeding treatment reduced the body size, mobility, brood size, and life span of adult animals. Embryos escaping ppXyl-T and Gal-T2 RNAi lethality develop to adulthood but have cytokinesis-deficient offspring, suggesting that glycosyltransferases in the proteoglycan pathway are maternal proteins in the early embryo. Gal-T2::GFP fusions and anti-Gal-T2 antibodies revealed a perinuclear staining pattern, consistent with the localization of the Golgi apparatus. RNAi in green fluorescent protein (GFP)-tagged strains to follow tubulin, PIE-1, and chromatin showed that deficient proteoglycan biosynthesis uncouples the stability of newly formed cell membranes from cytokinesis, whereas cleavage furrow initiation, mitotic spindle function, karyokinesis, and partitioning of intrinsic components are intact.
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Lax, Carlos, Ghizlane Tahiri, José Alberto Patiño-Medina, José T. Cánovas-Márquez, José A. Pérez-Ruiz, Macario Osorio-Concepción, Eusebio Navarro, and Silvia Calo. "The Evolutionary Significance of RNAi in the Fungal Kingdom." International Journal of Molecular Sciences 21, no. 24 (December 8, 2020): 9348. http://dx.doi.org/10.3390/ijms21249348.
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RNA interference (RNAi) was discovered at the end of last millennium, changing the way scientists understood regulation of gene expression. Within the following two decades, a variety of different RNAi mechanisms were found in eukaryotes, reflecting the evolutive diversity that RNAi entails. The essential silencing mechanism consists of an RNase III enzyme called Dicer that cleaves double-stranded RNA (dsRNA) generating small interfering RNAs (siRNAs), a hallmark of RNAi. These siRNAs are loaded into the RNA-induced silencing complex (RISC) triggering the cleavage of complementary messenger RNAs by the Argonaute protein, the main component of the complex. Consequently, the expression of target genes is silenced. This mechanism has been thoroughly studied in fungi due to their proximity to the animal phylum and the conservation of the RNAi mechanism from lower to higher eukaryotes. However, the role and even the presence of RNAi differ across the fungal kingdom, as it has evolved adapting to the particularities and needs of each species. Fungi have exploited RNAi to regulate a variety of cell activities as different as defense against exogenous and potentially harmful DNA, genome integrity, development, drug tolerance, or virulence. This pathway has offered versatility to fungi through evolution, favoring the enormous diversity this kingdom comprises.
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Watson, Samir, Lisanne Knol, Jeroen Witteveldt, and Sara Macias. "Crosstalk Between Mammalian Antiviral Pathways." Non-Coding RNA 5, no. 1 (March 22, 2019): 29. http://dx.doi.org/10.3390/ncrna5010029.
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As part of their innate immune response against viral infections, mammals activate the expression of type I interferons to prevent viral replication and dissemination. An antiviral RNAi-based response can be also activated in mammals, suggesting that several mechanisms can co-occur in the same cell and that these pathways must interact to enable the best antiviral response. Here, we will review how the classical type I interferon response and the recently described antiviral RNAi pathways interact in mammalian cells. Specifically, we will uncover how the small RNA biogenesis pathway, composed by the nucleases Drosha and Dicer can act as direct antiviral factors, and how the type-I interferon response regulates the function of these. We will also describe how the factors involved in small RNA biogenesis and specific small RNAs impact the activation of the type I interferon response and antiviral activity. With this, we aim to expose the complex and intricate network of interactions between the different antiviral pathways in mammals.
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Schnettler, Esther, Hans Hemmes, Rob Goldbach, and Marcel Prins. "The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells." Journal of General Virology 89, no. 1 (January 1, 2008): 336–40. http://dx.doi.org/10.1099/vir.0.83293-0.
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The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is described here that NS3 is also active in mammalian cells. This activity is independent of the inducer molecule used. Using either synthetic siRNAs or a short hairpin RNA construct, NS3 was able to significantly suppress the RNAi-mediated silencing of luciferase expression in both monkey (Vero) and human (HEK293) cells. These results support the proposed mode of action of NS3 to act by sequestering siRNAs, the key molecules of the RNAi pathway conserved in all eukaryotes. The possible applications of this protein in modulating RNAi and investigating the proposed antiviral RNAi response in mammalian cell systems are discussed.
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Bonning, Bryony C., and Maria-Carla Saleh. "The Interplay Between Viruses and RNAi Pathways in Insects." Annual Review of Entomology 66, no. 1 (January 7, 2021): 61–79. http://dx.doi.org/10.1146/annurev-ento-033020-090410.
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As an overarching immune mechanism, RNA interference (RNAi) displays pathogen specificity and memory via different pathways. The small interfering RNA (siRNA) pathway is the primary antiviral defense mechanism against RNA viruses of insects and plays a lesser role in defense against DNA viruses. Reflecting the pivotal role of the siRNA pathway in virus selection, different virus families have independently evolved unique strategies to counter this host response, including protein-mediated, decoy RNA–based, and microRNA-based strategies. In this review, we outline the interplay between insect viruses and the different pathways of the RNAi antiviral response; describe practical application of these interactions for improved expression systems and for pest and disease management; and highlight research avenues for advancement of the field.
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Bonning, Bryony C., and Maria-Carla Saleh. "The Interplay Between Viruses and RNAi Pathways in Insects." Annual Review of Entomology 66, no. 1 (January 7, 2021): 61–79. http://dx.doi.org/10.1146/annurev-ento-033020-090410.
Full textAbstract:
As an overarching immune mechanism, RNA interference (RNAi) displays pathogen specificity and memory via different pathways. The small interfering RNA (siRNA) pathway is the primary antiviral defense mechanism against RNA viruses of insects and plays a lesser role in defense against DNA viruses. Reflecting the pivotal role of the siRNA pathway in virus selection, different virus families have independently evolved unique strategies to counter this host response, including protein-mediated, decoy RNA–based, and microRNA-based strategies. In this review, we outline the interplay between insect viruses and the different pathways of the RNAi antiviral response; describe practical application of these interactions for improved expression systems and for pest and disease management; and highlight research avenues for advancement of the field.
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Mehrabadi, Mohammad, Mazhar Hussain, Leila Matindoost, and Sassan Asgari. "The Baculovirus Antiapoptotic p35 Protein Functions as an Inhibitor of the Host RNA Interference Antiviral Response." Journal of Virology 89, no. 16 (May 27, 2015): 8182–92. http://dx.doi.org/10.1128/jvi.00802-15.
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ABSTRACTRNA interference (RNAi) is considered an ancient antiviral defense in diverse organisms, including insects. Virus infections generate double-strand RNAs (dsRNAs) that trigger the RNAi machinery to process dsRNAs into virus-derived short interfering RNAs (vsiRNAs), which target virus genomes, mRNAs, or replication intermediates. Viruses, in turn, have evolved viral suppressors of RNAi (VSRs) to counter host antiviral RNAi. Following recent discoveries that insects mount an RNAi response against DNA viruses, in this study, we found thatAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) infection similarly induces an RNAi response inSpodoptera frugiperdacells by generating a large number of vsiRNAs postinfection. Interestingly, we found that AcMNPV expresses a potent VSR to counter RNAi. The viralp35gene, which is well known as an inhibitor of apoptosis, was found to be responsible for the suppression of RNAi in diverse insect and mammalian cells. The VSR activity of p35 was further confirmed by ap35-null AcMNPV that did not suppress the response. In addition, our results showed that the VSR activity is not due to inhibition of dsRNA cleavage by Dicer-2 but acts downstream in the RNAi pathway. Furthermore, we found that the VSR activity is not linked to the antiapoptotic activity of the protein. Overall, our results provide evidence for the existence of VSR activity in a double-stranded DNA virus and identify the responsible gene, which is involved in the inhibition of RNAi as well as apoptosis.IMPORTANCEOur findings demonstrate the occurrence of an insect RNAi response against a baculovirus (AcMNPV) that is highly utilized in microbial control, biological and biomedical research, and protein expression. Moreover, our investigations led to the identification of a viral suppressor of RNAi activity and the gene responsible for the activity. Notably, this gene is also a potent inhibitor of apoptosis. The outcomes signify the dual role of a virus-encoded protein in nullifying two key antiviral responses, apoptosis and RNAi.
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Li, Hai-Bo, Qi-Sheng You, Li-Xin Xu, Li-Xin Sun, Aman Shah Abdul Majid, Xiao-Bo Xia, and Dan Ji. "Long Non-Coding RNA-MALAT1 Mediates Retinal Ganglion Cell Apoptosis Through the PI3K/Akt Signaling Pathway in Rats with Glaucoma." Cellular Physiology and Biochemistry 43, no. 5 (2017): 2117–32. http://dx.doi.org/10.1159/000484231.
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Background/Aims: The aim of the present study is to investigate the effect of long non-coding RNA-MALAT1 (LncRNA-MALAT1) on retinal ganglion cell (RGC) apoptosis mediated by the PI3K/Akt signaling pathway in rats with glaucoma. Methods: RGCs were isolated and cultured, and monoclonal antibodies (anti-rat Thy-1, Brn3a and RBPMS) were examined by immunocytochemistry. An overexpression vector MALAT1-RNA activation (RNAa), gene knockout vector MALAT1-RNA interference (RNAi), and control vector MALAT1-negative control (NC) were constructed. A chronic high intraocular pressure (IOP) rat model of glaucoma was established by episcleral vein cauterization. The RGCs were divided into the RGC control, RGC pressure, RGC pressure + MALAT1-NC, RGC pressure + MALAT1-RNAi and RGC pressure + MALAT1-RNAa groups. Sixty Sprague-Dawley (SD) rats were randomly divided into the normal, high IOP, high IOP + MALAT1-NC, high IOP + MALAT1-RNAa and high IOP + MALAT1-RNAi groups. qRT-PCR and western blotting were used to detect the expression levels of LncRNA-MALAT1 and PI3K/Akt. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and flow cytometry were used to detect RGC apoptosis. Results: Immunocytochemistry revealed that the cultured RGCs reached 90% purity. Compared with the RGC pressure + MALAT1-NC group, the RGC pressure + MALAT1-RNAa group exhibited elevated expression levels of MALAT1, lower total protein levels of PI3K and Akt and decreased RGC apoptosis, while these expression levels were reversed in the RGC pressure + MALAT1-RNAi group. RGC numbers and PI3K/Akt expression levels in the high IOP model groups were lower than those in the normal group. In the high IOP + MALAT1-RNAa group, the mRNA and protein expression levels of PI3K/Akt were reduced but higher than those in the other three high IOP model groups. Additionally, RGC numbers in the high IOP + MALAT1-RNAa group were lower than those in the normal group but higher than those in the other three high IOP model groups. Conclusion: Our study provides evidence that LncRNA-MALAT1 could inhibit RGC apoptosis in glaucoma through activation of the PI3K/Akt signaling pathway.
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Demeter, Tomas, Michaela Vaskovicova, Radek Malik, Filip Horvat, Josef Pasulka, Eliska Svobodova, Matyas Flemr, and Petr Svoboda. "Main constraints for RNAi induced by expressed long dsRNA in mouse cells." Life Science Alliance 2, no. 1 (February 2019): e201800289. http://dx.doi.org/10.26508/lsa.201800289.
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RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.
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Petrocca, Fabio, and Judy Lieberman. "Promise and Challenge of RNA Interference–Based Therapy for Cancer." Journal of Clinical Oncology 29, no. 6 (February 20, 2011): 747–54. http://dx.doi.org/10.1200/jco.2009.27.6287.
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Cancer therapeutics still fall far short of our goals for treating patients with locally advanced or metastatic disease. Until recently, almost all cancer drugs were crude cytotoxic agents that discriminate poorly between cancer cells and normally dividing cells. The development of targeted biologics that recognize tumor cell surface antigens and of specific inhibitors of pathways dysregulated in cancer cells or normal cellular pathways on which a cancer cell differentially depends has provided hope for converting our increasing understanding of cellular transformation into intelligently designed anticancer therapeutics. However, new drug development is painfully slow, and the pipeline of new therapeutics is thin. The discovery of RNA interference (RNAi), a ubiquitous cellular pathway of gene regulation that is dysregulated in cancer cells, provides an exciting opportunity for relatively rapid and revolutionary approaches to cancer drug design. Small RNAs that harness the RNAi machinery may become the next new class of drugs for treating a variety of diseases. Although it has only been 9 years since RNAi was shown to work in mammalian cells, about a dozen phase I to III clinical studies have already been initiated, including four for cancer. So far there has been no unexpected toxicity and suggestions of benefit in one phase II study. However, the obstacles for RNAi-based cancer therapeutics are substantial. This article will discuss how the endogenous RNAi machinery might be harnessed for cancer therapeutics, why academic researchers and biotech and pharmaceutical companies are so excited, and what the obstacles are and how they might be overcome.
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Chu, Yongjun, Shinnichi Yokota, Jing Liu, Audrius Kilikevicius, Krystal C. Johnson, and David R. Corey. "Argonaute binding within human nuclear RNA and its impact on alternative splicing." RNA 27, no. 9 (June 9, 2021): 991–1003. http://dx.doi.org/10.1261/rna.078707.121.
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Mammalian RNA interference (RNAi) is often linked to the regulation of gene expression in the cytoplasm. Synthetic RNAs, however, can also act through the RNAi pathway to regulate transcription and splicing. While nuclear regulation by synthetic RNAs can be robust, a critical unanswered question is whether endogenous functions for nuclear RNAi exist in mammalian cells. Using enhanced crosslinking immunoprecipitation (eCLIP) in combination with RNA sequencing (RNA-seq) and multiple AGO knockout cell lines, we mapped AGO2 protein binding sites within nuclear RNA. The strongest AGO2 binding sites were mapped to micro RNAs (miRNAs). The most abundant miRNAs were distributed similarly between the cytoplasm and nucleus, providing no evidence for mechanisms that facilitate localization of miRNAs in one compartment versus the other. Beyond miRNAs, most statistically significant AGO2 binding was within introns. Splicing changes were confirmed by RT-PCR and recapitulated by synthetic miRNA mimics complementary to the sites of AGO2 binding. These data support the hypothesis that miRNAs can control gene splicing. While nuclear RNAi proteins have the potential to be natural regulatory mechanisms, careful study will be necessary to identify critical RNA drivers of normal physiology and disease.
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Bhinu, V. S., R. Li, J. Huang, S. Kaminskyj, A. Sharpe, and A. Hannoufa. "Perturbation of lignin biosynthesis pathway in Brassica napus (canola) plants using RNAi." Canadian Journal of Plant Science 89, no. 3 (May 1, 2009): 441–53. http://dx.doi.org/10.4141/cjps08164.
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Brassica napus meal contains high levels of lignin, which is one of the most important compositional factors affecting feed utilization by ruminants. We attempted to modify the concentration and composition of lignin in B. napus plants using the RNAi approach. Four genes were targeted for silencing by this approach either independently or in combination; caffeic acid O-methyltransferase (COMT), cinnamate 4-hydroxylase (C4H); coumarate 3-hydroxylase (C3H); ferulic acid 5-hydroxylase (F5H). We successfully developed transgenic B. napus lines expressing CaMV35S:C3H-C4H RNAi, CaMV35S:F5H-COMT RNAi, and Cruciferin:COMT RNAi that contained up to 40% less seed lignin in the transgenic seeds compared to the control. Despite successfully achieving suppression of these lignin biosynthesis genes and reduction in lignin content in B. napus seeds, we observed minor phenotypic effects on the transgenic plants. In lines carrying the cruciferin:COMT RNAi construct we observed a decrease in lignin content (40%) in the seed and anatomical variations when stem sections were examined. While our silencing had no major negative effect on plant growth it resulted in deformation of vessel elements, and minor changes in S-units. Taken together, these results clearly show that by employing RNAi strategy, it is possible to alter seed lignin content and composition in a manner non-detrimental to B. napus plants.Key words: Brassica napus, cruciferin, lignin, COMT, RNAi
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Tzelos, T., J. B. Matthews, B. Whitelaw, and D. P. Knox. "Marker genes for activation of the RNA interference (RNAi) pathway in the free-living nematode Caenorhabditis elegans and RNAi development in the ovine nematode Teladorsagia circumcincta." Journal of Helminthology 89, no. 2 (December 18, 2013): 208–16. http://dx.doi.org/10.1017/s0022149x13000801.
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AbstractThe nematode Teladorsagiacircumcincta is a major cause of parasitic gastroenteritis in sheep in temperate regions. The development of resistance to the major anthelmintic classes used for its control is a threat to small ruminant farming sustainability. Vaccination is a potential alternative control method for this nematode. Gene datasets can be exploited to identify potential vaccine candidates and these validated further by methods such as RNA interference (RNAi) prior to vaccine trials. Previous reports indicate that RNAi in parasitic nematodes is inconsistent and, to date, there are no internal controls that indicate activation of the RNAi pathway in response to double-stranded RNA (dsRNA). The present aims were to determine whether or not the transcription levels of potential marker genes in the RNAi pathway could indicate activation of the pathway in Caenorhabditis elegans and to develop an RNAi platform in T. circumcincta. In C. elegans, transcript levels of three candidate marker genes, Ce-dcr-1 (Dicer), Ce-ego-1 (Enhancer of Glp-One family member) and Ce-rsd-3 (RNAi Spreading Defective), were analysed and results indicated that activation of the pathway had no effect on transcript levels of these genes. In T. circumcincta, two vaccine candidate genes from the Activation-associated Secreted Protein (ASP) family were targets for knockdown. RNAi experiments showed successful silencing of both targets, although inconsistencies in efficacy were observed. After testing a number of parameters that might affect variability, it was found that the length of the storage period of the larvae plays an important role in the consistency of the RNAi results.
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Wang, Fangwei, Naoki Koyama, Hiroko Nishida, Tokuko Haraguchi, Walter Reith, and Toshiro Tsukamoto. "The Assembly and Maintenance of Heterochromatin Initiated by Transgene Repeats Are Independent of the RNA Interference Pathway in Mammalian Cells." Molecular and Cellular Biology 26, no. 11 (June 1, 2006): 4028–40. http://dx.doi.org/10.1128/mcb.02189-05.
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ABSTRACT A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.
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Zhang, Yinqiao, Hu Li, Juan Du, Junzheng Zhang, Jie Shen, and Wanzhi Cai. "Three Melanin Pathway Genes, TH, yellow, and aaNAT, Regulate Pigmentation in the Twin-Spotted Assassin Bug, Platymeris biguttatus (Linnaeus)." International Journal of Molecular Sciences 20, no. 11 (June 3, 2019): 2728. http://dx.doi.org/10.3390/ijms20112728.
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Pigmentation plays a vital role in insect survival and reproduction. Many melanin pathway genes have been studied in holometabolous insects; however, they have only been studied in two hemimetabolous insect genera, Oncopeltus and Periplaneta. Here we analyzed three melanin pathway genes (TH, yellow, and aaNAT) using RNA interference (RNAi) in another hemimetabolous insect, namely the twin-spotted assassin bug, Platymeris biguttatus. TH was highly expressed in freshly molted nymphs and adults. TH RNAi resulted in a complete loss of black pigment, with yellow coloration maintained. Therefore, black pigment in this assassin bug is solely generated from the melanin pathway, whereas yellow pigment is generated from other unknown pigmentation pathways. yellow and aaNAT were highly expressed in the white spot of the hemelytra. Downregulation of yellow caused a brown phenotype with high mortality, indicating an important role of yellow functions in cuticle formation and in the process of converting melanin from brown to black. Interestingly, aaNAT RNAi caused not only loss of white pigment, but also loss of yellow and red pigments. This phenotype of aaNAT has not been reported in other insects. Our results provide new information for understanding the melanin pathway in which aaNAT is essential for the formation of colorless patterns.
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Lingel, Andreas, and Michael Sattler. "Novel modes of protein–RNA recognition in the RNAi pathway." Current Opinion in Structural Biology 15, no. 1 (February 2005): 107–15. http://dx.doi.org/10.1016/j.sbi.2005.01.010.
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Bantounas, I., L. A. Phylactou, and J. B. Uney. "RNA interference and the use of small interfering RNA to study gene function in mammalian systems." Journal of Molecular Endocrinology 33, no. 3 (December 2004): 545–57. http://dx.doi.org/10.1677/jme.1.01582.
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In the past 2 years, extraordinary developments in RNA interference (RNAi)-based methodologies have seen small interfering RNAs (siRNA) become the method of choice for researchers wishing to target specific genes for silencing. In this review, an historic overview of the biochemistry of the RNAi pathway is described together with the latest advances in the RNAi field. Particular emphasis is given to strategies by which siRNAs are used to study mammalian gene function. In this regard, the use of plasmid-based and viral vector-based systems to mediate long-term RNAi in vitro and in vivo are described. However, recent work has shown that non-specific silencing effects and activation of the interferon response may occur following the use of some siRNA and delivery vector combinations. Future goals must therefore be to understand the mechanisms by which siRNA delivery leads to unwanted gene silencing effects in cells and, in this way, RNAi technology can reach its tremendous potential as a scientific tool and ultimately be used for therapeutic purposes.
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Choudhary, Swati, Heng-Chi Lee, Mekhala Maiti, Qun He, Ping Cheng, Qinghua Liu, and Yi Liu. "A Double-Stranded-RNA Response Program Important for RNA Interference Efficiency." Molecular and Cellular Biology 27, no. 11 (March 19, 2007): 3995–4005. http://dx.doi.org/10.1128/mcb.00186-07.
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ABSTRACT When recognized by the RNA interference (RNAi) pathway, double-stranded RNA (dsRNA) produced in eukaryotic cells results in posttranscriptional gene silencing. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that dsRNA, but not short interfering RNA (siRNA), induces the expression of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for normal gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 posttranscriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. Finally, a genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes in Neurospora. Together, our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections.
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Marmonier, Aurélie, Laetitia Perfus-Barbeoch, Corinne Rancurel, Sylvaine Boissinot, Bruno Favery, Gérard Demangeat, and Véronique Brault. "In Vitro Acquisition of Specific Small Interfering RNAs Inhibits the Expression of Some Target Genes in the Plant Ectoparasite Xiphinema index." International Journal of Molecular Sciences 20, no. 13 (July 3, 2019): 3266. http://dx.doi.org/10.3390/ijms20133266.
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Xiphinema index is an important plant parasitic nematode that induces direct damages and specifically transmits the Grapevine fanleaf virus, which is particularly harmful for grapevines. Genomic resources of this nematode species are still limited and no functional gene validation technology is available. RNA interference (RNAi) is a powerful technology to study gene function and here we describe the application of RNAi on several genes in X. index. Soaking the nematodes for 48 h in a suspension containing specific small interfering RNAs resulted in a partial inhibition of the accumulation of some targeted mRNA. However, low reproducible silencing efficiency was observed which could arise from X. index silencing pathway deficiencies. Indeed, essential accustomed proteins for these pathways were not found in the X. index proteome predicted from transcriptomic data. The most reproducible silencing effect was obtained when targeting the piccolo gene potentially involved in endo-exocytosis of synaptic molecules. This represents the first report of gene silencing in a nematode belonging to the Longidoridae family.
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Zhou, Xiao-Long, Gang Chen, Meng-Xue Li, Heng-Xue Wang, Jia-Wei Hong, Jun-Yu Shen, Qi Wang, Xing Ge, Zhen Ding, and Li-Chun Xu. "Targeting YOD1 by RNA Interference Inhibits Proliferation and Migration of Human Oral Keratinocytes through Transforming Growth Factor-β3 Signaling Pathway." BioMed Research International 2018 (September 13, 2018): 1–8. http://dx.doi.org/10.1155/2018/6254308.
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Objective. We have identified a gene YOD1 encoding deubiquitinating enzyme (DUB) responsible for nonsyndromic cleft lip with or without cleft palate (NSCL/P). We aimed to determine the effects of YOD1 RNA interference (RNAi) on cell proliferation and migration, playing an important role in lip and palate formation, and to clarify whether the mechanisms involved TGF-β3 signaling associated with NSCL/P. Methods. RNAi was applied to construct vectors expressing YOD1 small interference RNAs (siRNAs). The vectors were transfected into the human oral keratinocytes (HOK) cells. The cell proliferation and migration were evaluated by the cell counting kit-8 (CCK-8) assay and wound healing assay, respectively. The mRNA levels were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The protein levels were investigated by western blotting. Results. The proliferation of YOD1 siRNA-transfected HOK cells was remarkably inhibited. The migration rate was significantly decreased in the YOD1 siRNA-transfected HOK cells. The TGF-β3 mRNA and protein levels were decreased significantly by siRNA-mediated knockdown of YOD1. YOD1 RNAi reduced the phosphor-Smad2/3 levels significantly. Conclusions. YOD1 RNAi may inhibit cell proliferation and migration associated with the pathogenesis of NSCL/P through TGF-β3 signaling. The study indicates a novel role of YOD1 in regulating TGF-β3 signaling to affect cell proliferation and migration resulting in NSCL/P.
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Shi, Huafang, Nathalie Chamond, Christian Tschudi, and Elisabetta Ullu. "Selection and Characterization of RNA Interference-Deficient Trypanosomes Impaired in Target mRNA Degradation." Eukaryotic Cell 3, no. 6 (December 2004): 1445–53. http://dx.doi.org/10.1128/ec.3.6.1445-1453.2004.
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ABSTRACT Genetic analysis of the RNA interference (RNAi) pathway in Trypanosoma brucei has so far revealed one essential component, namely, TbAGO1, encoding a member of the Argonaute protein family. To gain further insight into the RNAi mechanism and its biological significance, we selected RNAi-deficient trypanosomes by using repeated cycles of electroporation with α-tubulin double-stranded RNA, a treatment that blocks cytokinesis in wild-type cells. Two independent clones, termed RiD-1 (for RNAi-deficient clone 1) and RiD-2, were characterized. At the cellular level, only RiD-1 trypanosomes showed a significant increase in doubling time with the concomitant accumulation of cells defective in the completion of cytokinesis. At the RNA level, both clones accumulated wild-type amounts of small interfering RNAs and displayed elevated levels of retroposon transcripts, the hallmark of RNAi deficiency in T. brucei. Importantly, both RiD-1 and RiD-2 clones were defective in the degradation of target mRNA, suggesting an impairment of the activity of AGO1, the putative RNAi endonuclease. Since in RiD cells the AGO1 gene was not mutated and was expressed at wild-type levels, we propose that in trypanosomes the cleavage of mRNA by AGO1 is regulated by the interaction with another factor(s).
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Dunoyer, Patrice, Christopher A. Brosnan, Gregory Schott, Yu Wang, Florence Jay, Abdelmalek Alioua, Christophe Himber, and Olivier Voinnet. "An endogenous, systemic RNAi pathway in plants." EMBO Journal 29, no. 10 (April 22, 2010): 1699–712. http://dx.doi.org/10.1038/emboj.2010.65.
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Azimzadeh Jamalkandi, Sadegh, and Ali Masoudi-Nejad. "RNAi pathway integration in Caenorhabditis elegans development." Functional & Integrative Genomics 11, no. 3 (July 22, 2011): 389–405. http://dx.doi.org/10.1007/s10142-011-0236-1.
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Sarkies, Peter, and Eric A. Miska. "RNAi pathways in the recognition of foreign RNA: antiviral responses and host–parasite interactions in nematodes." Biochemical Society Transactions 41, no. 4 (July 18, 2013): 876–80. http://dx.doi.org/10.1042/bst20130021.
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The nematode Caenorhabditis elegans was the first animal for which RNAi (RNA interference) in response to exogenous triggers was shown experimentally and subsequently the molecular components of the RNAi pathway have been characterized in some detail. However, the function of RNAi in the life cycle of nematodes in the wild is still unclear. In the present article, we argue that RNAi could be used in nematodes as a mechanism to sense and respond to foreign RNA that the animal might be exposed to either through viral infection or through ingestion of food sources. This could be of potential importance to the life cycle of parasitic nematodes as they ingest RNA from different hosts at different points during their life cycle. We postulate that RNA ingested from the host could be used by the parasite to regulate its own genes, through the amplification mechanism intrinsic to the nematode RNAi pathway.
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Lim, S., E. Tsend-Ayush, R. Kortschak, C. Ricciardelli, M. Oehler, and F. Grutzner. "144. MAELSTROM - A PROTEIN THAT IS ESSENTIAL FOR SPERMATOGENESIS AND TRANSPOSABLE REPRESSION IS EXPRESSED IN ADULT OVARY OF MAMMALS AND BIRDS." Reproduction, Fertility and Development 22, no. 9 (2010): 62. http://dx.doi.org/10.1071/srb10abs144.
Full textAbstract:
Maelstrom (MAEL) is a highly evolutionarily conserved protein located at the perinuclear structure of animal germ cells called nuage. The MAEL protein contains HMG and Tudor domains and associates with components of the piRNA and RNAi pathways and chromatin remodelling factors. Recent work has shown that MAEL is required for the differentiation of the germ-line stem cell lineage and for the retroposon repression. In mouse, Mael is expressed in male germ cells and is essential for spermatogenesis and retroposon suppression. We have investigated the evolution of the Mael gene in mammals and birds. As expected the gene is highly conserved in all three mammalian lineages and in chicken. Interestingly, the platypus MAEL has exclusive changes in the DnaQ-H 3’-5’ exonuclease domain and computational modelling suggested that these changes may affect the folding of the protein. Expression analysis revealed that the Mael gene is transcribed in testis but also in adult ovaries of chicken, platypus, mouse and human. In situ hybridisation of the Mael transcript on ovary sections of mouse and platypus shows that gene expression is found in pre-antral and antral follicles. The data so far also showed some differences in the expression pattern between mouse and platypus. In mouse, we detected transcript in oocyte, granulosa cells and cumulus cells whereas in the platypus we only observed expression in oocyte. Earlier work demonstrated that Drosophila Mael mutant ovaries had mislocalisation of the RNAi pathway proteins, Dicer and Argonaute2. It is well known that RNAi pathyway is involved in the repression of transposon in the testis and ovary across animal kingdom. As a key component of the RNAi pathway, MAEL is reported to co-localise and interact with MILI and MIWI proteins. These finding may suggest a role of MAEL in retroposon control in ovary and folliculogenesis.
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JARONCZYK, Katarzyna, Jon B. CARMICHAEL, and Tom C. HOBMAN. "Exploring the functions of RNA interference pathway proteins: some functions are more RISCy than others?" Biochemical Journal 387, no. 3 (April 26, 2005): 561–71. http://dx.doi.org/10.1042/bj20041822.
Full textAbstract:
PPD (PAZ Piwi domain) proteins and the Dicer family have been the subjects of intense study over the last 6 years. These proteins have well-established roles in RNAi (RNA interference), a process that relies on siRNAs (small interfering RNAs) or miRNAs (microRNAs) to mediate specificity. The development of techniques for applying RNAi as a laboratory tool and a molecular therapeutic technique has rapidly outpaced our understanding of the biology of this process. However, over the last 2 years, great strides have been made towards elucidating how PPD proteins and Dicer regulate gene-silencing at the pre- and post-transcriptional levels. In addition, evidence is beginning to emerge that suggests that these proteins have additional siRNA-independent roles as cell-cycle regulators. In the present review, we summarize the well-known roles of these two classes of proteins in gene-silencing pathways, as well as explore the evidence for novel roles of PPD and Dicer proteins.
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Ugolini, Ilaria, and Mario Halic. "Fidelity in RNA-based recognition of transposable elements." Philosophical Transactions of the Royal Society B: Biological Sciences 373, no. 1762 (November 5, 2018): 20180168. http://dx.doi.org/10.1098/rstb.2018.0168.
Full textAbstract:
Genomes are under constant threat of invasion by transposable elements and other genomic parasites. How can host genomes recognize these elements and target them for degradation? This requires a system that is highly adaptable, and at the same time highly specific. Current data suggest that perturbation of transcription patterns by transposon insertions could be detected by the RNAi surveillance pathway. Multiple transposon insertions might generate sufficient amounts of primal small RNAs to initiate generation of secondary small RNAs and silencing. At the same time primal small RNAs need to be constantly degraded to reduce the level of noise small RNAs below the threshold required for initiation of silencing. Failure in RNA degradation results in loss of fidelity of small RNA pathways and silencing of ectopic targets. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.
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Schuster, Susan, Pascal Miesen, and Ronald P. van Rij. "Antiviral RNAi in Insects and Mammals: Parallels and Differences." Viruses 11, no. 5 (May 16, 2019): 448. http://dx.doi.org/10.3390/v11050448.
Full textAbstract:
The RNA interference (RNAi) pathway is a potent antiviral defense mechanism in plants and invertebrates, in response to which viruses evolved suppressors of RNAi. In mammals, the first line of defense is mediated by the type I interferon system (IFN); however, the degree to which RNAi contributes to antiviral defense is still not completely understood. Recent work suggests that antiviral RNAi is active in undifferentiated stem cells and that antiviral RNAi can be uncovered in differentiated cells in which the IFN system is inactive or in infections with viruses lacking putative viral suppressors of RNAi. In this review, we describe the mechanism of RNAi and its antiviral functions in insects and mammals. We draw parallels and highlight differences between (antiviral) RNAi in these classes of animals and discuss open questions for future research.
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Garcia, Stephan, Agnès Billecocq, Jean-Marc Crance, Marcel Prins, Daniel Garin, and Michèle Bouloy. "Viral suppressors of RNA interference impair RNA silencing induced by a Semliki Forest virus replicon in tick cells." Journal of General Virology 87, no. 7 (July 1, 2006): 1985–89. http://dx.doi.org/10.1099/vir.0.81827-0.
Full textAbstract:
It was recently shown that infection of ISE6 tick cells by a recombinant Semliki Forest virus (SFV) expressing a heterologous gene induced small interfering RNAs (siRNAs) and silencing of the gene. To gain information on RNA interference (RNAi) in ticks, three known viral inhibitors that act in different ways, the NS1 protein of Influenza virus, NSs of Tospovirus Tomato spotted wilt virus and HC-Pro of Zucchini yellow mosaic virus were expressed and investigated to determine if they antagonize induced RNAi. Using the recombinant SFV replicon expressing firefly luciferase, silencing was induced and the suppressor activity of these inhibitors during or after initiation of siRNA synthesis was tested, to determine which step of the RNAi pathway is impaired. It was found that these proteins, identified in mammalian or plant systems, also display activity in tick cells. These data suggest that ticks utilize a mechanism similar to the one found in other eukaryotes.