Artykuły w czasopismach: „RNAi-INDUCED TRANSCRIPTIONAL SILENCING”

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Swaminathan, Sanjay, Chantelle L. Hood, Kazuo Suzuki i Anthony D. Kelleher. "RNA duplexes in transcriptional regulation". BioMolecular Concepts 1, nr 3-4 (1.10.2010): 285–96. http://dx.doi.org/10.1515/bmc.2010.021.

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AbstractTranscriptional regulation by small RNA molecules, including small interfering RNA and microRNA, has emerged as an important gene expression modulator. The regulatory pathways controlling gene expression, post-transcriptional gene silencing and transcriptional gene silencing (TGS) have been demonstrated in yeast, plants and more recently in human cells. In this review, we discuss the currents models of transcriptional regulation and the main components of the RNA-induced silencing complex and RNA-induced transcriptional silencing complex machinery, as well as confounding off-target effects and gene activation. We also discuss RNA-mediated TGS within the NF-κB motif of the human immunodeficiency virus type 1 5′ long tandem repeat promoter region and the associated epigenetic modifications. Finally, we outline the current RNA interference (RNAi) delivery methods and describe the current status of human trials investigating potential RNAi therapeutics for several human diseases.

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Abdellatef, Eltayb, Nasrein Mohamed Kamal i Hisashi Tsujimoto. "Tuning Beforehand: A Foresight on RNA Interference (RNAi) and In Vitro-Derived dsRNAs to Enhance Crop Resilience to Biotic and Abiotic Stresses". International Journal of Molecular Sciences 22, nr 14 (19.07.2021): 7687. http://dx.doi.org/10.3390/ijms22147687.

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Crop yield is severely affected by biotic and abiotic stresses. Plants adapt to these stresses mainly through gene expression reprogramming at the transcriptional and post-transcriptional levels. Recently, the exogenous application of double-stranded RNAs (dsRNAs) and RNA interference (RNAi) technology has emerged as a sustainable and publicly acceptable alternative to genetic transformation, hence, small RNAs (micro-RNAs and small interfering RNAs) have an important role in combating biotic and abiotic stresses in plants. RNAi limits the transcript level by either suppressing transcription (transcriptional gene silencing) or activating sequence-specific RNA degradation (post-transcriptional gene silencing). Using RNAi tools and their respective targets in abiotic stress responses in many crops is well documented. Many miRNAs families are reported in plant tolerance response or adaptation to drought, salinity, and temperature stresses. In biotic stress, the spray-induced gene silencing (SIGS) provides an intelligent method of using dsRNA as a trigger to silence target genes in pests and pathogens without producing side effects such as those caused by chemical pesticides. In this review, we focus on the potential of SIGS as the most recent application of RNAi in agriculture and point out the trends, challenges, and risks of production technologies. Additionally, we provide insights into the potential applications of exogenous RNAi against biotic stresses. We also review the current status of RNAi/miRNA tools and their respective targets on abiotic stress and the most common responsive miRNA families triggered by stress conditions in different crop species.

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Zhang, Junlong. "Special delivery: Small RNAs silencing gene expression". Biochemist 26, nr 5 (1.10.2004): 20–23. http://dx.doi.org/10.1042/bio02605020.

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RNA interference (RNAi), which refers to RNA-induced transcriptional gene silencing, is a natural phenomenon that exists widely in living organisms. Recent advances in RNAi research indicate that RNAi technology is a powerful tool in studying gene function and has a great potential in gene therapy. Although many methods, including viral and non-viral vectors, have been used to deliver small interference RNA molecules into cells and animals, development of better delivery methods is still crucial for the application of RNAi technology in both basic research and gene therapy.

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Das, Panchashree, i Satyabrata Nanda. "Host-delivered-RNAi-mediated resistance in bananas against biotic stresses". Journal of Experimental Biology and Agricultural Sciences 10, nr 5 (31.10.2022): 953–59. http://dx.doi.org/10.18006/2022.10(5).953.959.

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Both the biotic and abiotic stressors restrict the yield potential of many crops, including bananas. Bananas belong to the genus Musa and are the world’s most popular and widely produced fruit for their nutritional and industrial importance. The demand for bananas is growing each day worldwide. However, different pest infestations are hampering the production of bananas, making it a matter of concern for global food security. Several biotechnological tools and applications including RNA interference (RNAi) have been employed to enhance the biotic stress resistance in plants. The capacity to silence targeted genes at transcriptional and post-transcriptional levels makes the RNAi technique a popular choice for gene knock-down and functional genomics studies in crops. Silencing of different suppressor molecule coding genes through RNAi helps crops to combat the detrimental effects of plant pathogens. The host-induced gene silencing (HIGS) technology, also known as the host-delivered RNAi (HD-RNAi), is nowadays gaining popularity due to its ability to target an array of pathogens, comprising bacteria, nematodes, fungi, viruses, and insects. This methodology is employed to manage disease pest outbreaks in a diverse range of crop species, including bananas. Besides HIGS, virus-induced and spray-induced gene silencing (VIGS and SIGS, respectively) are the potential approaches where RNAi technology is exploited to control plant-pathogenic diseases. The current review emphasizes the different kinds of diseases of bananas and the potential of HD-RNAi, a new-age and promising technology to build a barrier against significant crop and economic loss.

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Kajitani, Takuya, Hiroaki Kato, Yuji Chikashige, Chihiro Tsutsumi, Yasushi Hiraoka, Hiroshi Kimura, Yasuyuki Ohkawa, Chikashi Obuse, Damien Hermand i Yota Murakami. "Ser7 of RNAPII-CTD facilitates heterochromatin formation by linking ncRNA to RNAi". Proceedings of the National Academy of Sciences 114, nr 52 (13.12.2017): E11208—E11217. http://dx.doi.org/10.1073/pnas.1714579115.

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Some long noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (RNAPII) are retained on chromatin, where they regulate RNAi and chromatin structure. The molecular basis of this retention remains unknown. We show that in fission yeast serine 7 (Ser7) of the C-terminal domain (CTD) of RNAPII is required for efficient siRNA generation for RNAi-dependent heterochromatin formation. Surprisingly, Ser7 facilitates chromatin retention of nascent heterochromatic RNAs (hRNAs). Chromatin retention of hRNAs and siRNA generation requires both Ser7 and an RNA-binding activity of the chromodomain of Chp1, a subunit of the RNA-induced transcriptional silencing (RITS) complex. Furthermore, RITS associates with RNAPII in a Ser7-dependent manner. We propose that Ser7 promotes cotranscriptional chromatin retention of hRNA by recruiting the RNA-chromatin connector protein Chp1, which facilitates RNAi-dependent heterochromatin formation. Our findings reveal a function of the CTD code: linking ncRNA transcription to RNAi for heterochromatin formation.

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Suprun, Andrey R., Konstantin V. Kiselev i Alexandra S. Dubrovina. "Exogenously Induced Silencing of Four MYB Transcription Repressor Genes and Activation of Anthocyanin Accumulation in Solanum lycopersicum". International Journal of Molecular Sciences 24, nr 11 (26.05.2023): 9344. http://dx.doi.org/10.3390/ijms24119344.

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RNA interference (RNAi) is a natural post-transcriptional regulatory mechanism that can be artificially induced by exogenous application of double-stranded RNAs (dsRNAs) to the plant surfaces. Recent studies show that it is possible to silence plant genes and change plant properties using plant RNA spraying and other approaches for dsRNA delivery. In this study, we investigated the effect of exogenous gene-specific dsRNAs on the silencing of four tomato genes encoding MYB-family transcription repressors of anthocyanin biosynthesis in the leaves of tomato Solanum lycopersicum L. We found that the exogenous application of dsRNAs encoding for the SlMYBATV1, SlMYB32, SlMYB76, and SlTRY genes downregulated mRNA levels of these endogenous repressors of anthocyanin production, upregulated the expression of anthocyanin biosynthesis-related genes, and enhanced anthocyanin content in the leaves of S. lycopersicum. The data demonstrated that exogenous gene-specific dsRNAs can induce post-transcriptional gene silencing in tomato leaves by direct foliar application of dsRNAs. This approach may be used for plant secondary metabolism induction and as a silencing tool for gene function studies without the need to produce genetically modified plants.

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Mao, Kai, Peter Breen i Gary Ruvkun. "Mitochondrial dysfunction induces RNA interference in C. elegans through a pathway homologous to the mammalian RIG-I antiviral response". PLOS Biology 18, nr 12 (2.12.2020): 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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Buck, Amy H., i Mark Blaxter. "Functional diversification of Argonautes in nematodes: an expanding universe". Biochemical Society Transactions 41, nr 4 (18.07.2013): 881–86. http://dx.doi.org/10.1042/bst20130086.

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In the last decade, many diverse RNAi (RNA interference) pathways have been discovered that mediate gene silencing at epigenetic, transcriptional and post-transcriptional levels. The diversity of RNAi pathways is inherently linked to the evolution of Ago (Argonaute) proteins, the central protein component of RISCs (RNA-induced silencing complexes). An increasing number of diverse Agos have been identified in different species. The functions of most of these proteins are not yet known, but they are generally assumed to play roles in development, genome stability and/or protection against viruses. Recent research in the nematode Caenorhabditis elegans has expanded the breadth of RNAi functions to include transgenerational epigenetic memory and, possibly, environmental sensing. These functions are inherently linked to the production of secondary siRNAs (small interfering RNAs) that bind to members of a clade of WAGOs (worm-specific Agos). In the present article, we review briefly what is known about the evolution and function of Ago proteins in eukaryotes, including the expansion of WAGOs in nematodes. We postulate that the rapid evolution of WAGOs enables the exceptional functional plasticity of nematodes, including their capacity for parasitism.

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Andika, Ida Bagus, Hideki Kondo i Nobuhiro Suzuki. "Dicer functions transcriptionally and posttranscriptionally in a multilayer antiviral defense". Proceedings of the National Academy of Sciences 116, nr 6 (23.01.2019): 2274–81. http://dx.doi.org/10.1073/pnas.1812407116.

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In antiviral RNA interference (RNAi), Dicer plays a primary role in processing double-stranded RNA (dsRNA) molecules into small-interfering RNAs (siRNAs) that guide Argonaute effectors to posttranscriptional suppression of target viral genes. Here, we show a distinct role for Dicer in the siRNA-independent transcriptional induction of certain host genes upon viral infection in a filamentous fungus. Previous studies have shown that the two key players, dicer-like 2 (dcl2) and argonaute-like 2 (agl2), of antiviral RNAi in a phytopathogenic ascomycete,Cryphonectria parasitica, are highly transcriptionally induced upon infection with certain RNA mycoviruses, including the positive-stranded RNA hypovirus mutant lacking the RNAi suppressor (Cryphonectriahypovirus 1-Δp69, CHV1-Δp69). This induction is regulated by the Spt–Ada–Gcn5 acetyltransferase (SAGA) complex, a well-known transcriptional coactivator. The present study shows that diverse host genes, in addition todcl2andagl2, were up-regulated more than 10-fold by SAGA upon infection with CHV1-Δp69. Interestingly, DCL2, but not AGL2, was essential for SAGA-mediated global gene up-regulation. Moreover, deletion of certain virus-induced genes enhanced a CHV1-Δp69 symptom (growth rate) but not its accumulation. Constitutive, modest levels ofdcl2expression drastically reduced viral siRNA accumulation but were sufficient for full-scale up-regulation of host genes, suggesting that high induction ofdcl2and siRNA production are not essential for the transcriptional up-regulation function of DCL2. These data clearly demonstrate the dual functionality of DCL2: as a dsRNA-specific nuclease in posttranscriptional antiviral RNA silencing and as a key player in SAGA-mediated host gene induction, which independently represses viral replication and alleviates virus-induced symptom expression.

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Betti, Federico, Maria José Ladera-Carmona, Pierdomenico Perata i Elena Loreti. "RNAi Mediated Hypoxia Stress Tolerance in Plants". International Journal of Molecular Sciences 21, nr 24 (10.12.2020): 9394. http://dx.doi.org/10.3390/ijms21249394.

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Small RNAs regulate various biological process involved in genome stability, development, and adaptive responses to biotic or abiotic stresses. Small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs). MicroRNAs (miRNAs) are regulators of gene expression that affect the transcriptional and post-transcriptional regulation in plants and animals through RNA interference (RNAi). miRNAs are endogenous small RNAs that originate from the processing of non-coding primary miRNA transcripts folding into hairpin-like structures. The mature miRNAs are incorporated into the RNA-induced silencing complex (RISC) and drive the Argonaute (AGO) proteins towards their mRNA targets. siRNAs are generated from a double-stranded RNA (dsRNA) of cellular or exogenous origin. siRNAs are also involved in the adaptive response to biotic or abiotic stresses. The response of plants to hypoxia includes a genome-wide transcription reprogramming. However, little is known about the involvement of RNA signaling in gene regulation under low oxygen availability. Interestingly, miRNAs have been shown to play a role in the responses to hypoxia in animals, and recent evidence suggests that hypoxia modulates the expression of various miRNAs in plant systems. In this review, we describe recent discoveries on the impact of RNAi on plant responses to hypoxic stress in plants.

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Nie, Jing, Nan Shan, Huan Liu, Xuehui Yao, Ziwei Wang, Ruoxue Bai, Yicong Guo, Ying Duan, Changlin Wang i Xiaolei Sui. "Transcriptional control of local auxin distribution by the CsDFB1-CsPHB module regulates floral organogenesis in cucumber". Proceedings of the National Academy of Sciences 118, nr 8 (18.02.2021): e2023942118. http://dx.doi.org/10.1073/pnas.2023942118.

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Plant cystatins are cysteine proteinase inhibitors that play key roles in defense responses. In this work, we describe an unexpected role for the cystatin-like protein DEFORMED FLORAL BUD1 (CsDFB1) as a transcriptional regulator of local auxin distribution in cucumber (Cucumis sativus L.). CsDFB1 was strongly expressed in the floral meristems, floral primordia, and vasculature. RNA interference (RNAi)-mediated silencing of CsDFB1 led to a significantly increased number of floral organs and vascular bundles, together with a pronounced accumulation of auxin. Conversely, accompanied by a decrease of auxin, overexpression of CsDFB1 resulted in a dramatic reduction in floral organ number and an obvious defect in vascular patterning, as well as organ fusion. CsDFB1 physically interacted with the cucumber ortholog of PHABULOSA (CsPHB), an HD-ZIP III transcription factor whose transcripts exhibit the same pattern as CsDFB1. Overexpression of CsPHB increased auxin accumulation in shoot tips and induced a floral phenotype similar to that of CsDFB1-RNAi lines. Furthermore, genetic and biochemical analyses revealed that CsDFB1 impairs CsPHB-mediated transcriptional regulation of the auxin biosynthetic gene YUCCA2 and the auxin efflux carrier PIN-FORMED1, and thus plays a pivotal role in auxin distribution. In summary, we propose that the CsDFB1-CsPHB module represents a regulatory pathway for local auxin distribution that governs floral organogenesis and vascular differentiation in cucumber.

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Shen, Jia Z., i Charles Spruck. "Targeting FBXO44/SUV39H1 elicits tumor cell-specific DNA replication stress and viral mimicry". Cell Stress 5, nr 3 (8.03.2021): 37–39. http://dx.doi.org/10.15698/cst2021.03.245.

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Repetitive elements (REs) are normally transcriptionally silenced in somatic cells by repressive epigenetic modifications, which are thought to include DNA methylation and histone modifications such as deacetylation, H3K9me3, and H4K20me3. Although, it is unclear how RE silencing is maintained through DNA replication cycles in rapidly growing cancer cells. On the other hand, the reactivation of endogenous retroelements beyond a threshold level of tolerance in cancer cells, such as by treatment with DNA demethylating agents or HDAC or LSD1 inhibitors, can induce viral mimicry responses that augment certain cancer therapies, including immunotherapy. However, these agents can also affect normal cells presenting obvious side effects. Therefore, uncovering cancer cell-specific RE silencing mechanisms could provide a basis for the development of a new generation of cancer immunotherapy drugs. In our study (Shen et al. (2020), Cell, doi: 10.1016/j.cell.2020.11.042), through a high-content RNAi screen we identified FBXO44 as a key regulator of H3K9me3-mediated transcriptional silencing of REs in cancer cells. Inhibition of FBXO44 or its co-factor SUV39H1 stimulated antiviral pathways and interferon (IFN) signaling and induced replication stress and DNA double-strand breaks (DSBs) in cancer cells, leading to restricted tumor growth and synergy with anti-PD-1 therapy (Figure 1).

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Mendes, Antonio M., Parfait H. Awono-Ambene, Sandrine E. Nsango, Anna Cohuet, Didier Fontenille, Fotis C. Kafatos, George K. Christophides, Isabelle Morlais i Dina Vlachou. "Infection Intensity-Dependent Responses of Anopheles gambiae to the African Malaria Parasite Plasmodium falciparum". Infection and Immunity 79, nr 11 (15.08.2011): 4708–15. http://dx.doi.org/10.1128/iai.05647-11.

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ABSTRACTMalaria remains a devastating disease despite efforts at control and prevention. Extensive studies using mostly rodent infection models reveal that successfulPlasmodiumparasite transmission by the African mosquito vectorAnopheles gambiaedepends on finely tuned vector-parasite interactions. Here we investigate the transcriptional response ofA. gambiaeto geographically relatedPlasmodium falciparumpopulations at various infection intensities and different infection stages. These responses are compared with those of mosquitoes infected with the rodent parasitePlasmodium berghei. We demonstrate that mosquito responses are largely dependent on the intensity of infection. A major transcriptional suppression of genes involved in the regulation of midgut homeostasis is detected in low-intensityP. falciparuminfections, the most common type of infection in Africa. Importantly, genes transcriptionally induced during these infections tend to be phylogenetically unique toA. gambiae. These data suggest that coadaptation between vectors and parasites may act to minimize the impact of infection on mosquito fitness by selectively suppressing specific functional classes of genes. RNA interference (RNAi)-mediated gene silencing provides initial evidence for important roles of the mosquito G protein-coupled receptors (GPCRs) in controlling infection intensity-dependent antiparasitic responses.

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Upadhyay, Udita, Suchita Srivastava, Indu Khatri, Jagpreet Singh Nanda, Srikrishna Subramanian, Amit Arora i Jagmohan Singh. "Ablation of RNA interference and retrotransposons accompany acquisition and evolution of transposases to heterochromatin protein CENPB". Molecular Biology of the Cell 28, nr 8 (15.04.2017): 1132–46. http://dx.doi.org/10.1091/mbc.e16-07-0485.

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Inactivation of retrotransposons is accompanied by the emergence of centromere-binding protein-B (CENPB) in Schizosaccharomyces, as well as in metazoans. The RNA interference (RNAi)-induced transcriptional silencing (RITS) complex, comprising chromodomain protein-1 (Chp1), Tas3 (protein with unknown function), and Argonaute (Ago1), plays an important role in RNAi-mediated heterochromatinization. We find that whereas the Ago1 subunit of the RITS complex is highly conserved, Tas3 is lost and Chp1 is truncated in Schizosaccharomyces cryophilus and Schizosaccharomyces octosporus. We show that truncated Chp1 loses the property of heterochromatin localization and silencing when transformed in Schizosaccharomyces pombe. Furthermore, multiple copies of CENPB, related to Tc1/mariner and Tc5 transposons, occur in all Schizosaccharomyces species, as well as in humans, but with loss of transposase function (except Schizosaccharomyces japonicus). We propose that acquisition of Tc1/mariner and Tc5 elements by horizontal transfer in S. pombe (and humans) is accompanied by alteration of their function from a transposase/endonuclease to a heterochromatin protein, designed to suppress transposon expression and recombination. The resulting redundancy of RITS may have eased the selection pressure, resulting in progressive loss or truncation of tas3 and chp1 genes in S. octosporus and S. cryophilus and triggered similar evolutionary dynamics in the metazoan orthologues.

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Shivakumara, Tagginahalli N., Pradeep K. Papolu, Tushar K. Dutta, Divya Kamaraju, Sonam Chaudhary i Uma Rao. "RNAi-induced silencing of an effector confers transcriptional oscillation in another group of effectors in the root-knot nematode, Meloidogyne incognita". Nematology 18, nr 7 (2016): 857–70. http://dx.doi.org/10.1163/15685411-00003003.

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The sophisticated parasitic tactic of sedentary endoparasitic nematodes seems to involve the simultaneous alteration of the expression of multitude of its effector genes in order to hijack the plant metabolic and developmental pathway. In concordance with this hypothesis, we have targeted some candidate effector genes of Meloidogyne incognita to understand the possible interaction among those effectors for successful infection of the host plant. In vitro RNAi strategy was used to knock down M. incognita-specific pioneer effector genes, such as msp-18, msp-20, msp-24, msp-33 and msp-16 (known to interact with plant transcription factor), to investigate their possible effect on the expression of key cell wall-degrading enzymes (CWDE) and vice versa. Supported by the phenotypic data, intriguingly our study revealed that induced suppression of these pioneer genes cause transcriptional alteration of CWDE genes in M. incognita. This remarkable finding may provide some useful links for future research on nematode effector interaction.

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Tiengwe, Calvin, Abigail E. N. A. Brown i James D. Bangs. "Unfolded Protein Response Pathways in Bloodstream-Form Trypanosoma brucei?" Eukaryotic Cell 14, nr 11 (28.08.2015): 1094–101. http://dx.doi.org/10.1128/ec.00118-15.

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ABSTRACT The unfolded protein response (UPR) is a stress mechanism to cope with misfolded proteins in the early secretory pathway, the hallmark being transcriptional upregulation of endoplasmic reticulum (ER) molecular chaperones such as BiP and protein disulfide isomerase. Despite the lack of transcriptional regulation and the absence of the classical UPR machinery, African trypanosomes apparently respond to persistent ER stress by a UPR-like response, including upregulation of BiP, and a related spliced leader silencing (SLS) response whereby SL RNA transcription is shut down. Initially observed by knockdown of the secretory protein translocation machinery, both responses are also induced by chemical agents known to elicit UPR in mammalian cells (H. Goldshmidt, D. Matas, A. Kabi, A. Carmi, R. Hope, S. Michaeli, PLoS Pathog 6:e1000731, 2010, http://dx.doi.org/10.1371/journal.ppat.1000731 ). As these findings were generated primarily in procyclic-stage trypanosomes, we have investigated both responses in pathogenic bloodstream-stage parasites. RNA interference (RNAi) silencing of the core translocon subunit Trypanosoma brucei Sec61α (TbSec61α) failed to induce either response. Interestingly, cell growth halted within 16 h of silencing, but sufficient TbSec61α remained to allow full competence for translocation of nascent secretory proteins for up to 24 h, indicating that replication is finely coupled with the capacity to synthesize and transport secretory cargo. Tunicamycin and thapsigargin at concentrations compatible with short-term (4 h) and long-term (24 h) viability also failed to induce any of the indicators of UPR-like or SLS responses. Dithiothreitol (DTT) was lethal at all concentrations tested. These results indicate that UPR-like and SLS responses to persistent ER stress do not occur in bloodstream-stage trypanosomes.

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Crowder, Chun, Øyvind Dahle, R. Eric Davis, Odd S. Gabrielsen i Stuart Rudikoff. "PML mediates IFN-α–induced apoptosis in myeloma by regulating TRAIL induction". Blood 105, nr 3 (1.02.2005): 1280–87. http://dx.doi.org/10.1182/blood-2004-04-1614.

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AbstractInterferon (IFN) induces expression of proapoptotic genes and has been used in the clinical treatment of multiple myeloma. The promyelocytic leukemia (PML) gene is an IFN-induced target that encodes a tumor suppressor protein. PML protein is typically localized within discrete speckled nuclear structures termed PML nuclear bodies (NBs). Multiple myeloma cells demonstrate differential responses to IFN treatment, the mechanism of which is largely unknown. Herein, we show that growth inhibition effects of IFN-α in myeloma cells correlate with PML NBs and tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) induction, whereas known IFN targets including signal transducer and activator of transcription-1 (STAT1), STAT3, p38, and Daxx cannot account for these differential responses. RNAi silencing of PML blocks IFN-α–induced apoptosis in myeloma cells and correspondingly down-regulates TRAIL expression. Similarly, stable expression of a dominant negative TRAIL receptor DR5 partially blocks IFN-induced cell death. These results demonstrate that PML and TRAIL play important roles in IFN-induced apoptosis and identify TRAIL as a novel downstream transcriptional target of PML. Identification of PML and PML NBs as effectors of IFN responses provides insights into mechanisms by which tumor cells exhibit resistance to this class of agents and may prove useful in assessing treatment regimens.

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Chicas, Agustin, Emma C. Forrest, Silvia Sepich, Carlo Cogoni i Giuseppe Macino. "Small Interfering RNAs That Trigger Posttranscriptional Gene Silencing Are Not Required for the Histone H3 Lys9 Methylation Necessary for Transgenic Tandem Repeat Stabilization in Neurospora crassa". Molecular and Cellular Biology 25, nr 9 (1.05.2005): 3793–801. http://dx.doi.org/10.1128/mcb.25.9.3793-3801.2005.

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ABSTRACT In Neurospora crassa, the introduction of a transgene can lead to small interfering RNA (siRNA)-mediated posttranscriptional gene silencing (PTGS) of homologous genes. siRNAs can also guide locus-specific methylation of Lys9 of histone H3 (Lys9H3) in Schizosaccharomyces pombe. Here we tested the hypothesis that transgenically derived siRNAs may contemporaneously both activate the PTGS mechanism and induce chromatin modifications at the transgene and the homologous endogenous gene. We carried out chromatin immunoprecipitation using a previously characterized albino-1 (al-1) silenced strain but detected no alterations in the pattern of histone modifications at the endogenous al-1 locus, suggesting that siRNAs produced from the transgenic locus do not trigger modifications in trans of those histones tested. Instead, we found that the transgenic locus was hypermethylated at Lys9H3 in our silenced strain and remained hypermethylated in the quelling defective mutants (qde), further demonstrating that the PTGS machinery is dispensable for Lys9H3 methylation. However, we found that a mutant in the histone Lys9H3 methyltransferase dim-5 was unable to maintain PTGS, with transgenic copies being rapidly lost, resulting in reversion of the silenced phenotype. These results indicate that the defect in PTGS of the Δdim-5 strain is due to the inability to maintain the transgene in tandem, suggesting a role for DIM-5 in stabilizing such repeated sequences. We conclude that in Neurospora, siRNAs produced from the transgenic locus are used in the RNA-induced silencing complex-mediated PTGS pathway and do not communicate with an RNAi-induced initiation of transcriptional gene silencing complex to effect chromatin-based silencing.

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Gong, Yuping, Ling Gu i Ting Liu. "Study on RNA Interference Reversing Multidrug Resistance in K562/A02 Resistant Leukemia Cell." Blood 108, nr 11 (16.11.2006): 4362. http://dx.doi.org/10.1182/blood.v108.11.4362.4362.

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Abstract Multidrug-resistance (MDR) is a major hindrance to successful chemotherapy. Among different mechanisms responsible for MDR, cellular over-expression of P-glycoprotein (Pgp) plays an important role. Pgp is an energy dependent drug efflux pump that effectively transports a broad of commonly used chemotherapeutic drug out of the cells and leads to resistance to the chemotherapeutic drugs. Use of modulators is main approach to overcome MDR. Although, in vitro and in vivo preclinical studies have yielded encouraging results, the clinical reversal of resistance by common modulators such as verapamil, quinine, and cyclosporine A has been difficult due to the toxic side effects. Antisense technology offers an alternative approach to overcome MDR. RNA interference (RNAi) is the best one of all antisense technologies. RNAi acts through a post-transcriptional targeting of mRNA for degradation, resulting in sequence-specific post-transcriptional gene silencing. Short hairpin RNA (shRNA) vectors can be transfected into the cells to stably express siRNA. In our preliminary study, eukaryotic shRNA expression vectors aimed at mdr1 mRNA target sequences were cloned and transfected into drug resistance cell line K562/A02 by liposome-induced gene transfection. The mdr1 mRNA was identified by real time RT-PCR, the function of P-gp was measured by a daunorubicin (DNR) efflux assay, and the sensitivity of cell lines to doxorubicin (ADM) was detected by an MTT assay. Our results showed that two mdr1-targeted shRNAs could down-regulate mdr1 mRNA and P-gp expression, with mdr1 mRNA reduced by 86% and 88%. The intracellular DNR increased after RNAi treatment, with the daunorubicin efflux ratio at 60min were 13% and 22%, compared with control (40%~45%) (P<0.05). The MTT assay demonstrated the relative reversing efficiency to doxorubicin to be 84% and 77%. Conclusion: RNA interference can effectively reverse mdr1-mediated multidrug resistance in resistant leukemic cell line.

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Ma, Ming, Yunhe Zhou, Ruilin Sun, Jiahao Shi, Yutong Tan, Hua Yang, Mengjie Zhang i in. "STAT3 and AKT signaling pathways mediate oncogenic role of NRSF in hepatocellular carcinoma". Acta Biochimica et Biophysica Sinica 52, nr 10 (18.06.2020): 1063–70. http://dx.doi.org/10.1093/abbs/gmaa069.

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Abstract Neuron-restrictive silencer factor (NRSF) is a zinc finger protein that acts as a negative transcriptional regulator by recruiting histone deacetylases and other co-factors. It plays a crucial role in nervous system development and is recently reported to be involved in tumorigenesis in a tumor type-dependent manner; however, the role of NRSF in hepatocellular carcinoma (HCC) tumorigenesis remains unclear. Here, we found that NRSF expression was up-regulated in 27 of 49 human HCC tissue samples examined. Additionally, mice with conditional NRSF-knockout in the liver exhibited a higher tolerance against diethylnitrosamine (DEN)-induced acute liver injury and were less sensitive to DEN-induced HCC initiation. Our results showed that silencing NRSF in HepG2 cells using RNAi technology significantly inhibited HepG2 cell proliferation and severely hindered their migration and invasion potentials. Our results demonstrated that NRSF plays a pivotal role in promoting DEN-induced HCC initiation via a mechanism related to the STAT3 and AKT signaling pathways. Thus, NRSF could be a potential therapeutic target for treating human HCC.

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Petrie, Victoria J., Jeffrey D. Wuitschick, Cheryl D. Givens, Aaron M. Kosinski i Janet F. Partridge. "RNA Interference (RNAi)-Dependent and RNAi-Independent Association of the Chp1 Chromodomain Protein with Distinct Heterochromatic Loci in Fission Yeast". Molecular and Cellular Biology 25, nr 6 (15.03.2005): 2331–46. http://dx.doi.org/10.1128/mcb.25.6.2331-2346.2005.

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ABSTRACT The establishment of centromeric heterochromatin in the fission yeast Schizosaccharomyces pombe is dependent on the RNA interference (RNAi) pathway. Dicer cleaves centromeric transcripts to produce short interfering RNAs (siRNAs) that actively recruit components of heterochromatin to centromeres. Both centromeric siRNAs and the heterochromatin component Chp1 are components of the RITS (RNA-induced initiation of transcriptional gene silencing) complex, and the association of RITS with centromeres is linked to Dicer activity. In turn, centromeric binding of RITS promotes Clr4-mediated methylation of histone H3 lysine 9 (K9), recruitment of Swi6, and formation of heterochromatin. Similar to centromeres, the mating type locus (Mat) is coated in K9-methylated histone H3 and is bound by Swi6. Here we report that Chp1 associates with the mating type locus and telomeres and that Chp1 localization to heterochromatin depends on its chromodomain and the C-terminal domain of the protein. Another protein component of the RITS complex, Tas3, also binds to Mat and telomeres. Tas3 interacts with Chp1 through the C-terminal domain of Chp1, and this interaction is necessary for Tas3 stability. Interestingly, in cells lacking the Argonaute (Ago1) protein component of the RITS complex, or lacking Dicer (and hence siRNAs), Chp1 and Tas3 can still bind to noncentromeric loci, although their association with centromeres is lost. Thus, Chp1 and Tas3 exist as an Ago1-independent subcomplex that associates with noncentromeric heterochromatin independently of the RNAi pathway.

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Yu, Hai-Zhong, Ning-Yan Li, Yan-Xin Xie, Qin Zhang, Ying Wang i Zhan-Jun Lu. "Identification and Functional Analysis of Two Chitin Synthase Genes in the Common Cutworm, Spodoptera litura". Insects 11, nr 4 (17.04.2020): 253. http://dx.doi.org/10.3390/insects11040253.

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Chitin is one the main components of the insect cuticle, and chitin synthase (CHS) is an important enzyme required for chitin formation. CHS has been characterized in various insect species, but the structure and biochemical properties in Spodoptera litura have not been determined. In this study, we identified two CHS genes, SlCHS1 and SlCHS2, which encode proteins with 1565 and 1520 amino acid residues, respectively. Transcriptional analysis suggested that SlCHS1 has a high expression level in the integument whereas SlCHS2 showed the highest expression level in the midgut. During S. litura growth and development, SlCHS1 and SlCHS2 were both predominantly expressed in the fourth-instar larval stage. In addition, the expression of SlCHS1 and SlCHS2 could be induced by 20-hydroxyecdysone (20E). Silencing of SlCHS1 by RNA interference significantly inhibited the pupation and molting of S. litura larvae (RNAi), while knockdown of SlCHS2 had no significant effects on the S. litura phenotype. These results may provide a new molecular target for control of S. litura.

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23

Samakoglu, Selda, Yelena Usachenko, Tulin Budak-Alpdogan, Santina Acuto, Rosalba DiMarzo, Aurelio Maggio, John Tisdale, Isabelle Riviere i Michel Sadelain. "Genetic Correction of Sickle Cell Disease by Co-Regulated γ-Globin Transgene Expression and ßS Interference." Blood 106, nr 11 (16.11.2005): 1280. http://dx.doi.org/10.1182/blood.v106.11.1280.1280.

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Abstract RNA interference (RNAi) is a promising therapeutic strategy, but its application to stem cell-based gene therapy for the treatment of congenital or acquired disorders will require highly specific gene silencing. To ensure co-expression of a therapeutic transgene and a small interfering RNA (siRNA), we hypothesized that a promoter-less small hairpin RNA (shRNA) embedded within an intron could yield siRNA in tissue-specific fashion and thus achieve regulated RNAi. We demonstrate here that γ-globin expression and erythroid-specific siRNA generation can be achieved in mammalian cells, including human CD34+ cells. The shRNA was encoded under the transcriptional control of the human β-globin promoter, a prototypic tissue-specific Pol II promoter, and positioned at two different sites in the second intron or in the 5′-UTR of a recombinant human γ-globin gene. Three different genes were targeted in mouse erythroleukemia (MEL) cells, green fluorescent protein (EGFP), human sickle β-globin (β S) and endogenous mouse β-gobin. When cloned immediately upstream of the branch point, the siRNA was efficiently generated without altering γ-globin mRNA expression and processing, suggesting that hairpin positioning near the branch point is not detrimental to RNA splicing. When cloned near the 5′-end of the intron, the siRNA was structurally impaired, and the γ-globin mRNA levels greatly diminished. This strong effect of shRNA positioning is consistent with a quality control pathway of gene transcription, whereby introns harboring dsRNA stem loops are degraded if splicing is altered. The strong induction of interferon type I genes associated with the latter position but not the former correlated with the formation of small shRNA degradation products. Positioning of the shRNA in the 5′-UTR did not induce major interferon responses but severely compromised γ-globin expression. To further validate these findings in a clinically relevant model, we engineered an RNAi lentiviral vector in which the human sickle β-globin specific (β S) siRNA is embedded the second intron of a recombinant γ-globin gene. Following transduction of CD34+ cells from patients with sickle cell disease, γ-globin transgene expression was induced upon erythroid differentiation concomitant with a dramatic decrease of the β S transcripts. These findings fully support the principle of synergistic gene delivery and lariat-encoded RNAi in human CD34+ cells, demonstrating the feasibility of using lariat-embedded siRNA to potentiate globin gene transfer by reducing competition from endogenous β S globin chains. Importantly, a moderate decrease in β S expression may substantially improve SCD and abrogate the need for high level expression of the vector-encoded globin gene. This approach to regulate RNAi may find broad applicability in a wide range of disorders where the concomitant expression of a transgene and RNAi will enhance treatment safety and/or efficacy.

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Favetta, L. A., G. F. Mastromonaco, E. J. St. John, W. A. King i D. H. Betts. "218 SELECTIVE REDUCTION OF p66shc mRNA IN BOVINE OOCYTES/EMBRYOS BY RNA INTERFERENCE". Reproduction, Fertility and Development 17, nr 2 (2005): 260. http://dx.doi.org/10.1071/rdv17n2ab218.

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High embryo loss occurs during the first week of in vitro bovine embryo development with a high percentage of embryo death and arrest. These early stages of development are regulated by stored maternal mRNAs that accumulate in the oocyte cytoplasm during its growth phase. In our in vitro production (IVP) system approximately 13.5% of embryos arrest at the 2–4-cell stage, displaying no characteristics of apoptosis. We hypothesized that these arrested embryos enter an oxidative stress-induced senescence-like state. We previously showed that elevated levels of reactive oxygen species and increased levels of the stress-adaptor protein p66shc were associated with this early embryonic arrest. The aim of this study was to selectively down-regulate p66shc mRNA levels in bovine oocytes, using post-transcriptional gene silencing by RNA interference (RNAi), to study the effects of p66shc mRNA “knock-down” on early arrest of IVP bovine embryos. Approximately 12,000 short hairpin (sh) RNAi molecules specific for p66shc were microinjected into bovine germinal vesicle (GV) oocytes. Experiments consisted of a control group undergoing IVF alone, and two groups microinjected with and without p66shc shRNAi molecules. Quantification of p66shc mRNA by real-time PCR was used to assure down-regulation of p66shc mRNA in 2-cell embryos collected at 35 hours post-insemination (hpi). The amount of p66shc mRNA detected in control IVF, vehicle-injected, and p66shc RNAi-injected groups was 0.92 ± 0.02 × 10-5 pg, 1.2 ± 0.20 × 10-5 pg, and 0.46 ± 0.06 × 10-5 pg, respectively. This statistically significant (P < 0.001) reduction in p66shc mRNA levels by 54% upon p66shc shRNAi microinjection was selective for p66shc mRNA, as both histone H2a and p53 mRNA levels were not altered. Percentage of 2–4-cell arrest was evaluated at Day 8 post-insemination and related to p66shc mRNA down-regulation. While there were no significant differences in the percentage of 2–4-cell arrested embryos between the control (13.3 ± 0.8%) and vehicle-microinjected (10.8 ± 0.7%) embryos, there was a significant decrease (P < 0.001) in the incidence of arrest in p66shc shRNAi-microinjected embryos (0.9 ± 0.9%). Quantification by real time PCR in blastocysts from the three groups showed no significant differences in p66shc mRNA levels (P = 0.314) among control IVF, vehicle, or p66shc hairpin RNAi microinjected at 0.59 ± 0.1 × 10-5 pg, 0.57 ± 0.2 × 10-5 pg, and 0.51 ± 0.2 × 10-5 pg, respectively. This suggests that p66shc down-regulation by RNAi might be temporary. Thus, using this novel approach of RNAi and microinjection of oocytes at the GV stage, we were able to selectively down-regulate the expression of p66shc mRNA and correlate this down-regulation with a significant decrease in 2–4 cell arrest. These data reinforce our hypothesis that p66shc is involved in a stress-induced pathway that executes a senescent-like embryonic arrest at the 2–4-cell stage, thereby postulating the possible role of p66shc as a new molecular marker for developmental competence. Statistics performed using ANOVA with Fisher LSD test for multiple comparisons. This work was funded by NSERC, CIHR, OGS, and OMAFRA.

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Derenzini, Enrico, Patrizia Mondello, Yuxuan Liu, Mary Scallion, Zahra Asgari, John Philip, Patrick Hilden i in. "MYC-Dependent PI3K and MCL-1 Feedbacks Attenuate BET Inhibitors Activity in Diffuse Large B-Cell Lymphoma". Blood 128, nr 22 (2.12.2016): 294. http://dx.doi.org/10.1182/blood.v128.22.294.294.

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Abstract MYC overexpression is a poor prognostic predictor in Diffuse Large B-Cell Lymphoma (DLBCL). MYC-targeting with bromodomain and extraterminal protein family (BET) inhibitors is a promising strategy for the treatment of MYC-driven cancers, including lymphomas. However, preclinical and emerging data from early clinical trials demonstrated a modest antiproliferative activity in vitro and in vivo. We hypothesized that BET inhibition may induce feedback survival mechanisms preventing or attenuating cell death that could be exploited for designing future, more effective, combination strategies. In a high-throughput combinatorial drug screening experiment, we found that phosphatidylinositol 3-kinase (PI3K) pathway inhibitors enhanced the antiproliferative effects of BET inhibitors (JQ1, I-BET 151, CPI-203) with a strong class effect. JQ1 upregulated the mRNA expression of several upstream components of the PI3K pathway, including PIK3CA, PIK3R1, PDK1 in a large panel of DLBCL and Burkitt lymphoma cell lines. These effects translated in increased pathway activation as demonstrated by increased levels of the phosphorylated forms of downstream targets GSK3α/β, TSC2, P70S6K, and by increased concentrations of chemokines known to be regulated by PI3K in cell culture supernatants (CCL3 and CCL4). This effect was reversed by submicromolar doses of the PI3K inhibitor BKM-120. MYC silencing recapitulated the effects of BET inhibitors on PI3K pathway gene expression, activation and chemokine secretion. These data indicate that BET inhibition induces PI3K activation by a MYC-dependent feedback. We also observed transcriptional upregulation of the antiapoptotic gene Myeloid Leukemia 1 (MCL-1) following BET inhibition or MYC depletion, suggesting a second MYC-dependent mechanism. RNAi-mediated MCL-1 silencing or co-treatment with a small molecule MCL-1 inhibitor (UMI-77) enhanced the effects of BET inhibitors in DLBCL cell lines by inducing apoptosis. Using SILAC-based quantitative mass spectrometry, we found that BET inhibitors at submicromolar doses downregulated several E2 ubiquitin conjugating enzymes including UBE2C. RNAi mediated UBE2C knockdown induced MCL-1 upregulation in DLBCL cells. The enhanced in vitro effect of combining BETi and PI3Ki was reproduced in TMD8 mouse xenografts. To our knowledge, this is the first study demonstrating MYC-dependent regulation of the PI3K pathway, MCL-1 and the ubiquitin system upon BET inhibition. Our study revealed previously unknown mechanisms of action of BET inhibitors uncovering novel MYC-dependent survival feedback loops, and providing a framework for future combination strategies. Disclosures Zelenetz: Gilead Sciences: Research Funding.

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Surdziel, Ewa, Maciej Cabanski, Iris Dallmann, Marcin Lyszkiewicz, Andreas Krueger, Arnold Ganser, Michaela Scherr i Matthias Eder. "Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways". Blood 117, nr 16 (21.04.2011): 4338–48. http://dx.doi.org/10.1182/blood-2010-06-289058.

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Abstract MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-, maturation-, and disease-specific miRNA expression has been described, miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics, biochemical analysis, and unbiased and hypothesis-driven miRNA target prediction, we show that lentivirally over-expressed miR-125b blocks G-CSF–induced granulocytic differentiation and enables G-CSF–dependent proliferation of murine 32D cells. In primary lineage-negative cells, miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression, respectively. However, gene-specific RNAi reveals that this reduction, alone and in combination, is not sufficient to block G-CSF–dependent differentiation. STAT3 protein expression, DNA-binding, and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression, indicating miR-125b–mediated reduction of one or more STAT3 cofactors. Indeed, we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly, gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells.

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Hawku, Mehari Desta, Fuxin He, Xingxuan Bai, Md Ashraful Islam, Xueling Huang, Zhensheng Kang i Jun Guo. "A R2R3 MYB Transcription Factor, TaMYB391, Is Positively Involved in Wheat Resistance to Puccinia striiformis f. sp. tritici". International Journal of Molecular Sciences 23, nr 22 (15.11.2022): 14070. http://dx.doi.org/10.3390/ijms232214070.

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A biotrophic fungus, Puccinia striiformis f.sp. tritici (Pst), which causes stripe rust disease in wheat is the most yield-limiting factor in wheat production. Plants have complex defense mechanisms against invading pathogens. Hypersensitive response (HR), a kind of programmed cell death (PCD) at the infection site, is among these defense mechanisms. Transcription factors (TFs) play a crucial role in plant defense response against invading pathogens. Myeloblastosis (MYB) TFs are among the largest TFs families that are involved in response to both biotic and abiotic stresses. However, little is known about the mechanisms of MYB TFs during the interaction between wheat and the stripe rust fungus. Here, we identified an R2R3 MYB TF from wheat, designated as TaMYB391, and characterized its functional role during wheat–Pst interaction. Our data indicated that TaMYB391 is induced by Pst infection and exogenous application of salicylic acid (SA) and abscisic acid (ABA). TaMYB391 is localized in the nucleus of both wheat and Nicotiana benthamiana. Transient overexpression of TaMYB391 in N. benthamiana triggered HR-related PCD accompanied by increased electrolyte leakage, high accumulation of reactive oxygen species (ROS), and transcriptional accumulation of SA defense-related genes and HR-specific marker genes. Overexpression of TaMYB391 in wheat significantly enhanced wheat resistance to stripe rust fungus through the induction of pathogenesis-related (PR) genes, ROS accumulation and hypersensitive cell death. On the other hand, RNAi-mediated silencing of TaMYB391 decreased the resistance of wheat to Pst accompanied by enhanced growth of the pathogen. Together our findings demonstrate that TaMYB391 acts as a positive regulator of HR-associated cell death and positively contributes to the resistance of wheat to the stripe rust fungus by regulating certain PR genes, possibly through SA signaling pathways.

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Koldehoff, Michael, Nina K. Steckel, Rudolf Trenschel, Dietrich W. Beelen i Ahmet H. Elmaagacli. "VEGF Specific siRNAs Act Synergistically with Bortezomib and Steroids in Inhibiting VEGF Gene Expression, Proliferation and Induction of Apoptosis in Multiple Myeloma Cells." Blood 104, nr 11 (16.11.2004): 5266. http://dx.doi.org/10.1182/blood.v104.11.5266.5266.

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Abstract Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of malignant plasma cells within the bone marrow (BM). Vascular endothelial growth factor (VEGF), a glycoprotein produced by normal and neoplastic cells is an important regulator of physiological and pathological angiogenesis. MM cells secrete VEGF, which promotes production of cytokines in bone marrow stromal cells, as well as migration and proliferation of the tumor cells. Inhibition of VEGF activity or disabling the function of its receptors has been shown to inhibit both tumor growth and spread of metastases in a variety of animal tumor models. RNA interference (RNAi) is rapidly being established as a post-transcriptional gene silencing method and holds promise to specifically inhibit gene expression in mammals. Another novel class of antitumor agents is based on the inhibition of the ubiquitin-proteosomal system which represents the major nonlysosomal pathway through which intracellular proteins are degraded in eukaryotic cells. Bortezomib, a reversible proteosome inhibitor, shows remarkable anticancer activity in various malignant cell types, including MM cells that are resistant to conventional therapies. We studied the effect of transfection with small interfering RNA (siRNA) targeting VEGF in MM cells in terms of proliferation, induction of apoptosis, and cell differentiation. Further, we evaluated if the effects of post-transcriptional gene silencing by VEGF specific siRNA can be augmented by bortezomib and/or steroids in the cell line OPM-2. A mean reduction of VEGF gene expression to 38% as determined by real-time PCR was observed with 0.8 ug VEGF siRNA in OPM-2 cells compared to controls (controls were set up to 100%). Simultaneous administration of bortezomib and siRNA was able to reduce VEGF gene expression down to 23% compared to VEGF siRNA alone demonstrating a synergistic effect of combined bortezomib and siRNA treatment. We found a 2.5-fold increase in induced apoptosis in OPM-2 cells subsequent to VEGF siRNA administration but we saw no additional stimulation of apoptosis after combination of VEGF siRNA with bortezomib and/or steroids. Proliferation in OPM-2 cells was strongly inhibited (about 91%) following combination treatment as opposed to only 62% after administration of VEGF siRNA alone. The transfection of VEGF siRNA in OPM-2 cells had no influence on the expression levels of differentiation markers such as CD38, CD138, CD19, CD34, CD45, and CD7AAD. Our findings suggest that synergistic effects of VEGF siRNA with bortezomib and dexamethason may offer new therapeutic options in the treatment of MM.

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Kashyap, Trinayan, Boris Klebanov, Christian Argueta, Margaret S. Lee, Sharon Shacham, Michael Kauffman, Yosef Landesman i William Senapedis. "Efficacy of Selinexor Is Dependent on IκB-α Expression and NF-Kb Deactivation in Multiple Myeloma Cells". Blood 128, nr 22 (2.12.2016): 5660. http://dx.doi.org/10.1182/blood.v128.22.5660.5660.

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Abstract Background: Selinexor is a Selective Inhibitor of Nuclear Export (SINE) compound that binds to and inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) including p53, pRB, and IκB-α. Selinexor has therapeutic benefit both pre-clinically and clinically (NCT01607892, NCT02336815) in multiple myeloma (MM). It has been previously demonstrated that the extent of NF-κB transcriptional inhibition is one of the critical mechanisms contributing to the efficacy and/or resistance to selinexor in cells. However, the mechanism leading to NF-κB inhibition after selinexor treatment is not fully understood. We hypothesized that the level of the cellular inhibitor of NF-κB, IκB-α, and its compartmental localization play an important role in NF-κB transactivation and response to selinexor. In this study, we investigate the effect of selinexor treatment on MM cells with low expression of IκB-α and high NF-kB activity in order to understand the mechanism of NF-κB inhibition by selinexor. Methods: IM9 and RPMI-8226cells were treated with selinexor in the presence or absence of 20 ng/mL tumor necrosis factor α (TNFα; inflammatory mimic) and whole protein lysates were analyzed by immunoblotting. Cytotoxic effects of selinexor were evaluated using standard viability assays. IκB-α knockdown was performed using transfection of specific siRNA duplexes. NF-κB transcriptional activity was analyzed using an ELISA assay. Results: Selinexor induces nuclear localization of IκB-α in MM cells. RNAi of IκB-α in MM cells reduced the cytotoxic effects of selinexor by 10-fold. In addition, knockdown of IκB-α reduces the synergy of the selinexor plus proteasome inhibitor (bortezomib or carfilzomib) combination. This data suggests that in MM, IκB-α plays a major role in cellular sensitivity to selinexor potentially through NF-κB activity. Selinexor inhibited NF-kB transcriptional activity in IM9 and RPMI-8226 cells with IC50 of 1079 nM and 591 nM respectively. Although the difference in NF-κB activity IC50 is only 2-fold between the two MM cell lines (MTT IC50s are ~100 nM), IM9 cells have a 100-fold higher basal NF-kB activity when compared to RPMI-8226 cells. Under TNFα stimulation NF-kB activity was induced by 1.5- and 35-fold in IM9 and RPMI-8226, respectively. We observed that selinexor treatment caused a dose dependent inhibition of IκB kinase (IKK)-mediated phosphorylation of serine 32/36 on IκB-α and serine 536 on the NF-κB p65 subunit (RelA) upon TNFα stimulation in both cell lines. In RPMI-8226 cells, selinexor reduced TNFα-induced IκB-α phosphorylation in a dose dependent manner and protected IκB-α from degradation. In IM9 cells that have high basal NF-κB activity, TNFα did not induce NF-kB activity or cause IκB-α degradation. However, selinexor treatment inhibited NF-kB activity below its basal level (70% reduction) which resulted in dose dependent reduction in the level of IκB-α protein perhaps through inhibition of NF-κB transcriptional control of IκB-α mRNA expression. Conclusions: IκB-α plays a major role in the cellular cytotoxicity of selinexor in cancer cells. Multiple myeloma cells lose sensitivity to selinexor treatment upon IκB-α silencing, which in turn reduces the cytotoxicity of selinexor. TNFα stimulation induces the phosphorylation of NF-κB p65 subunit and IκB-α through increased IKK activity resulting in IκB-α degradation and NF-κB activation. In RPMI-8226 cells, selinexor treatment blocked TNFα-induced degradation of IκB-α. However, in IM9 cells TNFα alone did not have any significant effect on IκB-α which might be due to the high basal NF-kB activity. Interestingly, IκB-α is also a transcriptional target of NF-kB. In IM9 cells, selinexor treatment reduces NF-kB activity below the high basal level in a dose dependent manner resulting in near complete inhibition of NF-kB-controlled IκB-α mRNA transcription and a loss of the IκB-α protein. Ultimately, selinexor treatment inhibits cell viability and NF-kB transcriptional activity regardless of basal NF-κB activity in MM cells. Because of this IκB-α/NF-kB transcriptional mechanism, selinexor treatment can inhibit both chronic (unresponsive to TNFα) and acute (TNFα-simulated) inflammatory signaling which makes selinexor an applicable therapy to cancer cells with a variety of aberrant signaling pathways. Disclosures Kashyap: Karyopharm Therapeutics: Employment, Equity Ownership. Klebanov:Karyopharm Therapeutics: Employment, Equity Ownership. Argueta:Karyopharm Therapeutics: Employment, Equity Ownership. Lee:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Senapedis:Karyopharm Therapeutics: Employment, Equity Ownership.

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Akbar, Sehrish, Yao Wei i Mu-Qing Zhang. "RNA Interference: Promising Approach to Combat Plant Viruses". International Journal of Molecular Sciences 23, nr 10 (10.05.2022): 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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Giordano, Ennio, Rosaria Rendina, Ivana Peluso i Maria Furia. "RNAi Triggered by Symmetrically Transcribed Transgenes in Drosophila melanogaster". Genetics 160, nr 2 (1.02.2002): 637–48. http://dx.doi.org/10.1093/genetics/160.2.637.

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Abstract Specific silencing of target genes can be induced in a variety of organisms by providing homologous double-stranded RNA molecules. In vivo, these molecules can be generated either by transcription of sequences having an inverted-repeat (IR) configuration or by simultaneous transcription of sense-antisense strands. Since IR constructs are difficult to prepare and can stimulate genomic rearrangements, we investigated the silencing potential of symmetrically transcribed sequences. We report that Drosophila transgenes whose sense-antisense transcription was driven by two convergent arrays of Gal4-dependent UAS sequences can induce specific, dominant, and heritable repression of target genes. This effect is not dependent on a mechanism based on homology-dependent DNA/DNA interactions, but is directly triggered by transcriptional activation and is accompanied by specific depletion of the endogenous target RNA. Tissue-specific induction of these transgenes restricts the target gene silencing to selected body domains, and spreading phenomena described in other cases of post-transcriptional gene silencing (PTGS) were not observed. In addition to providing an additional tool useful for Drosophila functional genomic analysis, these results add further strength to the view that events of sense-antisense transcription may readily account for some, if not all, PTGS-cosuppression phenomena and can potentially play a relevant role in gene regulation.

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Asokan, R., G. Sharath Chandra, M. Manamohan i N. K. Krishna Kumar. "Effect of diet delivered various concentrations of double-stranded RNA in silencing a midgut and a non-midgut gene of Helicoverpa armigera". Bulletin of Entomological Research 103, nr 5 (5.04.2013): 555–63. http://dx.doi.org/10.1017/s0007485313000138.

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AbstractRibonucleic acid interference (RNAi) is a sequence-specific gene silencing mechanism induced by double-stranded RNA (dsRNA). Recently, RNAi has gained popularity as a reverse genetics tool owing to its tremendous potential in insect pest management, which includes Helicoverpa armigera. However, its efficiency is mainly governed by dsRNA concentration, frequency of application, target gene, etc. Therefore, to obtain a robust RNAi response in H. armigera, we evaluated various concentrations of dsRNA and its frequency of applications delivered through diet in silencing a midgut gene, chymotrypsin and a non-midgut gene, juvenile hormone acid methyl transferase (jhamt) of H. armigera. The extent of target gene silencing was determined by employing reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Our study revealed four significant findings: (i) single application of dsRNA elicited a delayed and transient silencing, while multiple applications resulted in early and persistent silencing of the above genes; (ii) silencing of the non-midgut gene (jhamt) through diet delivered dsRNA revealed prevalence of systemic silencing probably due to communication of silencing signals in this pest; (iii) the extent of silencing of chymotrypsin was positively correlated with dsRNA concentration and was negatively correlated with jhamt; (iv) interestingly, over-expression (15–18 folds) of an upstream gene, farnesyl diphosphate synthase (fpps), in juvenile hormone (JH) biosynthetic pathway at higher concentrations of jhamt dsRNA was the plausible reason for lesser silencing of jhamt. This study provides an insight into RNAi response of target genes, which is essential for RNAi design and implementation as a pest management strategy.

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Thibaut, Olivier, i Bragard Claude. "Innate Immunity Activation and RNAi Interplay in Citrus Exocortis Viroid—Tomato Pathosystem". Viruses 10, nr 11 (26.10.2018): 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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Lou, Qiang, Yanzhong Hu, Yanfang Ma i Zheng Dong. "RNA interference may suppress stress granule formation by preventing argonaute 2 recruitment". American Journal of Physiology-Cell Physiology 316, nr 1 (1.01.2019): 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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Haider, Saba, Muhammad Ajmal Bashir, Umer Habib, Yike Gao, Muhammad Rashid Shaheen, Rashid Hussain i Fan Min. "Phenotypic Characterization and RT-qPCR Analysis of Flower Development in F1 Transgenics of Chrysanthemum × grandiflorum". Plants 10, nr 8 (16.08.2021): 1681. http://dx.doi.org/10.3390/plants10081681.

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Gene silencing is the epigenetic regulation of any gene in order to prevent gene expression at the transcription or translation levels. Among various gene silencing techniques, RNA silencing (RNAi) is notable gene regulation technique that involves sequence-specific targeting and RNA degradation. However, the effectiveness of transgene-induced RNAi in F1 generation of chrysanthemum has not been studied yet. In the current study, we used RNAi-constructed CmTFL1 (white-flowered) and CmSVP overexpressed (yellow flowered) transgenic plants of previously conducted two studies for our experiment. Cross hybridization was performed between these intergeneric transgenic and non-transgenic plants of the winter-growing chrysanthemum selection “37” (light pink flowered). The transgene CmSVP was confirmed in F1 hybrids by RT-PCR analysis, whereas hybrids of CmTFL1 parental plants were non-transgenic. Besides this, quantitative real-time PCR (qPCR) was used to explain the molecular mechanism of flower development using reference genes. Intergeneric and interspecific hybrids produced different colored flowers unlike their respective parents. These results suggest that generic traits of CmSVP overexpressed plants can be transferred into F1 generations when crossed with mutant plants. This study will aid in understanding the breeding phenomenon among intergeneric hybrids of chrysanthemum plants at an in vivo level, and such transgenics will also be more suitable for sustainable flower yield under a low-light production system.

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Atanassov, Hristo, Jonathan Parrilla, Caroline Artault, Jérémy Verbeke, Thomas Schneider, Jonas Grossmann, Bernd Roschitzki i Rossitza Atanassova. "Grape ASR-Silencing Sways Nuclear Proteome, Histone Marks and Interplay of Intrinsically Disordered Proteins". International Journal of Molecular Sciences 23, nr 3 (28.01.2022): 1537. http://dx.doi.org/10.3390/ijms23031537.

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In order to unravel the functions of ASR (Abscisic acid, Stress, Ripening-induced) proteins in the nucleus, we created a new model of genetically transformed grape embryogenic cells by RNAi-knockdown of grape ASR (VvMSA). Nuclear proteomes of wild-type and VvMSA-RNAi grape cell lines were analyzed by quantitative isobaric tagging (iTRAQ 8-plex). The most significantly up- or down-regulated nuclear proteins were involved in epigenetic regulation, DNA replication/repair, transcription, mRNA splicing/stability/editing, rRNA processing/biogenesis, metabolism, cell division/differentiation and stress responses. The spectacular up-regulation in VvMSA-silenced cells was that of the stress response protein VvLEA D-29 (Late Embryogenesis Abundant). Both VvMSA and VvLEA D-29 genes displayed strong and contrasted responsiveness to auxin depletion, repression of VvMSA and induction of VvLEA D-29. In silico analysis of VvMSA and VvLEA D-29 proteins highlighted their intrinsically disordered nature and possible compensatory relationship. Semi-quantitative evaluation by medium-throughput immunoblotting of eighteen post-translational modifications of histones H3 and H4 in VvMSA-knockdown cells showed significant enrichment/depletion of the histone marks H3K4me1, H3K4me3, H3K9me1, H3K9me2, H3K36me2, H3K36me3 and H4K16ac. We demonstrate that grape ASR repression differentially affects members of complex nucleoprotein structures and may not only act as molecular chaperone/transcription factor, but also participates in plant responses to developmental and environmental cues through epigenetic mechanisms.

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Yu, Jisuk, Kyung-Mi Lee, Won Kyong Cho, Ju Yeon Park i Kook-Hyung Kim. "Differential Contribution of RNA Interference Components in Response to Distinct Fusarium graminearum Virus Infections". Journal of Virology 92, nr 9 (7.02.2018): e01756-17. http://dx.doi.org/10.1128/jvi.01756-17.

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ABSTRACT The mechanisms of RNA interference (RNAi) as a defense response against viruses remain unclear in many plant-pathogenic fungi. In this study, we used reverse genetics and virus-derived small RNA profiling to investigate the contributions of RNAi components to the antiviral response against Fusarium graminearum viruses 1 to 3 (FgV1, -2, and -3). Real-time reverse transcription-quantitative PCR (qRT-PCR) indicated that infection of Fusarium graminearum by FgV1, -2, or -3 differentially induces the gene expression of RNAi components in F. graminearum. Transcripts of the DICER-2 and AGO-1 genes of F. graminearum (FgDICER-2 and FgAGO-1) accumulated at lower levels following FgV1 infection than following FgV2 or FgV3 infection. We constructed gene disruption and overexpression mutants for each of the Argonaute and dicer genes and for two RNA-dependent RNA polymerase (RdRP) genes and generated virus-infected strains of each mutant. Interestingly, mycelial growth was significantly faster for the FgV1-infected FgAGO-1 overexpression mutant than for the FgV1-infected wild type, while neither FgV2 nor FgV3 infection altered the colony morphology of the gene deletion and overexpression mutants. FgV1 RNA accumulation was significantly decreased in the FgAGO-1 overexpression mutant. Furthermore, the levels of induction of FgAGO-1, FgDICER-2, and some of the FgRdRP genes caused by FgV2 and FgV3 infection were similar to those caused by hairpin RNA-induced gene silencing. Using small RNA sequencing analysis, we documented different patterns of virus-derived small interfering RNA (vsiRNA) production in strains infected with FgV1, -2, and -3. Our results suggest that the Argonaute protein encoded by FgAGO-1 is required for RNAi in F. graminearum, that FgAGO-1 induction differs in response to FgV1, -2, and -3, and that FgAGO-1 might contribute to the accumulation of vsiRNAs in FgV1-infected F. graminearum. IMPORTANCE To increase our understanding of how RNAi components in Fusarium graminearum react to mycovirus infections, we characterized the role(s) of RNAi components involved in the antiviral defense response against Fusarium graminearum viruses (FgVs). We observed differences in the levels of induction of RNA silencing-related genes, including FgDICER-2 and FgAGO-1, in response to infection by three different FgVs. FgAGO-1 can efficiently induce a robust RNAi response against FgV1 infection, but FgDICER genes might be relatively redundant to FgAGO-1 with respect to antiviral defense. However, the contribution of this gene in the response to the other FgV infections might be small. Compared to previous studies of Cryphonectria parasitica, which showed dicer-like protein 2 and Argonaute-like protein 2 to be important in antiviral RNA silencing, our results showed that F. graminearum developed a more complex and robust RNA silencing system against mycoviruses and that FgDICER-1 and FgDICER-2 and FgAGO-1 and FgAGO-2 had redundant roles in antiviral RNA silencing.

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Zhang, Jie, Guangming Yang, Yu Zhu, Xiaoyong Peng, Tao Li i Liangming Liu. "Relationship of Cx43 regulation of vascular permeability to osteopontin-tight junction protein pathway after sepsis in rats". American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 314, nr 1 (1.01.2018): R1—R11. http://dx.doi.org/10.1152/ajpregu.00443.2016.

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Our previous study demonstrated that connexin (Cx)43 participated in the regulation of vascular permeability in severe sepsis. Osteopontin (OPN) has been demonstrated to participate in the occurrence of atherosclerosis, inflammation, as well as the adhesion and migration of cells. It is not clear whether OPN is involved in Cx43 regulating vascular permeability after sepsis and if it is related to tight-junction proteins. with the use of cecal ligation and puncture (CLP)-induced septic rats and lipopolysaccharide (LPS)-treated pulmonary vein vascular endothelial cells (VECs), the role of zona occuldens 1 (ZO-1) and claudin-5 in Cx43 regulation of vascular permeability and its relationship to OPN were investigated in the present study. The results showed that the expression of ZO-1 and claudin-5 in pulmonary vein were decreased in CLP rats and LPS-treated pulmonary vein VECs. Cx43-overexpressed lentivirus induced the degradation of ZO-1 and claudin-5, while Cx43 RNAi lentivirus abrogated the degradation of ZO-1 and claudin-5 induced by LPS. The vascular permeability and expression of OPN in pulmonary veins were significantly increased in CLP rats and LPS-treated pulmonary vein VECs. Silencing OPN by OPN RNAi lentivirus inhibited the vascular hyperpermeability induced by LPS. Overexpressed Cx43 lentivirus increased the expression of OPN and vascular permeability and downregulated the expression of ZO-1 and claudin-5 in pulmonary vein VECs. Silencing OPN by OPN RNAi lentivirus inhibited the effects of Cx43-overexpressed lentivirus on downregulation of ZO-1 and claudin-5 and vascular hyperpermeability in pulmonary vein VECs. Transfection of specific double-stranded RNA targeting to β-catenin and T-cell factor-4 (Tcf-4) abolished the upregulation of OPN induced by Cx43 overexpression. These results suggest that OPN participates in the regulation of vascular permeability by Cx43 after sepsis. Cx43 upregulation of OPN is via the Tcf-4/β-catenin transcription pathway; OPN increases vascular permeability by downregulating the expression of the tight junction proteins ZO-1 and claudin-5.

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Hong, Hao, Chunli Wang, Ying Huang, Min Xu, Jiaoling Yan, Mingfeng Feng, Jia Li i in. "Antiviral RISC mainly targets viral mRNA but not genomic RNA of tospovirus". PLOS Pathogens 17, nr 7 (28.07.2021): e1009757. http://dx.doi.org/10.1371/journal.ppat.1009757.

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Antiviral RNA silencing/interference (RNAi) of negative-strand (-) RNA plant viruses (NSVs) has been studied less than for single-stranded, positive-sense (+)RNA plant viruses. From the latter, genomic and subgenomic mRNA molecules are targeted by RNAi. However, genomic RNA strands from plant NSVs are generally wrapped tightly within viral nucleocapsid (N) protein to form ribonucleoproteins (RNPs), the core unit for viral replication, transcription and movement. In this study, the targeting of the NSV tospoviral genomic RNA and mRNA molecules by antiviral RNA-induced silencing complexes (RISC) was investigated, in vitro and in planta. RISC fractions isolated from tospovirus-infected N. benthamiana plants specifically cleaved naked, purified tospoviral genomic RNAs in vitro, but not genomic RNAs complexed with viral N protein. In planta RISC complexes, activated by a tobacco rattle virus (TRV) carrying tospovirus NSs or Gn gene fragments, mainly targeted the corresponding viral mRNAs and hardly genomic (viral and viral-complementary strands) RNA assembled into RNPs. In contrast, for the (+)ssRNA cucumber mosaic virus (CMV), RISC complexes, activated by TRV carrying CMV 2a or 2b gene fragments, targeted CMV genomic RNA. Altogether, the results indicated that antiviral RNAi primarily targets tospoviral mRNAs whilst their genomic RNA is well protected in RNPs against RISC-mediated cleavage. Considering the important role of RNPs in the replication cycle of all NSVs, the findings made in this study are likely applicable to all viruses belonging to this group.

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Diao, Pengfei, Qimeng Zhang, Hongyu Sun, Wenjie Ma, Aiping Cao, Ruonan Yu, Jiaojiao Wang, Yiding Niu i Hada Wuriyanghan. "miR403a and SA Are Involved in NbAGO2 Mediated Antiviral Defenses Against TMV Infection in Nicotiana benthamiana". Genes 10, nr 7 (12.07.2019): 526. http://dx.doi.org/10.3390/genes10070526.

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RNAi (RNA interference) is an important defense response against virus infection in plants. The core machinery of the RNAi pathway in plants include DCL (Dicer Like), AGO (Argonaute) and RdRp (RNA dependent RNA polymerase). Although involvement of these RNAi components in virus infection responses was demonstrated in Arabidopsis thaliana, their contribution to antiviral immunity in Nicotiana benthamiana, a model plant for plant-pathogen interaction studies, is not well understood. In this study, we investigated the role of N. benthamiana NbAGO2 gene against TMV (Tomato mosaic virus) infection. Silencing of NbAGO2 by transient expression of an hpRNA construct recovered GFP (Green fluorescent protein) expression in GFP-silenced plant, demonstrating that NbAGO2 participated in RNAi process in N. benthamiana. Expression of NbAGO2 was transcriptionally induced by both MeSA (Methylsalicylate acid) treatment and TMV infection. Down-regulation of NbAGO2 gene by amiR-NbAGO2 transient expression compromised plant resistance against TMV infection. Inhibition of endogenous miR403a, a predicted regulatory microRNA of NbAGO2, reduced TMV infection. Our study provides evidence for the antiviral role of NbAGO2 against a Tobamovirus family virus TMV in N. benthamiana, and SA (Salicylic acid) mediates this by induction of NbAGO2 expression upon TMV infection. Our data also highlighted that miR403a was involved in TMV defense by regulation of target NbAGO2 gene in N. Benthamiana.

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Wang, Xueli, Yike Ding, Xiangyang Lu, Danqian Geng, Shan Li, Alexander S. Raikhel i Zhen Zou. "The ecdysone-induced protein 93 is a key factor regulating gonadotrophic cycles in the adult female mosquito Aedes aegypti". Proceedings of the National Academy of Sciences 118, nr 8 (16.02.2021): e2021910118. http://dx.doi.org/10.1073/pnas.2021910118.

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Repeated blood feedings are required for adult female mosquitoes to maintain their gonadotrophic cycles, enabling them to be important pathogen carriers of human diseases. Elucidating the molecular mechanism underlying developmental switches between these mosquito gonadotrophic cycles will provide valuable insight into mosquito reproduction and could aid in the identification of targets to disrupt these cycles, thereby reducing disease transmission. We report here that the transcription factor ecdysone-induced protein 93 (E93), previously implicated in insect metamorphic transitions, plays a key role in determining the gonadotrophic cyclicity in adult females of the major arboviral vector Aedes aegypti. Expression of the E93 gene in mosquitoes is down-regulated by juvenile hormone (JH) and up-regulated by 20-hydroxyecdysone (20E). We find that E93 controls Hormone Receptor 3 (HR3), the transcription factor linked to the termination of reproductive cycles. Moreover, knockdown of E93 expression via RNAi impaired fat body autophagy, suggesting that E93 governs autophagy-induced termination of vitellogenesis. E93 RNAi silencing prior to the first gonadotrophic cycle affected normal progression of the second cycle. Finally, transcriptomic analysis showed a considerable E93-dependent decline in the expression of genes involved in translation and metabolism at the end of a reproductive cycle. In conclusion, our data demonstrate that E93 acts as a crucial factor in regulating reproductive cycle switches in adult female mosquitoes.

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Wen, Dan, Yan-Fang Zou, Yao-Hui Gao, Qian Zhao, Yin-Yin Xie, Ping-Yan Shen, Yao-Wen Xu i in. "Inhibitor of DNA Binding 1 Is Induced during Kidney Ischemia-Reperfusion and Is Critical for the Induction of Hypoxia-Inducible Factor-1α". BioMed Research International 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/4634386.

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In this study, rat models of acute kidney injury (AKI) induced by renal ischemia-reperfusion (I/R) and HK-2 cell models of hypoxia-reoxygenation (H/R) were established to investigate the expression of inhibitor of DNA binding 1 (ID1) in AKI, and the regulation relationship between ID1 and hypoxia-inducible factor 1 alpha (HIF-1α). Through western blot, quantitative real-time PCR, immunohistochemistry, and other experiment methods, the induction of ID1 after renal I/R in vivo was observed, which was expressed mainly in renal tubular epithelial cells (TECs). ID1 expression was upregulated in in vitro H/R models at both the protein and mRNA levels. Via RNAi, it was found that ID1 induction was inhibited with silencing of HIF-1α. Moreover, the suppression of ID1 mRNA expression could lead to decreased expression and transcription of HIF-1αduring hypoxia and reoxygenation. In addition, it was demonstrated that both ID1 and HIF-1αcan regulate the transcription of twist. This study demonstrated that ID1 is induced in renal TECs during I/R and can regulate the transcription and expression of HIF-1α.

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Xu, You-Ping, Yuan Zhao, Xiao-Yi Song, Yun-Feng Ye, Ren-Gang Wang, Zi-Li Wang, Xue-Liang Ren i Xin-Zhong Cai. "Ubiquitin Extension Protein UEP1 Modulates Cell Death and Resistance to Various Pathogens in Tobacco". Phytopathology® 109, nr 7 (lipiec 2019): 1257–69. http://dx.doi.org/10.1094/phyto-06-18-0212-r.

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Ubiquitin (Ub) extension proteins (UEPs) are fusion proteins of a Ub at the N terminus to a ribosomal protein. They are the main source of Ub and the only source of extension ribosomal protein. Although important roles of the Ub-26S proteasome system in various biological processes have been well established, direct evidence for the role of UEP genes in plant defense is rarely reported. In this study, we cloned a Ub-S27a-type UEP gene (NbUEP1) from Nicotiana benthamiana and demonstrated its function in cell death and disease resistance. Virus-induced gene silencing of NbUEP1 led to intensive cell death, culminating in whole-seedling withering. Transient RNA interference (RNAi) of NbUEP1 caused strong cell death in infiltrated areas, while stable NbUEP1-RNAi tobacco plants constitutively formed necrotic lesions in leaves. NbUEP1-RNAi plants exhibited increased resistance to the oomycete Pythium aphanidermatum and viruses Tobacco mosaic virus and Cucumber mosaic virus while displaying decreased resistance to the nematode Meloidogyne incognita compared with non-RNAi control plants. Transcription profiling analysis indicated that jasmonate and ethylene pathways, lipid metabolism, copper amine oxidase-mediated active species generation, glycine-rich proteins, vacuolar processing enzyme- and RD21-mediated cell death and defense regulation, and autophagy might be associated with NbUEP1-mediated cell death and resistance. Our results provided evidence for the important roles of plant UEPs in modulating plant cell death and disease resistance.

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Dejjuy, Duangnapa, Chavaboon Dechsukhum, Kovit Pattanapanyasat, Egarit Noulsri, Gregory A. Dissen i Wilairat Leeanansaksiri. "Novel WT1 Target Genes: IL-2, IL-2RB, and IL-2RG Discovered during WT1 Silencing Using Lentiviral-Based RNAi in Myeloid Leukemia Cells". BioMed Research International 2020 (14.10.2020): 1–10. http://dx.doi.org/10.1155/2020/7851414.

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Wilms’ tumor 1 (WT1) is a transcription factor which plays a major role in cell proliferation, differentiation, survival, and apoptosis. WT1 was first identified as a tumor suppressor gene in Wilms’ tumor. However, overexpression of WT1 has been detected in several types of malignancy including some types of leukemia. To investigate the molecular mechanism underlying WT1-mediated leukemogenesis, lentiviral-based siRNA was employed as a tool to suppress WT1 expression in the myeloid leukemia cell line, K562. Successfully, both WT1 RNA and protein levels were downregulated in the leukemia cells. The silencing of WT1 resulted in significant growth inhibition in WT1-siRNA-treated cells for 40 ± 7.0 % , 44 ± 9.5 % , and 88 ± 9.1 % at 48, 72, and 96 hours posttransduction as compared with the control cells, respectively. By using apoptosis detection assays (caspase-3/7 activity and Annexin V-FITC/PI assays), WT1 silencing induced a higher degree of early and late apoptosis in siRNA-treated K562 as compared with the control cells. Interestingly, the expression of survival signaling genes, IL-2, IL-2RB, and IL-2RG, was also suppressed after WT1-siRNA treatment. In addition, the WT1 silencing also inhibited the S phase of the cell cycle and induced cell death. Our results indicated that WT1 silencing by siRNA can suppress cellular proliferation, induce apoptosis, and reduce S phase fraction of K562 cells. Moreover, transcriptional modulation of IL-2, IL-2RB, and IL2-2RG expression by WT1 was likely involved in this phenotypic change. Overall, this study confirmed the oncogenic role of WT1 in myeloid leukemia and discovered the new target genes of WT1 which are likely involved in WT1-mediated leukemogenesis.

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Leung-Hagesteijn, Chungyee, Brad Wouters i Rodger E. Tiedemann. "ERN1/IRE1 and XBP1 Are Dispensable for Myeloma Cell Survival but Are Required for the Anti-Myeloma Activity of Bortezomib". Blood 118, nr 21 (18.11.2011): 986. http://dx.doi.org/10.1182/blood.v118.21.986.986.

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Abstract Abstract 986 We have previously conducted kinome- (Tiedemann et al., Blood 2010) and genome- (Zhu et al., Blood 2011) wide small interfering RNA (siRNA) studies in human myeloma cells in the absence and presence of titrated bortezomib (BTZ) in order to identify genes that modulate BTZ sensitivity or resistance. From high throughput RNAi screening, endoplasmic reticulum-to-nucleus-signaling-1 (ERN1), the human homolog of yeast IRE-1, was identified with multiple siRNA as the kinase most potently required for BTZ activity, with genetic silencing associated with BTZ resistance. ERN1/IRE1 is an endoplasmic reticulum (ER)-resident transmembrane protein and a core component of the unfolded protein response (UPR). By gene expression profiling and western blot IRE1 is ubiquitously expressed across primary myeloma tumors and a panel of 14 human myeloma tumor lines (HMCL). Significantly, silencing of IRE1 with lentiviral shRNA induced BTZ resistance in 9/9 HMCL. IRE1 silencing in the absence of BTZ was surprisingly well tolerated in the majority of HMCL tested, with no loss of viability; however in 2/9 HMCL (JJN3, 8226) silencing of IRE1 resulted in reduced proliferation. Multiple RNAi yielded identical results. In response to unfolded protein accumulation within the ER lumen, IRE1 oligomerizes and auto-phosphorylates to reveal an endonuclease activity that splices X-box protein 1 (XBP1) mRNA to activate the potent bZIP transcription factor, XBP1s. XBP1 is essential for physiologic differentiation of B cells to plasma cells and its absence results in profound plasma cell deficiency (Reimold et al., 2001). Despite it's essential role in plasma cell differentiation, we find that XBP1, like IRE1, is redundant for myeloma cell survival under standard growth conditions. Lentiviral knockdown of XBP1 using multiple distinct RNAi failed to induce HMCL cytotoxicity; and resulted in reduced growth in only 2/9 HMCL, closely mirroring results observed with IRE1 inhibition. Significantly, XBP1 silencing also closely reproduced the BTZ resistance observed with IRE1 silencing. As plasmacytoma in patients grow under hypoxic conditions we next evaluated myeloma cell growth in response to hypoxia (0.2% O2) and concurrent UPR signaling disruption. At 48hrs, viability was comparable in IRE1 knockouts and parental cells. In contrast, HMCL with IRE1 silencing showed marked vulnerability to protein synthesis inhibition induced by puromycin, with selection against IRE1 silenced cells in mixed cultures. Notably, ER stress is signaled by 3 different transmembrane transducers (IRE1, PERK and ATF6), each contributing to distinct UPR pathways. As induced BTZ resistance from IRE1 or XBP1 inhibition might conceivably result either from loss of a death signal transmitted via IRE1-XBP1 following PI-induced ER stress, or from a compensatory increase in PERK or ATF6 homeostasis signaling in response to IRE1-XBP1 silencing, we generated single, double and triple knockdown HMCL with suppressed expression of 1, 2 or all 3 ER stress transducers (IRE1, PERK, ATF6). Individual silencing of either IRE1 or PERK in HMCL had no effect on levels of other ER stress transducers by western blot, however silencing of ATF6 caused compensatory increases in PERK while XBP1 silencing resulted in reduced IRE1. Importantly, HMCL lacking IRE1 and PERK or ATF6, showed no loss of BTZ resistance induced by either IRE1 or XBP1 knockdown, demonstrating that secondary increases in signaling via these alternative ER stress pathways do not account for the BTZ resistance induced by IRE1-XBP1 pathway inhibition. Consistent with the hypothesis that increased UPR signaling (via IRE1-XBP1) causes myeloma cell death, knock down of the ER luminal chaperone BiP (HSPA5), which negatively regulates all 3 stress transducers, proved cytotoxic in multiple HMCL. Overall, these data suggest that the anti-myeloma activity of BTZ involves a specific IRE1-XBP1 death signal generated in response to proteasome inhibition and unfolded protein ER stress, rather than ER failure from unfolded protein. As IRE1-XBP1 signaling is dispensable for myeloma cell survival under many growth conditions, targeted small molecule inhibitors of IRE1 currently under development may prove ineffective as single agent therapy for myeloma; moreover such agents may prove antagonist to PI therapy. Finally, disruption of the IRE1-XBP1 axis in primary tumors may mediate bortezomib resistance in patients. Disclosures: No relevant conflicts of interest to declare.

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Samarskaya, Viktoriya O., Nadezhda Spechenkova, Nikolay Markin, Tatyana P. Suprunova, Sergey K. Zavriev, Andrew J. Love, Natalia O. Kalinina i Michael Taliansky. "Impact of Exogenous Application of Potato Virus Y-Specific dsRNA on RNA Interference, Pattern-Triggered Immunity and Poly(ADP-ribose) Metabolism". International Journal of Molecular Sciences 23, nr 14 (18.07.2022): 7915. http://dx.doi.org/10.3390/ijms23147915.

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In this work we developed and exploited a spray-induced gene silencing (SIGS)-based approach to deliver double-stranded RNA (dsRNA), which was found to protect potato against potato virus Y (PVY) infection. Given that dsRNA can act as a defence-inducing signal that can trigger sequence-specific RNA interference (RNAi) and non-specific pattern-triggered immunity (PTI), we suspected that these two pathways may be invoked via exogeneous application of dsRNA, which may account for the alterations in PVY susceptibility in dsRNA-treated potato plants. Therefore, we tested the impact of exogenously applied PVY-derived dsRNA on both these layers of defence (RNAi and PTI) and explored its effect on accumulation of a homologous virus (PVY) and an unrelated virus (potato virus X, PVX). Here, we show that application of PVY dsRNA in potato plants induced accumulation of both small interfering RNAs (siRNAs), a hallmark of RNAi, and some PTI-related gene transcripts such as WRKY29 (WRKY transcription factor 29; molecular marker of PTI), RbohD (respiratory burst oxidase homolog D), EDS5 (enhanced disease susceptibility 5), SERK3 (somatic embryogenesis receptor kinase 3) encoding brassinosteroid-insensitive 1-associated receptor kinase 1 (BAK1), and PR-1b (pathogenesis-related gene 1b). With respect to virus infections, PVY dsRNA suppressed only PVY replication but did not exhibit any effect on PVX infection in spite of the induction of PTI-like effects in the presence of PVX. Given that RNAi-mediated antiviral immunity acts as the major virus resistance mechanism in plants, it can be suggested that dsRNA-based PTI alone may not be strong enough to suppress virus infection. In addition to RNAi- and PTI-inducing activities, we also showed that PVY-specific dsRNA is able to upregulate production of a key enzyme involved in poly(ADP-ribose) metabolism, namely poly(ADP-ribose) glycohydrolase (PARG), which is regarded as a positive regulator of biotic stress responses. These findings offer insights for future development of innovative approaches which could integrate dsRNA-induced RNAi, PTI and modulation of poly(ADP-ribose) metabolism in a co-ordinated manner, to ensure a high level of crop protection.

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Khan, Adnan, Muhammad Zahoor Khan, Jinhuan Dou, Saqib Umer, Huitao Xu, Abdul Sammad, Hua-Bin Zhu i Yachun Wang. "RNAi-Mediated Silencing of Catalase Gene Promotes Apoptosis and Impairs Proliferation of Bovine Granulosa Cells under Heat Stress". Animals 10, nr 6 (19.06.2020): 1060. http://dx.doi.org/10.3390/ani10061060.

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Heat stress in dairy cattle is recognized to compromise fertility by altering the functions of ovarian follicle-enclosed cells, e.g., oocyte and granulosa cells (GCs). Catalase is an antioxidant enzyme that plays a significant role in cellular protection against oxidative damage by the degradation of hydrogen peroxide to oxygen and water. In this study, the role and mechanism of CAT on the heat stress (HS)-induced apoptosis and altered proliferation of bovine GCs were studied. The catalase gene was knocked-down successfully in bovine GCs at both the transcriptional and translational levels. After a successful knockdown using siRNA, GCs were divided into HS (40 °C + NC and 40 °C + CAT siRNA) and 38 °C + NC (NC) groups. The GCs were then examined for ROS, viability, mitochondrial membrane potential (MMP), cell cycle, and biosynthesis of progesterone (P4) and estrogen (E2) hormones. The results indicated that CAT silencing promoted ROS production and apoptosis by up-regulating the Bcl-2-associated X protein (BAX) and Caspase-3 genes both at the transcriptional and translational levels. Furthermore, the knockdown of CAT markedly disrupted the MMP, impaired the production of P4 and E2, altered the progression of the G1 phase of the cell cycle, and decreased the number of cells in the S phase. This was further verified by the down-regulation of proliferating cell nuclear antigen (PCNA), CyclinB1, steroidogenic acute regulatory protein (STAR), and cytochrome P450 family 11 subfamily A member 1 (Cyp11A1) genes. Our study presented a novel strategy to characterize how CAT can regulate cell proliferation and apoptosis in GCs under HS. We concluded that CAT is a broad regulatory marker in GCs by regulating apoptosis, cellular progression, and simultaneously by vital fluctuations in hormonal signaling. Our findings infer a crucial evidence of how to boost the fertility of heat-stressed cows.

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Dzitoyeva, Svetlana, i Hari Manev. "Reduction of Cellular Lipid Content by a Knockdown ofDrosophila PDP1γand Mammalian Hepatic Leukemia Factor". Journal of Lipids 2013 (2013): 1–7. http://dx.doi.org/10.1155/2013/297932.

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In exploring the utility of double-stranded RNA (dsRNA) injections for silencing thePAR-domain protein 1 (Pdp1)gene in adultDrosophila, we noticed a dramatic loss of fat tissue lipids. To verify that our RNAi approach produced the expectedPdp1knockdown, the abdominal fat tissues sections were stained with PDP1 antibodies. PDP1 protein immunostaining was absent in flies injected with dsRNA targeting a sequence common to all knownPdp1isoforms. Subsequent experiments revealed that lipid staining is reduced in flies injected with dsRNA against Pdp1γ(fat body specific) and not againstPdp1ε(predominantly involved in circadian mechanisms).DrosophilaPDP1γprotein shows a high homology to mammalian thyrotroph embryonic factor (TEF), albumin D site-binding protein (DBP), and hepatic leukemia factor (HLF) transcription factors. In an in vitro model of drug- (olanzapine-) induced adiposity in mouse 3T3-L1 cells, the mRNA content of HLF but not TEF and DBP was increased by the drug treatment. A knockdown of the HLF mRNA by transfecting the cultures with HLF dsRNA significantly reduced their lipid content. Furthermore, the HLF RNAi prevented olanzapine from increasing the cell lipid content. These results suggest that the PDP1/HLF system may play a role in physiological and drug-influenced lipid regulation.

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Höhn, Britta, Harris S. Soifer, Stephen J. Forman i John J. Rossi. "Bifunctional siRNA Design Targeting Non-Hodgkin’s Lymphoma." Blood 110, nr 11 (16.11.2007): 2357. http://dx.doi.org/10.1182/blood.v110.11.2357.2357.

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Abstract Non-Hodgkin’s lymphomas comprise a group of heterogeneous lymphoid malignancies that represent the fifth most common form of cancer in the United States. A hallmark of many types of B-cell lymphomas is the constitutive expression of oncogenes such as the transcription factors Bcl-6, STAT3 and c-Myc and the anti-apoptotic protein Bcl-2. Over expression of these genes causes uncontrolled proliferation and survival of malignant cells, making knockdown of these genes by RNA interference (RNAi) a rational strategy for therapeutic intervention. RNAi is a conserved endogenous mechanism in which small interfering RNAs (siRNAs) suppress target-specific gene expression by promoting mRNA degradation. We have designed potent Dicer-substrate siRNAs using different computer algorithms to predict accessible target sites in the mRNAs of B-cell lymphoma oncogene targets. The Dicer-substrate 27mers are designed asymmetrically, so that Dicer processing yields a predicted 21mer siRNA duplex for entry into RNA induced silencing complex (RISC). Dicer-substrate siRNAs show improved efficacy at lower concentrations compared with conventional 21mer siRNAs, suggesting the reduction of potential off-target effects. In addition, we have designed bifunctional siRNA duplexes that contain two fully target-complimentary antisense strands against two different target mRNAs, but that are only partially complementary to each other. In vitro cleavage assays indicate that our bifunctional siRNAs have sufficient complementarity to form stable duplexes and can be processed into smaller molecules by recombinant Dicer. When delivered to the Burkitt’s lymphoma cell line Raji by electroporation, the most effective siRNAs reduced target mRNA levels by ∼80% as determined by quantitative RT-PCR and immunoblot analysis. Silencing of transcription factors affected the expression of downstream target genes, indicating a relevant effect on growth on survival of lymphoma cells through oncogene down regulation by RNAi. One concern with RNAi-mediated therapy is the possible recognition of siRNA duplex by cell’s own response to double-stranded RNA (dsRNA) that could trigger an unwanted interferon response. To determine whether our Dicer-substrate siRNAs cause an interferon response, we monitored induction of the cellular dsRNA pathway by measuring gene expression of p56, OAS1 and interferon by quantitative RT-PCR after transfection of siRNAs in different cell lines. None of the analyzed siRNAs show a significant increase in the expression of interferon pathway related genes indicating that our selected siRNAs are powerful silencers of gene expression without inducing an interferon response. In future studies, these new identified siRNAs will be incorporated in nanoparticles or attached to antibodies / aptamers for cell-specific delivery to lymphoma cell lines to evaluate their potential alone or in combination with chemotherapeutic drugs in therapy for lymphoma.

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Filip, Anca T., Ovidiu Balacescu, Catalin Marian i Andrei Anghel. "Microbiota Small RNAs in Inflammatory Bowel Disease". Journal of Gastrointestinal and Liver Diseases 25, nr 4 (1.12.2016): 509–16. http://dx.doi.org/10.15403/jgld.2014.1121.254.lip.

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MiRNAs are a class of potential gene regulators of critical importance in Inflammatory Bowel Disease (IBD). This review aims to present the connection between gut microbiota, probiotics administration and microRNA (miRNA) expression in IBD. It also brings into question cross-kingdom RNAi (RNA interference). Not only that gut host cells garden the intestinal microbiome via miRNA, but also strong evidence supports the idea that different species of bacteria have an impact on the intestinal immune response by modulating miRNA expression. Cross-kingdom RNAi refers to RNA silencing signals that travel between two unrelated, interacting organisms. RNAs communication between prokaryotes and eukaryotes (bacteria and nematodes) via RNAs transfer has been proved. Some authors also support the idea that non-coding RNAs are being transferred by bacterial pathogens to the host cells as part of the intracellular infection process. Further studies are required in order to clarify whether the mechanism by which bacteria modulate miRNA expression concerns RNAs transfer. These findings may lead to a different approach to IBD therapy in the future. Abbreviations: AChE: Acetylcholinesterase; AIEC: Adherent-invasive E coli; ATF: Activating transcription factor; Bcl-2: B-cell lymphoma 2; BMDC: Bone marrow-derived dendritic cells; C elegans: Caenorhabditis elegans; CCL: Chemokine C-C motif ligand; CD: Crohn’s disease; CDC42: Cell division control protein 42 homolog; CXCL: Chemokine (C-X-C motif) ligand; DC: Dendritic cells; E Coli: Escherichia coli; EcN: E coli Nissle; EPEC: Entheropathogenic E coli; FOXO3: Forkhead box protein O3; GF: Germ-free; IBD: Inflammatory bowel disease; IECs: Intestinal epithelial cells; IGLC: Immunoglobulin Lambda Constant Region; IkB: Inhibitor of NF-Kb; IL: Interleukin; IRGM: Immunity-related GTPase family M protein; L del: Lactobacillus delbrueckii; LGG: Lactobacillus rhamnosus GG; MAPK: Mitogen-activated protein kinases; miRNA: MicroRNA; mRNA: Messenger RNA; MyD88: Myeloid differentiation primary response gene 88; NF-kB: Nuclear factor kappa B; NOD2: Nucleotide-binding oligomerization domain-containing protein 2; PAR: Partitioning defective protein; RhoB: Ras Homolog Family Member B; RISC: RNA induced silencing complex; RNAi: RNA interference; SHH: Sonic hedgehog (gene); SPF: Specific-pathogen-free; SRNA: Small RNA; STAT3: Signal transducer and activator of transcription 3; TGF: Transforming growth factor; Th17: T helper 17 cells; TJ: Tight junction; TLR: Toll like receptor; TNF: Tumor necrosis factor; UC: Ulcerative colitis; Xcv: Xanthomonas campestris pv. Vesicatoria; ZO-2: Zonula occludens-2.

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