RDE-2 interacts with MUT-7 to mediate RNA interference in Caenorhabditis elegans.

Science.gov (United States)

Tops, Bastiaan B J; Tabara, Hiroaki; Sijen, Titia; Simmer, Femke; Mello, Craig C; Plasterk, Ronald H A; Ketting, René F

2005-01-01

In Caenorhabditis elegans, the activity of transposable elements is repressed in the germline. One of the mechanisms involved in this repression is RNA interference (RNAi), a process in which dsRNA targets cleavage of mRNAs in a sequence-specific manner. The first gene found to be involved in RNAi and transposon silencing in C.elegans is mut-7, a gene encoding a putative exoribonuclease. Here, we show that the MUT-7 protein resides in complexes of approximately 250 kDa in the nucleus and in the cytosol. In addition, we find that upon triggering of RNAi the cytosolic MUT-7 complex increases in size. This increase is independent of the presence of target RNA, but does depend on the presence of RDE-1 and RDE-4, two proteins involved in small interfering RNA (siRNA) production. Finally, using a yeast two-hybrid screen, we identified RDE-2/MUT-8 as one of the other components of this complex. This protein is encoded by the rde-2/mut-8 locus, previously implicated in RNAi and transposon silencing. Using genetic complementation analysis, we show that the interaction between these two proteins is required for efficient RNAi in vivo. Together these data support a role for the MUT-7/RDE-2 complex downstream of siRNA formation, but upstream of siRNA mediated target RNA recognition, possibly indicating a role in the siRNA amplification step.

[Construction of lentiviral mediated CyPA siRNA and its functions in non-small cell lung cancer].

Science.gov (United States)

FENG, Yan-ming; WU, Yi-ming; TU, Xin-ming; XU, Zheng-shun; WU, Wei-dong

2010-02-01

To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer. First, four target sequences were selected according to CyPA mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA, and it was confirmed by PCR and sequencing. Next, it was cotransfected by Lipofectamine 2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles. At the same time, the packed virus infected non-small cell lung cancer cell (A549), the level of CyPA protein at 5 d after infection was detected by Western Blot to screen the target of CyPA. A549 were infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice. Cell cycle and apoptosis were measured by FCM. It was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully. The titer of concentrated virus were 1 x 10(7) TU/ml. Flow cytometric analysis demonstrated G2-M phase (11.40% +/- 0.68%) was decreased relatively in A549/LvshCyPA compared with control groups (14.52% +/- 1.19%) (Ppathways may lead to new targeted therapies for non-small cell lung cancer.

The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

International Nuclear Information System (INIS)

Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule; Rustin, Pierre

2011-01-01

Highlights: → NDUFB6 is required for activity of mitochondrial complex I in human cell lines. → Lentivirus based RNA interference results in frequent off target insertions. → Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

Energy Technology Data Exchange (ETDEWEB)

Loublier, Sandrine; Bayot, Aurelien; Rak, Malgorzata; El-Khoury, Riyad; Benit, Paule [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France); Rustin, Pierre, E-mail: pierre.rustin@inserm.fr [Inserm U676, Hopital Robert Debre, F-75019 Paris (France); Universite Paris 7, Faculte de medecine Denis Diderot, IFR02 Paris (France)

2011-10-22

Highlights: {yields} NDUFB6 is required for activity of mitochondrial complex I in human cell lines. {yields} Lentivirus based RNA interference results in frequent off target insertions. {yields} Flp-In recombinase mediated miRNA insertion allows gene-specific extinction. -- Abstract: Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.

Understanding the core of RNA interference: The dynamic aspects of Argonaute-mediated processes

KAUST Repository

Zhu, Lizhe

2016-10-05

At the core of RNA interference, the Argonaute proteins (Ago) load and utilize small guide nucleic acids to silence mRNAs or cleave foreign nucleic acids in a sequence specific manner. In recent years, based on extensive structural studies of Ago and its interaction with the nucleic acids, considerable progress has been made to reveal the dynamic aspects of various Ago-mediated processes. Here we review these novel insights into the guide-strand loading, duplex unwinding, and effects of seed mismatch, with a focus on two representative Agos, the human Ago 2 (hAgo2) and the bacterial Thermus thermophilus Ago (TtAgo). In particular, comprehensive molecular simulation studies revealed that although sharing similar overall structures, the two Agos have vastly different conformational landscapes and guide-strand loading mechanisms because of the distinct rigidity of their L1-PAZ hinge. Given the central role of the PAZ motions in regulating the exposure of the nucleic acid binding channel, these findings exemplify the importance of protein motions in distinguishing the overlapping, yet distinct, mechanisms of Ago-mediated processes in different organisms.

CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

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Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

2014-05-06

In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K, E-mail: Jamboor.vishwanatha@unthsc.edu [Department of Molecular Biology and Immunology and Institute for Cancer Research, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

2011-11-04

Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high ({approx}97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

Science.gov (United States)

Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.

2011-11-01

Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

International Nuclear Information System (INIS)

Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K

2011-01-01

Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (∼97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

The rde-1 gene, RNA interference, and transposon silencing in C. elegans.

Science.gov (United States)

Tabara, H; Sarkissian, M; Kelly, W G; Fleenor, J; Grishok, A; Timmons, L; Fire, A; Mello, C C

1999-10-15

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

Lentivirus-mediated Knockdown of HDAC1 Uncovers Its Role in Esophageal Cancer Metastasis and Chemosensitivity

OpenAIRE

Song, Min; He, Gang; Wang, Yan; Pang, Xueli; Zhang, Bo

2016-01-01

Histone deacetylationase 1 (HDAC1) is ubiquitously expressed in various cell lines and tissues and play an important role of regulation gene expression. Overexpression of HDAC1 has been observed in various types of cancers, which indicated that it might be a target for cancer therapy. To test HDAC1 inhibition for cancer treatment, the gene expression of HDAC1 was knockdown mediated by a lentivirus system. Our data showed the gene expression of HDAC1 could be efficiently knockdown by RNAi medi...

Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system

Directory of Open Access Journals (Sweden)

Verhaagen Joost

2010-02-01

Full Text Available Abstract Background After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs and several axon guidance molecules, including all members of the secreted (class 3 Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV mediated expression of short hairpin RNAs (shRNAs to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1 and Neuropilin 2 (Npn-2. Results We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents. Conclusions RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.

The Role of Repeat Administration of Adventitial Delivery of Lentivirus-shRNA-Vegf-A in Arteriovenous Fistula to Prevent Venous Stenosis Formation.

Science.gov (United States)

Janardhanan, Rajiv; Yang, Binxia; Kilari, Sreenivasulu; Leof, Edward B; Mukhopadhyay, Debabrata; Misra, Sanjay

2016-04-01

To determine if a second dose of a lentivirus mediated small hairpin RNA that inhibits Vegf-A gene expression (LV-shRNA-Vegf-A) can improve lumen vessel area (LVA) of the outflow vein of an arteriovenous fistula (AVF) and decrease venous neointimal hyperplasia. Chronic kidney disease was created in C57BL/6 mice; 28 days later, an AVF was created by connecting the right carotid artery to the ipsilateral jugular vein. Immediately after AVF creation, 5 × 10(6) plaque-forming units of LV-shRNA-Vegf-A or control shRNA was administered to the adventitia of the outflow vein, and a second dose of the same treatment was administered 14 days later. Animals were sacrificed at 21 days, 28 days, and 42 days after AVF creation for reverse transcription polymerase chain reaction and histomorphometric analyses. By day 21, there was a 125% increase in the average LVA (day 21, P = .11), with a decrease in cell proliferation (day 21, P = .0079; day 28, P = .28; day 42, P = .5), decrease in α-smooth muscle cell actin staining (day 21, P < .0001; day 28, P < .05; day 42, P = .59), and decrease in hypoxic stress (day 21, P < .001; day 28, P = .28; day 42, P = .46) in LV versus control shRNA vessels. A second dose of LV-shRNA-Vegf-A administration results in a moderate improvement in LVA at day 21. Copyright © 2016 SIR. Published by Elsevier Inc. All rights reserved.

RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells

Directory of Open Access Journals (Sweden)

Liu X

2016-04-01

Full Text Available Xiaoxia Liu, Guiling Sun, Xiuju Sun Department of Nephrology, Affiliated Hospital of Weifang Medical University, Weifang, People’s Republic of China Abstract: This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP gene on renal cell cancer (RCC cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte–macrophage colony-stimulating factor and E-cadherin was significantly increased (P<0.05. The relevant signaling pathways were the integrin-mediated cell surface interactions pathway and extracellular matrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial

Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.

Science.gov (United States)

Reed, Jonathan C; Westergreen, Nick; Barajas, Brook C; Ressler, Dylan T B; Phuong, Daryl J; Swain, John V; Lingappa, Vishwanath R; Lingappa, Jaisri R

2018-05-01

During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here we asked whether feline immunodeficiency virus (FIV), a nonprimate lentivirus, also forms RNA granule-derived capsid assembly intermediates. First, we showed that the released FIV immature capsid and a large FIV Gag-containing intracellular complex are unstable during analysis, unlike for HIV-1. We identified harvest conditions, including in situ cross-linking, that overcame this problem, revealing a series of FIV Gag-containing complexes corresponding in size to HIV-1 assembly intermediates. Previously, we showed that assembly-defective HIV-1 Gag mutants are arrested at specific assembly intermediates; here we identified four assembly-defective FIV Gag mutants, including three not previously studied, and demonstrated that they appear to be arrested at the same intermediate as the cognate HIV-1 mutants. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from coimmunoprecipitations demonstrating that endogenous ABCE1 and the RNA granule protein DDX6 are associated with FIV Gag, as shown previously for HIV-1 Gag, but are not associated with a ribosomal protein, at steady state. Additionally, we showed that FIV Gag associates with another RNA granule protein, DCP2. Finally, we validated the FIV Gag-ABCE1 and FIV Gag-DCP2 interactions with proximity ligation assays demonstrating colocalization in situ Together, these data support a model in which primate and nonprimate lentiviruses form intracellular capsid assembly intermediates derived from nontranslating host RNA granules. IMPORTANCE Like HIV-1 Gag, FIV Gag assembles into immature capsids; however, it is not known whether

A primitive endogenous lentivirus in a colugo: insights into the early evolution of lentiviruses.

Science.gov (United States)

Han, Guan-Zhu; Worobey, Michael

2015-01-01

Lentiviruses infect a wide range of mammal species. Much remains unknown about their deep history and host distribution. Here, we report the discovery of an endogenous lentivirus within the genome of the Sunda flying lemur (Galeopterus variegatus) (which we designate "Galeopterus variegatus endogenous lentivirus" [GvaELV]). We estimate the GvaELV genome invasion to have occurred more than 14 Ma, supporting an ancient origin of the lentivirus clade and an ancient lentiviral infection in colugo. Phylogenetic analyses show that GvaELV is a sister group of all previously known lentiviruses. The GvaELV genome appears to possess some primitive genomic features of a lentivirus, encoding not only a trans-activator of transcription (tat) gene but also two additional putative accessory genes that share no discernible similarity with other lentiviral accessory genes. The discovery of GvaELV provides novel insights into the prehistory and host distribution of lentivirus. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The Role of RNA Interference (RNAi in Arbovirus-Vector Interactions

Directory of Open Access Journals (Sweden)

Carol D. Blair

2015-02-01

Full Text Available RNA interference (RNAi was shown over 18 years ago to be a mechanism by which arbovirus replication and transmission could be controlled in arthropod vectors. During the intervening period, research on RNAi has defined many of the components and mechanisms of this antiviral pathway in arthropods, yet a number of unexplored questions remain. RNAi refers to RNA-mediated regulation of gene expression. Originally, the term described silencing of endogenous genes by introduction of exogenous double-stranded (dsRNA with the same sequence as the gene to be silenced. Further research has shown that RNAi comprises three gene regulation pathways that are mediated by small RNAs: the small interfering (siRNA, micro (miRNA, and Piwi-interacting (piRNA pathways. The exogenous (exo-siRNA pathway is now recognized as a major antiviral innate immune response of arthropods. More recent studies suggest that the piRNA and miRNA pathways might also have important roles in arbovirus-vector interactions. This review will focus on current knowledge of the role of the exo-siRNA pathway as an arthropod vector antiviral response and on emerging research into vector piRNA and miRNA pathway modulation of arbovirus-vector interactions. Although it is assumed that arboviruses must evade the vector’s antiviral RNAi response in order to maintain their natural transmission cycles, the strategies by which this is accomplished are not well defined. RNAi is also an important tool for arthropod gene knock-down in functional genomics studies and in development of arbovirus-resistant mosquito populations. Possible arbovirus strategies for evasion of RNAi and applications of RNAi in functional genomics analysis and arbovirus transmission control will also be reviewed.

Accumulation of dsRNA in endosomes contributes to inefficient RNA interference in the fall armyworm, Spodoptera frugiperda.

Science.gov (United States)

Yoon, June-Sun; Gurusamy, Dhandapani; Palli, Subba Reddy

2017-11-01

RNA interference (RNAi) efficiency varies among insects studied. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. We recently showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into small interference RNAs (siRNAs) because they are trapped in acidic bodies. In the current study, we focused on the identification of acidic bodies in which dsRNAs accumulate in Sf9 cells. Time-lapse imaging studies showed that dsRNAs enter Sf9 cells and accumulate in acidic bodies within 20 min after their addition to the medium. CypHer-5E-labeled dsRNA also accumulated in the midgut and fat body dissected from Spodoptera frugiperda larvae with similar patterns observed in Sf9 cells. Pharmacological inhibitor assays showed that the dsRNAs use clathrin mediated endocytosis pathway for transport into the cells. We investigated the potential dsRNA accumulation sites employing LysoTracker and double labeling experiments using the constructs to express a fusion of green fluorescence protein with early or late endosomal marker proteins and CypHer-5E-labeled dsRNA. Interestingly, CypHer-5E-labeled dsRNA accumulated predominantly in early and late endosomes. These data suggest that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi response in lepidopteran insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Immune modulation through RNA interference-mediated silencing of CD40 in dendritic cells.

Science.gov (United States)

Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Samiee, Shahram; Ataee, Zahra; Tabei, Seyyed Ziyaoddin; Moazzeni, Seyed Mohammad

2009-01-01

RNA interference (RNAi) is an exciting mechanism for knocking down any target gene in transcriptional level. It is now clear that small interfering RNA (siRNA), a 19-21nt long dsRNA, can trigger a degradation process (RNAi) that specifically silences the expression of a cognate mRNA. Our findings in this study showed that down regulation of CD40 gene expression in dendritic cells (DCs) by RNAi culminated to immune modulation. Effective delivery of siRNA into DCs would be a reasonable method for the blocking of CD40 gene expression at the cell surface without any effect on other genes and cell cytotoxicity. The effects of siRNA against CD40 mRNA on the function and phenotype of DCs were investigated. The DCs were separated from the mice spleen and then cultured in vitro. By the means of Lipofectamine2000, siRNA was delivered to the cells and the efficacy of transfection was estimated by flow cytometry. By Annexine V and Propidium Iodide staining, we could evaluate the transfected cells viability. Also, the mRNA expression and protein synthesis were assessed by real-time PCR and flow cytometry, respectively. Knocking down the CD40 gene in the DCs caused an increase in IL-4 production, decrease in IL-12 production and allostimulation activity. All together, these effects would stimulate Th2 cytokines production from allogenic T-cells in vitro.

Induction of RNA interference in dendritic cells.

Science.gov (United States)

Li, Mu; Qian, Hua; Ichim, Thomas E; Ge, Wei-Wen; Popov, Igor A; Rycerz, Katarzyna; Neu, John; White, David; Zhong, Robert; Min, Wei-Ping

2004-01-01

Dendritic cells (DC) reside at the center of the immunological universe, possessing the ability both to stimulate and inhibit various types of responses. Tolerogenic/regulatory DC with therapeutic properties can be generated through various means of manipulations in vitro and in vivo. Here we describe several attractive strategies for manipulation of DC using the novel technique of RNA interference (RNAi). Additionally, we overview some of our data regarding yet undescribed characteristics of RNAi in DC such as specific transfection strategies, persistence of gene silencing, and multi-gene silencing. The advantages of using RNAi for DC genetic manipulation gives rise to the promise of generating tailor-made DC that can be used effectively to treat a variety of immunologically mediated diseases.

RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells.

Science.gov (United States)

Liu, Xiaoxia; Sun, Guiling; Sun, Xiuju

2016-01-01

This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP) gene on renal cell cancer (RCC) cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA)-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte-macrophage colony-stimulating factor and E-cadherin was significantly increased (Pmatrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial growth factor receptor, matrix metallopeptidase-9, and vascular cell adhesion molecule, which are related to the integrin-mediated cell surface interactions and extracellular matrix organization signaling pathway.

A simple and robust vector-based shRNA expression system used for RNA interference.

Science.gov (United States)

Wang, Xue-jun; Li, Ying; Huang, Hai; Zhang, Xiu-juan; Xie, Pei-wen; Hu, Wei; Li, Dan-dan; Wang, Sheng-qi

2013-01-01

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

Targeting CCl4 -induced liver fibrosis by RNA interference-mediated inhibition of cyclin E1 in mice.

Science.gov (United States)

Bangen, Jörg-Martin; Hammerich, Linda; Sonntag, Roland; Baues, Maike; Haas, Ute; Lambertz, Daniela; Longerich, Thomas; Lammers, Twan; Tacke, Frank; Trautwein, Christian; Liedtke, Christian

2017-10-01

Initiation and progression of liver fibrosis requires proliferation and activation of resting hepatic stellate cells (HSCs). Cyclin E1 (CcnE1) is the regulatory subunit of the cyclin-dependent kinase 2 (Cdk2) and controls cell cycle re-entry. We have recently shown that genetic inactivation of CcnE1 prevents activation, proliferation, and survival of HSCs and protects from liver fibrogenesis. The aim of the present study was to translate these findings into preclinical applications using an RNA interference (RNAi)-based approach. CcnE1-siRNA (small interfering RNA) efficiently inhibited CcnE1 gene expression in murine and human HSC cell lines and in primary HSCs, resulting in diminished proliferation and increased cell death. In C57BL/6 wild-type (WT) mice, delivery of stabilized siRNA using a liposome-based carrier targeted approximately 95% of HSCs, 70% of hepatocytes, and 40% of CD45 + cells after single injection. Acute CCl 4 -mediated liver injury in WT mice induced endogenous CcnE1 expression and proliferation of surviving hepatocytes and nonparenchymal cells, including CD45 + leukocytes. Pretreatment with CcnE1-siRNA reverted CcnE1 induction to baseline levels of healthy mice, which was associated with reduced liver injury, diminished proliferation of hepatocytes and leukocytes, and attenuated overall inflammatory response. For induction of liver fibrosis, WT mice were challenged with CCl 4 for 4-6 weeks. Co-treatment with CcnE1-siRNA once a week was sufficient to continuously block CcnE1 expression and cell-cycle activity of hepatocytes and nonparenchymal cells, resulting in significantly ameliorated liver fibrosis and inflammation. Importantly, CcnE1-siRNA also prevented progression of liver fibrosis if applied after onset of chronic liver injury. Therapeutic targeting of CcnE1 in vivo using RNAi is feasible and has high antifibrotic activity. (Hepatology 2017;66:1242-1257). © 2017 by the American Association for the Study of Liver Diseases.

A lentivirus-free inducible CRISPR-Cas9 system for efficient targeting of human genes.

Science.gov (United States)

Bisht, Kamlesh; Grill, Sherilyn; Graniel, Jacqueline; Nandakumar, Jayakrishnan

2017-08-01

CRISPR-Cas9 is a cutting-edge tool for modifying genomes. The efficacy with which Cas9 recognizes its target has revolutionized the engineering of knockouts. However this efficacy complicates the knocking out of important genes in cultured cells. Unedited cells holding a survival advantage within an edited population can confound the knockout phenotype. Here we develop a HeLa-based system that overcomes this limitation, incorporating several attractive features. First, we use Flp-recombinase to generate clones stably integrated for Cas9 and guide RNAs, eliminating the possibility of unedited cells. Second, Cas9 can be induced uniformly in the clonal cultures using doxycycline to measure the knockout phenotype. Third, two genes can be simultaneously knocked out using this approach. Finally, by not involving lentiviruses, our method is appealing to a broad research audience. Using this methodology we generated an inducible AGO2-knockout cell line showing normal RNA interference in the absence of doxycycline. Upon induction of Cas9, the AGO2 locus was cleaved, the AGO2 protein was depleted, and RNA interference was compromised. In addition to generating inducible knockouts, our technology can be adapted to improve other applications of Cas9, including transcriptional/epigenetic modulation and visualization of cellular DNA loci. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Intervention of radiation‐induced skin fibrosis by RNA interference

DEFF Research Database (Denmark)

Nawroth, Isabel

‐α (TNFα) production by macrophages might promote RIF. RNA interference (RNAi) is an evolutionary conserved gene‐silencing mechanism capable of degrading mRNA containing a homologous sequence to an exogenously introduced double stranded small interfering RNA (siRNA). These siRNAs can induce RNAi...... and inhibit the expression of target proteins. Therefore, siRNAs are considered as promising therapeutics for treatment of various diseases including genetic and viral diseases, and cancer. In this study, the therapeutic potential of RNA interference was investigated as an intervention strategy for radiation......‐induced skin fibrosis. Chitosan‐based nanoparticles (or polyplexes) formed by self‐assembly with siRNA were applied to overcome extracellular and intracellular barriers and deliver siRNA site‐specific. In this work we show that intraperitoneal administration of chitosan/DsiRNA nanoparticles targeting TNFα...

RNA virus interference via CRISPR/Cas13a system in plants

KAUST Repository

Aman, Rashid

2017-11-04

CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produced interference against green fluorescent protein (GFP) expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. crRNAs targeting the HC-Pro and GFP sequences exhibited better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses, and for other RNA manipulations in plants.

Using RNA Interference to Study Protein Function

OpenAIRE

Curtis, Carol D.; Nardulli, Ann M.

2009-01-01

RNA interference can be extremely useful in determining the function of an endogenously-expressed protein in its normal cellular environment. In this chapter, we describe a method that uses small interfering RNA (siRNA) to knock down mRNA and protein expression in cultured cells so that the effect of a putative regulatory protein on gene expression can be delineated. Methods of assessing the effectiveness of the siRNA procedure using real time quantitative PCR and Western analysis are also in...

RNA virus interference via CRISPR/Cas13a system in plants

KAUST Repository

Aman, Rashid

2018-01-04

CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants.CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs.Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

A simple and robust vector-based shRNA expression system used for RNA interference.

Directory of Open Access Journals (Sweden)

Xue-jun Wang

Full Text Available BACKGROUND: RNA interference (RNAi mediated by small interfering RNAs (siRNAs or short hairpin RNAs (shRNAs has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. RESULTS: In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. CONCLUSION: This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes.

Science.gov (United States)

Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

2016-01-01

Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.

Small Molecule Modifiers of the microRNA and RNA Interference Pathway

OpenAIRE

Deiters, Alexander

2009-01-01

Recently, the RNA interference (RNAi) pathway has become the target of small molecule inhibitors and activators. RNAi has been well established as a research tool in the sequence-specific silencing of genes in eukaryotic cells and organisms by using exogenous, small, double-stranded RNA molecules of approximately 20 nucleotides. Moreover, a recently discovered post-transcriptional gene regulatory mechanism employs microRNAs (miRNAs), a class of endogenously expressed small RNA molecules, whic...

Lentivirus-mediated knockdown of NLK inhibits small-cell lung cancer growth and metastasis

Directory of Open Access Journals (Sweden)

Lv MT

2016-11-01

Full Text Available Mutian Lv,1 Yaming Li,1 Xin Tian,2 Shundong Dai,3,4 Jing Sun,5 Guojiang Jin,6 Shenyi Jiang7 1Department of Nuclear Medicine, 2Molecular Oncology Laboratory of Cancer Research Institute, The First Affiliated Hospital of China Medical University, 3Department of Pathology, The First Affiliated Hospital, College of Basic Medical Sciences of China Medical University, 4Department of Pathology, Institute of Pathology and Pathophysiology, 5Department of Immunology and Biotherapy, Liaoning Cancer Hospital and Institute, 6Department of Laboratory Medicine, 7Department of Rheumatology, The First Affiliated Hospital of China Medical University, Shenyang, People’s Republic of China Abstract: Nemo-like kinase (NLK, an evolutionarily conserved serine/threonine kinase, has been recognized as a critical regulator of various cancers. In this study, we investigated the role of NLK in human small-cell lung cancer (SCLC, which is the most aggressive form of lung cancer. NLK expression was evaluated by quantitative real-time polymerase chain reaction in 20 paired fresh SCLC tissue samples and found to be noticeably elevated in tumor tissues. Lentivirus-mediated RNAi efficiently suppressed NLK expression in NCI-H446 cells, resulting in a significant reduction in cell viability and proliferation in vitro. Moreover, knockdown of NLK led to cell cycle arrest at the S-phase via suppression of Cyclin A, CDK2, and CDC25A, which could contribute to cell growth inhibition. Furthermore, knockdown of NLK decreased the migration of NCI-H446 cells and downregulated matrix metalloproteinase 9. Treatment with NLK short hairpin RNA significantly reduced SCLC tumor growth in vivo. In conclusion, this study suggests that NLK plays an important role in the growth and metastasis of SCLC and may serve as a potential therapeutic target for the treatment of SCLC. Keywords: NLK, SCLC, RNAi, proliferation, migration

Neuroprotective effects of lentivirus-mediated cystathionine-beta-synthase overexpression against 6-OHDA-induced parkinson's disease rats.

Science.gov (United States)

Yin, Wei-Lan; Yin, Wei-Guo; Huang, Bai-Sheng; Wu, Li-Xiang

2017-09-14

Parkinson's disease (PD) is age-related neurodegenerative disorder by a progressive loss of dopaminergic(DA) neurons in the substantia nigra (SN) and striatum, which is at least partly associated with α-synuclein protein accumulation in these neurons. Hydrogen sulfide (H 2 S) plays an important role in the nervous system. Studies have shown that H 2 S has a protective effect on PD. However, as a kind of gas molecules, H 2 S is lively, volatile, and not conducive to scientific research and clinical application. Cystathionine-beta-synthase(CBS) is the main enzymes of synthesis of H 2 S in the brain. In order to examine the neuroprotective effects of CBS on PD, we detected the effects of CBS overexpression on 6-Hydroxydopamine (6-OHDA)-lesioned PD rats using lentivirus-mediated gene transfection techniques. In the injured SN of 6-OHDA-induced PD rats, the CBS expression and the endogenous H 2 S level markedly decreased, while administration of lentivirus-mediated CBS overexpression increased the CBS expression and the endogenous H 2 S production.CBS overexpression dramatically reversed apomorphine-induced rotation of the 6-OHDA model rats, decreased the number of TUNEL-positive neurons and the loss of the nigral DA neurons，specifically inhibited 6-OHDA-induced oxidase stress injury, and down-regulated the expression of α-synuclein(α-SYN) in the injured SN. NaHS (an H 2 S donor) had similar effects to CBS overexpression, while Amino-oxyacetate(AOAA, a CBS inhibitor) had opposite effects on PD rats. In summary, we demonstrated that CBS overexpression was able to provide neuroprotective on PD rats and improving the expression of CBS may be a potential therapeutic method for PD. Copyright © 2017. Published by Elsevier B.V.

Radiation enhancement effect of RNA interference for HIF-1α on the transplant tumor

International Nuclear Information System (INIS)

Ren Ruimei; Sun Xindong; Zhao Hanxi; Yan Qingxia; Huang Guangwu

2008-01-01

Objective: To determine and explore the radiation enhancement of RNA interference for HIF-1α on the transplant tumor using polycationic polyethylenimine (PEI), as a new kind of gene vector. Methods: SPCA-1 nude mouse model was used. 160 nude mice bearing SPCA-1 were randomly divided into 4 treated groups and 1 control groups, each group had 32 mice. The expression of HIF-1α was studied by immunohistochemical method after RNA interference for HIF-1α. The differences of the volume, weight, survival time of the transplant tumor were studied among the simple radiation group, the simple RNA interference for HIF- 1α group and the combination of radiation and RNA interference for HIF-1α. Results: The expression of HIF-1α was decreased after RNA interference for HIF-1α. RNA interference for HIF-1α combined with radiation decreased the volume, weight of the transplant tumor, and prolonged its survival time period significantly than other methods. Conclusions: RNA interference targeting HIF-1α might enhance the radiosensitivity of the transplant tumor using PEI as a new kind of gene vector in vitro. (authors)

Influence of silencing the MC4R gene by lentivirusmediated RNA ...

African Journals Online (AJOL)

Melanocortin receptor 4 (MC4R) is a key element in the mechanisms used to regulate both aspects of keeping the balance between energy uptake and energy expenditure. MC4R was knocked down by lentivirus-mediated shRNA expressing plasmids, which were controlled by the U6 promoter in bovine fibroblast cells, and ...

Effects of deoxycycline induced lentivirus encoding FasL gene on ...

African Journals Online (AJOL)

Abstract. Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in deletion of activated T cells. This study aimed to construct the lentivirus encoding FasL gene induced by deoxycycline and evaluate its effects on apoptosis of Th1 cells. A plasmid expression system encoding FasL was constructed through utilizing the ...

Study of RNA interference inhibiting rat ovarian androgen biosynthesis by depressing 17alpha-hydroxylase/17, 20-lyase activity in vivo

Directory of Open Access Journals (Sweden)

Yang Xing

2009-07-01

Full Text Available Abstract Background 17alpha-hydroxylase/17, 20-lyase encoded by CYP17 is the key enzyme in androgen biosynthesis pathway. Previous studies demonstrated the accentuation of the enzyme in patients with polycystic ovary syndrome (PCOS was the most important mechanism of androgen excess. We chose CYP17 as the therapeutic target, trying to suppress the activity of 17alpha-hydroxylase/17, 20-lyase and inhibit androgen biosynthesis by silencing the expression of CYP17 in the rat ovary. Methods Three CYP17-targeting and one negative control oligonucleotides were designed and used in the present study. The silence efficiency of lentivirus shRNA was assessed by qRT-PCR, Western blotting and hormone assay. After subcapsular injection of lentivirus shRNA in rat ovary, the delivery efficiency was evaluated by GFP fluorescence and qPCR. Total RNA was extracted from rat ovary for CYP17 mRNA determination and rat serum was collected for hormone measurement. Results In total, three CYP17-targeting lentivirus shRNAs were synthesized. The results showed that all of them had a silencing effect on CYP17 mRNA and protein. Moreover, androstenedione secreted by rat theca interstitial cells (TIC in the RNAi group declined significantly compared with that in the control group. Two weeks after rat ovarian subcapsular injection of chosen CYP17 shRNA, the GFP fluorescence of frozen ovarian sections could be seen clearly under fluorescence microscope. It also showed that the GFP DNA level increased significantly, and its relative expression level was 7.42 times higher than that in the control group. Simultaneously, shRNA treatment significantly decreased CYP17 mRNA and protein levels at 61% and 54%, respectively. Hormone assay showed that all the levels of androstenedione, 17-hydroxyprogesterone and testosterone declined to a certain degree, but progesterone levels declined significantly. Conclusion The present study proves for the first time that ovarian androgen

Neuron-specific RNA interference using lentiviral vectors

DEFF Research Database (Denmark)

Nielsen, Troels Tolstrup; Marion, Ingrid van; Hasholt, Lis

2009-01-01

BACKGROUND: Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise...... transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding...... the possible promoter choices and eventually allowing cell type specific down-regulation of target genes. METHODS: In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell culture and in the brain. RESULTS: We...

Prokaryotic Argonautes - variations on the RNA interference theme

Science.gov (United States)

van der Oost, John; Swarts, Daan C.; Jore, Matthijs M.

2014-01-01

The discovery of RNA interference (RNAi) has been a major scientific breakthrough. This RNA-guided RNA interference system plays a crucial role in a wide range of regulatory and defense mechanisms in eukaryotes. The key enzyme of the RNAi system is Argonaute (Ago), an endo-ribonuclease that uses a small RNA guide molecule to specifically target a complementary RNA transcript. Two functional classes of eukaryotic Ago have been described: catalytically active Ago that cleaves RNA targets complementary to its guide, and inactive Ago that uses its guide to bind target RNA to down-regulate translation efficiency. A recent comparative genomics study has revealed that Argonaute-like proteins are also encoded by prokaryotic genomes. Interestingly, there is a lot of variation among these prokaryotic Argonaute (pAgo) proteins with respect to domain architecture: some resemble the eukaryotic Ago (long pAgo) containing a complete or disrupted catalytic site, while others are truncated versions (short pAgo) that generally contain an incomplete catalytic site. Prokaryotic Agos with an incomplete catalytic site often co-occur with (predicted) nucleases. Based on this diversity, and on the fact that homologs of other RNAi-related protein components (such as Dicer nucleases) have never been identified in prokaryotes, it has been predicted that variations on the eukaryotic RNAi theme may occur in prokaryotes. PMID:28357239

Suppression of the expression of hypoxia-inducible factor-1α by RNA interference alleviates hypoxia-induced pulmonary hypertension in adult rats.

Science.gov (United States)

Li, Ying; Shi, Bo; Huang, Liping; Wang, Xin; Yu, Xiaona; Guo, Baosheng; Ren, Weidong

2016-12-01

Hypoxia-inducible factor-1α (HIF-1α) has been implicated in the pathogenesis of hypoxic pulmonary hypertension (PH). However, the potential clinical value of HIF-1α as a therapeutic target in the treatment of PH has not yet been evaluated. In this study, an animal model of hypoxia-induced PH was established by exposing adult rats to 10% O2 for 3 weeks, and the effects of the lentivirus-mediated delivery of HIF-1α short hairpin RNA (shRNA) by intratracheal instillation prior to exposure to hypoxia on the manifestations of hypoxia-induced PH were assessed. The successful delivery of HIF-1α shRNA into the pulmonary arteries effectively suppressed the hypoxia-induced upregulation of HIF-1α, accompanied by the prominent attenuation the symptoms associated with hypoxia-induced PH, including the elevation of pulmonary arterial pressure, hypertrophy and hyperplasia of pulmonary artery smooth muscle cells (PASMCs), as well as the muscularization of pulmonary arterioles. In addition, the knockdown of HIF-1α in cultured rat primary PASMCs significantly inhibited the hypoxia-induced acceleration of the cell cycle and the proliferation of the PASMCs, suggesting that HIF-1α may be a direct mediator of PASMC hyperplasia in hypoxia-induced PH. In conclusion, this study demonstrates the potent suppressive effects of HIF-1α shRNA on hypoxia-induced PH and PASMC hyperplasia, providing evidence for the potential application of HIF-1α shRNA in the treatment of hypoxic PH.

RNA virus interference via CRISPR/Cas13a system in plants

KAUST Repository

Aman, Rashid; Ali, Zahir; Butt, Haroon; Mahas, Ahmed; Aljedaani, Fatimah R.; Khan, Muhammad Zuhaib; Ding, Shouwei; Mahfouz, Magdy M.

2018-01-01

-crRNAs into functional crRNAs.Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.

Manipulation of saponin biosynthesis by RNA interference-mediated silencing of β-amyrin synthase gene expression in soybean.

Science.gov (United States)

Takagi, Kyoko; Nishizawa, Keito; Hirose, Aya; Kita, Akiko; Ishimoto, Masao

2011-10-01

Soybean seeds contain substantial amount of diverse triterpenoid saponins that influence the seed quality, although little is known about the physiologic functions of saponins in plants. We now describe the modification of saponin biosynthesis by RNA interference (RNAi)-mediated gene silencing targeted to β-amyrin synthase, a key enzyme in the synthesis of a common aglycon of soybean saponins. We identified two putative β-amyrin synthase genes in soybean that manifested distinct expression patterns with regard to developmental stage and tissue specificity. Given that one of these genes, GmBAS1, was expressed at a much higher level than the other (GmBAS2) in various tissues including the developing seeds, we constructed two RNAi vectors that encode self-complementary hairpin RNAs corresponding to the distinct regions of GmBAS1 under the control of a seed-specific promoter derived from the soybean gene for the α' subunit of the seed storage protein β-conglycinin. These vectors were introduced independently into soybean. Six independent transgenic lines exhibited a stable reduction in seed saponin content, with the extent of saponin deficiency correlating with the β-amyrin synthase mRNA depletion. Although some transgenic lines produced seeds almost devoid of saponins, no abnormality in their growth was apparent and the antioxidant activity of their seeds was similar to that of control seeds. These results suggest that saponins are not required for seed development and survival, and that soybean seeds may therefore be amenable to the modification of triterpenoid saponin content and composition through molecular biologic approaches.

RNA interference in designing transgenic crops.

Science.gov (United States)

Ali, Nusrat; Datta, Swapan K; Datta, Karabi

2010-01-01

RNA interference (RNAi) is a sequence specific gene silencing mechanism, triggered by the introduction of dsRNA leading to mRNA degradation. It helps in switching on and off the targeted gene, which might have significant impact in developmental biology. Discovery of RNAi represents one of the most promising and rapidly advancing frontiers in plant functional genomics and in crop improvement by plant metabolic engineering and also plays an important role in reduction of allergenicity by silencing specific plant allergens. In plants the RNAi technology has been employed successfully in improvement of several plant species- by increasing their nutritional value, overall quality and by conferring resistance against pathogens and diseases. The review gives an insight to the perspective use of the technology in designing crops with innovation, to bring improvement to crop productivity and quality.

Inhibition of virus replication by RNA interference

NARCIS (Netherlands)

Haasnoot, P. C. Joost; Cupac, Daniel; Berkhout, Ben

2003-01-01

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene

Persistent interferon transgene expression by RNA interference-mediated silencing of interferon receptors.

Science.gov (United States)

Takahashi, Yuki; Vikman, Elin; Nishikawa, Makiya; Ando, Mitsuru; Watanabe, Yoshihiko; Takakura, Yoshinobu

2010-09-01

The in vivo half-life of interferons (IFNs) is very short, and its extension would produce a better therapeutic outcome in IFN-based therapy. Delivery of IFN genes is one solution for providing a sustained supply. IFNs have a variety of functions, including the suppression of transgene expression, through interaction with IFN receptors (IFNRs). This suppression could prevent IFNs from being expressed from vectors delivered. Silencing the expression of IFNAR and IFNGR, the receptors for type I and II IFNs, respectively, in cells expressing IFNs may prolong transgene expression of IFNs. Mouse melanoma B16-BL6 cells or mouse liver were selected as a site expressing IFNs (not a target for IFN gene therapy) and IFN-expressing plasmid DNA was delivered with or without small interfering RNA (siRNA) targeting IFNRs. Transfection of B16-BL6 cells with siRNA targeting IFNAR1 subunit (IFNAR1) resulted in the reduced expression of IFNAR on the cell surface. This silencing significantly increased the IFN-beta production in cells that were transfected with IFN-beta-expressing plasmid DNA. Similar results were obtained with the combination of IFN-gamma and IFNGR. Co-injection of IFN-beta-expressing plasmid DNA with siRNA targeting IFNAR1 into mice resulted in sustained plasma concentration of IFN-beta. These results provide experimental evidence that the RNAi-mediated silencing of IFNRs in cells expressing IFN, such as hepatocytes, is an effective approach for improving transgene expression of IFNs when their therapeutic target comprises cells other than those expressing IFNs.

RNA interference: its use as antiviral therapy

NARCIS (Netherlands)

Haasnoot, J.; Berkhout, B.

2006-01-01

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism that has been proposed to function as a defence mechanism of eukaryotic cells against viruses and transposons. RNAi was first observed in plants in the form of a mysterious immune response to viral pathogens. But RNAi is more

Reversal of pathology in CHMP2B-mediated frontotemporal dementia patient cells using RNA interference

DEFF Research Database (Denmark)

Nielsen, Troels Tolstrup; Mizielinska, Sarah; Hasholt, Lis

2012-01-01

role in the pathogenesis of the disease. METHODS: In the present study, we used lentiviral vectors to efficiently knockdown CHMP2B by delivering microRNA embedded small hairpin RNAs. RESULTS: We show that CHMP2B can be efficiently knocked down in patient fibroblasts using an RNA interference approach......BACKGROUND: Frontotemporal dementia is the second most common form of young-onset dementia after Alzheimer's disease, and several genetic forms of frontotemporal dementia are known. A rare genetic variant is caused by a point mutation in the CHMP2B gene. CHMP2B is a component of the ESCRT......-III complex, which is involved in endosomal trafficking of proteins targeted for degradation in lysosomes. Mutations in CHMP2B result in abnormal endosomal structures in patient fibroblasts and patient brains, probably through a gain-of-function mechanism, suggesting that the endosomal pathway plays a central...

MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

Science.gov (United States)

Zhong, Bushuai; Zhang, Yanli; Yan, Yibo; Wang, Ziyu; Ying, Shijia; Huang, Mingrui; Wang, Feng

2014-01-01

Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi) mediated by microRNAs (miRNAs) is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF) was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN) response genes, such as interferon beta (IFN-β) and 2′-5′-oligoadenylate synthetase 2 (OAS2), were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats. PMID:25244645

MicroRNA-mediated myostatin silencing in caprine fetal fibroblasts.

Directory of Open Access Journals (Sweden)

Bushuai Zhong

Full Text Available Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi mediated by microRNAs (miRNAs is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN response genes, such as interferon beta (IFN-β and 2'-5'-oligoadenylate synthetase 2 (OAS2, were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats.

Downregulation of mouse CCR3 by lentiviral shRNA inhibits proliferation and induces apoptosis of mouse eosinophils.

Science.gov (United States)

Zhu, Xin-Hua; Liao, Bing; Xu, Yi; Liu, Ke; Huang, Yun; Huang, Quan-Long; Liu, Yue-Hui

2017-02-01

RNA interference has been considered as an effective gene silencing method in basic and preclinical investigations. The aims of the present study were to construct a lentiviral vector expressing a short hairpin RNA (shRNA) targeting the murine CC chemokine receptor 3 (mCCR3), and to investigate its effects on the proliferation and apoptosis of mouse eosinophils. A recombinant lentiviral vector expressing four fragments of mouse CCR3 shRNA (pLVX‑mCCR3‑1+2+3+4‑shRNA) was constructed using subcloning techniques. This novel lentivirus was then packaged into 293T cells by co‑transduction with plasmids, including Baculo p35, pCMV R8.2 and VSV. The interference effects of the vector were verified using polymerase chain reaction (PCR) and western blot analyses. The effects of the interference on the proliferation and apoptosis of mouse eosinophils were investigated using 3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxymethoxyphenyl)‑2‑(4‑sulfophenyl)‑2H‑tetrazolium and terminal deoxynucleotidyl transferase dUTP nick end labeling methods, respectively. The results of the PCR and western blot analyses confirmed that the novel recombinant vector, pLVX‑mCCR3‑1+2+3+4‑shRNA, had high efficiency in inhibiting the mRNA and protein expression levels of mCCR3 in mouse eosinophils. The downregulation of mCCR3 significantly inhibited proliferation of the eosinophils. Furthermore, the present study found that the downregulation of mCCR3 significantly promoted apoptosis of the eosinophils. Therefore, the downregulation of mCCR3 led to the inhibition of proliferation and induction of apoptosis in mouse eosinophils. The predominant characteristics of allergic rhinitis are eosinophil infiltration and release of inflammatory mediators, which appear in a variety of clinical manifestations. The results of the present study indicate that mCCR3 silencing may serve as a putative approach for the treatment of allergic rhinitis.

RNA interference and Register Machines (extended abstract

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Masahiro Hamano

2012-11-01

Full Text Available RNA interference (RNAi is a mechanism whereby small RNAs (siRNAs directly control gene expression without assistance from proteins. This mechanism consists of interactions between RNAs and small RNAs both of which may be single or double stranded. The target of the mechanism is mRNA to be degraded or aberrated, while the initiator is double stranded RNA (dsRNA to be cleaved into siRNAs. Observing the digital nature of RNAi, we represent RNAi as a Minsky register machine such that (i The two registers hold single and double stranded RNAs respectively, and (ii Machine's instructions are interpreted by interactions of enzyme (Dicer, siRNA (with RISC com- plex and polymerization (RdRp to the appropriate registers. Interpreting RNAi as a computational structure, we can investigate the computational meaning of RNAi, especially its complexity. Initially, the machine is configured as a Chemical Ground Form (CGF, which generates incorrect jumps. To remedy this problem, the system is remodeled as recursive RNAi, in which siRNA targets not only mRNA but also the machine instructional analogues of Dicer and RISC. Finally, probabilistic termination is investigated in the recursive RNAi system.

Feline glycoprotein A repetitions predominant anchors transforming growth factor beta on the surface of activated CD4(+)CD25(+) regulatory T cells and mediates AIDS lentivirus-induced T cell immunodeficiency.

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Miller, Michelle M; Fogle, Jonathan E; Ross, Peter; Tompkins, Mary B

2013-04-01

Using the feline immunodeficiency virus (FIV) model for AIDS-lentivirus infection, our laboratory has previously demonstrated that T regulatory (Treg) cell-mediated immune T and B cell dysfunction contributes to lentivirus persistence and chronic disease through membrane bound transforming growth factor beta (mTGFb). Studying Treg cells in the context of infection has been problematic as no inducible marker for activated Treg cells had been identified. However, recent reports in human Treg studies have described a novel protein, glycoprotein A repetitions predominant (GARP), as a unique marker of activated human Treg cells that anchors mTGFb. Herein we extend these studies to the feline Treg system, identifying feline GARP and demonstrating that human and feline GARP proteins are homologous in structure, expression pattern, and ability to form a complex with TGFb. We further demonstrate that GARP and TGFb form a complex on the surface of activated Treg cells and that these GARP(+)TGFb(+) Treg cells are highly efficient suppressor cells. Analysis of expression of this Treg activation marker in the FIV-AIDS model reveals an up-regulation of GARP expressing Treg cells during chronic FIV infection. We demonstrate that the GARP(+) Treg cells from FIV-infected cats suppress T helper cells in vivo and that blocking GARP or TGFb eliminates this suppression. These data suggest that GARP is expressed in complex with TGFb on the surface of activated Treg cells and plays an important role in TGFb(+) Treg-mediated T cell immune suppression during lentivirus infection.

Role of RNA interference (RNAi) in the moss Physcomitrella patens

KAUST Repository

Arif, Muhammad Asif; Frank, Wolfgang; Khraiwesh, Basel

2013-01-01

RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species. 2013 by the authors; licensee MDPI, Basel, Switzerland.

Role of RNA interference (RNAi) in the moss Physcomitrella patens

KAUST Repository

Arif, Muhammad Asif

2013-01-14

RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species. 2013 by the authors; licensee MDPI, Basel, Switzerland.

Domain motions of Argonaute, the catalytic engine of RNA interference

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Wall Michael E

2007-11-01

Full Text Available Abstract Background The Argonaute protein is the core component of the RNA-induced silencing complex, playing the central role of cleaving the mRNA target. Visual inspection of static crystal structures already has enabled researchers to suggest conformational changes of Argonaute that might occur during RNA interference. We have taken the next step by performing an all-atom normal mode analysis of the Pyrococcus furiosus and Aquifex aeolicus Argonaute crystal structures, allowing us to quantitatively assess the feasibility of these conformational changes. To perform the analysis, we begin with the energy-minimized X-ray structures. Normal modes are then calculated using an all-atom molecular mechanics force field. Results The analysis reveals low-frequency vibrations that facilitate the accommodation of RNA duplexes – an essential step in target recognition. The Pyrococcus furiosus and Aquifex aeolicus Argonaute proteins both exhibit low-frequency torsion and hinge motions; however, differences in the overall architecture of the proteins cause the detailed dynamics to be significantly different. Conclusion Overall, low-frequency vibrations of Argonaute are consistent with mechanisms within the current reaction cycle model for RNA interference.

RNA interference targeting raptor inhibits proliferation of gastric cancer cells

International Nuclear Information System (INIS)

Wu, William Ka Kei; Lee, Chung Wa; Cho, Chi Hin; Chan, Francis Ka Leung; Yu, Jun; Sung, Joseph Jao Yiu

2011-01-01

Mammalian target of rapamycin complex 1 (mTORC1) is dysregulated in gastric cancer. The biologic function of mTORC1 in gastric carcinogenesis is unclear. Here, we demonstrate that disruption of mTORC1 function by RNA interference-mediated downregulation of raptor substantially inhibited gastric cancer cell proliferation through induction of G 0 /G 1 -phase cell cycle arrest. The anti-proliferative effect was accompanied by concomitant downregulation of activator protein-1 and upregulation of Smad2/3 transcriptional activities. In addition, the expression of cyclin D 3 and p21 Waf1 , which stabilizes cyclin D/cdk4 complex for G 1 -S transition, was reduced by raptor knockdown. In conclusion, disruption of mTORC1 inhibits gastric cancer cell proliferation through multiple pathways. This discovery may have an implication in the application of mTORC1-directed therapy for the treatment of gastric cancer.

Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector-mediated RNAi.

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Patel, Utsav A; Patel, Amrutlal K; Joshi, Chaitanya G

2015-01-01

Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin-signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector-based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic-cell nuclear transfer (SCNT) studies. Sh-RNA positive cells were screened by puromycin selection. Using real-time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down-regulation in sh2 shRNA-treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin-targeting siRNA produced endogenously could efficiently down-regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus-mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals. © 2014 American Institute of Chemical Engineers.

cAMP/PKA-CREB-BDNF signaling pathway in hippocampus mediates cyclooxygenase 2-induced learning/memory deficits of rats subjected to chronic unpredictable mild stress.

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Luo, Ying; Kuang, Shengnan; Li, Huan; Ran, Dongzhi; Yang, Junqing

2017-05-30

To investigate the mechanism of cyclooxygenase 2 (COX2) in learning and memory impairments in rats subjected to chronic unpredictable mild stress (CUMS), meloxicam was used intragastrically to inhibit the activity of cyclooxygenase 2. Moreover, cyclooxygenase 2 over-expressing or RNA interfere lentivirus was injected intraventricularly to increase or decrease the enzyme's expression, respectively. The body weights and sucrose consumption were used to analyze depressive behaviors, while the Morris water maze and step-down-type passive avoidance tests were carried out to evaluate the learning-memory functions. The levels of inflammatory cytokines were measured to estimate inflammation and the contents of cyclic adenosine monophosphate (cAMP) were used to measure the levels of the second messenger. Changes in cyclooxygenase 2 mRNA levels were analyzed using reverse transcription polymerase chain reaction. Moreover, the expression of cyclooxygenase 2, brain-derived neurotrophic factor (BDNF), prostaglandins receptor 3 (EP3), protein kinase A (PKA), cAMP response element binding protein (CREB), and phosphorylated CREB were estimated using immunohistochemical staining or western blotting. The results showed that CUMS led to significant depressive-like behaviors and learning and memory dysfunctions. Also, the cAMP levels decreased significantly, while levels of inflammatory cytokines and prostaglandins E2 increased significantly. The expressions of PKA, BDNF, phosphorylated CREB/CREB declined and cyclooxygenase 2 was increased. Meloxicam and cyclooxygenase 2 RNA interfere lentivirus reversed the changes caused by CUMS while cyclooxygenase 2-overexpressing lentivirus worsened these abnormalities. The findings also showed that CUMS increased cyclooxygenase 2 expression, which can cause learning and memory impairments, mainly through activating the hippocampal neuronal cAMP/PKA-CREB-BDNF signaling pathways.

[Intramuscular injection of lentivirus-mediated EPAS1 gene improves hind limb ischemia and its mechanism in a rat model of peripheral artery vascular disease].

Science.gov (United States)

Wang, Zhihong; Gu, Hongbin; Yang, Fan; Xie, Huajie; Sheng, Lei; Li, Mingfei

2017-11-01

Objective To investigate the effect of over-expressed endothelial Per-Arnt-Sim domain protein 1 (EPAS1) on peripheral arterial disease (PAD) in a rat model. Methods PAD rat model was established by external iliac artery ligation followed by lentivirus-mediated EPAS1 gene injection into rat right adductor magnus. The models were evaluated by quantitative analysis of gait disturbance. The changes of blood flow in the posterior extremity of the rats were detected using laser Doppler. The expressions of EPAS1, hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) mRNAs were tested by real-time quantitative PCR. The expression of α-smooth muscle actin (αSMA) was detected by immunohistochemical staining. Results Compared with lenti-EGFP group, rat hind limb function and circulation got recovered obviously 7 days after lenti-EPAS1 injection. The mRNA expressions of EPAS1, HGF, bFGF, and VEGF were up-regulated in the lenti-EPAS1-treated sites.The expression of αSMA showed an obvious increase in the lenti-EPAS1-treated muscles. Conclusion Over-expressed lenti-EPAS1 can promote angiogenesis via the up-regulation of EPAS1-related angiogenic factors in the muscles of the affected hind limb and reduce gait disturbance.

Parallel germline infiltration of a lentivirus in two Malagasy lemurs.

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Clément Gilbert

2009-03-01

Full Text Available Retroviruses normally infect the somatic cells of their host and are transmitted horizontally, i.e., in an exogenous way. Occasionally, however, some retroviruses can also infect and integrate into the genome of germ cells, which may allow for their vertical inheritance and fixation in a given species; a process known as endogenization. Lentiviruses, a group of mammalian retroviruses that includes HIV, are known to infect primates, ruminants, horses, and cats. Unlike many other retroviruses, these viruses have not been demonstrably successful at germline infiltration. Here, we report on the discovery of endogenous lentiviral insertions in seven species of Malagasy lemurs from two different genera -- Cheirogaleus and Microcebus. Combining molecular clock analyses and cross-species screening of orthologous insertions, we show that the presence of this endogenous lentivirus in six species of Microcebus is the result of one endogenization event that occurred about 4.2 million years ago. In addition, we demonstrate that this lentivirus independently infiltrated the germline of Cheirogaleus and that the two endogenization events occurred quasi-simultaneously. Using multiple proviral copies, we derive and characterize an apparently full length and intact consensus for this lentivirus. These results provide evidence that lentiviruses have repeatedly infiltrated the germline of prosimian species and that primates have been exposed to lentiviruses for a much longer time than what can be inferred based on sequence comparison of circulating lentiviruses. The study sets the stage for an unprecedented opportunity to reconstruct an ancestral primate lentivirus and thereby advance our knowledge of host-virus interactions.

Endogenous lentivirus in Malayan colugo (Galeopterus variegatus), a close relative of primates.

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Hron, Tomáš; Fábryová, Helena; Pačes, Jan; Elleder, Daniel

2014-10-04

A significant fraction of mammalian genomes is composed of endogenous retroviral (ERV) sequences that are formed by germline infiltration of various retroviruses. In contrast to other retroviral genera, lentiviruses only rarely form ERV copies. We performed a computational search aimed at identification of novel endogenous lentiviruses in vertebrate genomes. Using the in silico strategy, we have screened 104 publicly available vertebrate genomes for the presence of endogenous lentivirus sequences. In addition to the previously described cases, the search revealed the presence of endogenous lentivirus in the genome of Malayan colugo (Galeopterus variegatus). At least three complete copies of this virus, denoted ELVgv, were detected in the colugo genome, and approximately one hundred solo LTR sequences. The assembled consensus sequence of ELVgv had typical lentivirus genome organization including three predicted accessory genes. Phylogenetic analysis placed this virus as a distinct subgroup within the lentivirus genus. The time of insertion into the dermopteran lineage was estimated to be more than thirteen million years ago. We report the discovery of the first endogenous lentivirus in the mammalian order Dermoptera, which is a taxon close to the Primates. Lentiviruses have infiltrated the mammalian germline several times across millions of years. The colugo virus described here represents possibly the oldest documented endogenization event and its discovery can lead to new insights into lentivirus evolution. This is also the first report of an endogenous lentivirus in an Asian mammal, indicating a long-term presence of this retrovirus family in Asian continent.

RNAi Knockdown of Hypoxia-Inducible Factor-1α Decreased the Proliferation, Migration, and Invasion of Hypoxic Hepatocellular Carcinoma Cells.

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Chen, ChengShi; Liu, Rong; Wang, JianHua; Yan, ZhiPing; Qian, Sheng; Zhang, Wei

2015-04-01

The obstruction of hepatic arterial blood flow results in tumor tissue hypoxia and elevated expression of hypoxia-inducible factor-1alpha (HIF-1α). Our study evaluated whether lentivirus-mediated short interference RNA against HIF-1α inhibits proliferation, invasion, and migration of hepatocellular carcinoma (HCC) cells under hypoxia. RNA interference knockdown of HIF-1α was achieved by HIF-1α-directed lentiviral shRNA, in a rat HCC cell line cultured under hypoxia condition for varying length of times. The expression levels of HIF-1α and vascular endothelial growth factor were examined using reverse transcription polymerase chain reaction and western blot analyses. Cell proliferation, migration, and invasion were measured by cell viability, transwell migration, and invasion assays, respectively. Inhibition of HIF-1α expression by shRNA suppressed vascular endothelial growth factor mRNA and protein levels under both normoxia and hypoxia. It also suppressed cell migration and invasion, which were enhanced under hypoxic conditions. RNAi knockdown of HIF-1α further suppressed hypoxia-mediated inhibition of the cell proliferation. These data suggest that shRNA of HIF-1α could antagonize the hypoxia-mediated increase in hepatic cancer cell migration and invasion, and synergize with hypoxia to inhibit the cell proliferation in HCC cells.

A large-scale RNA interference screen identifies genes that regulate autophagy at different stages.

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Guo, Sujuan; Pridham, Kevin J; Virbasius, Ching-Man; He, Bin; Zhang, Liqing; Varmark, Hanne; Green, Michael R; Sheng, Zhi

2018-02-12

Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays revealed that 57 autophagy-regulating genes suppressed autophagy initiation, whereas 21 candidates promoted autophagy maturation. Our RNA interference screen identifies identified genes that regulate autophagy at different stages, which helps decode autophagy regulation in cancer and offers novel avenues to develop autophagy-related therapies for cancer.

RNA interference prevents lipopolysaccharide-induced preprotachykinin gene expression

International Nuclear Information System (INIS)

Lai, Y.-L.; Yu, S.C.; Chen, M.-J.

2003-01-01

We showed previously that lipopolysaccharide (LPS) induces noncholinergic airway hyperreactivity to capsaicin via an upregulation of tachykinin synthesis. This study was designed to test whether double-stranded preprotachykinin (ds PPT) RNA, RNA interference (RNAi), prevents the LPS-induced alterations. First, cultured primary nodose ganglial cells of newborn Brown-Norway rats were divided into four groups: control; LPS; LPS+RNAi; and LPS+RNAi+liposome. Second, young Brown-Norway rats for the in vivo study were divided into three groups (control; LPS; and LPS+RNAi), and ds PPT RNA was microinjected bilaterally into the nodose ganglia in the LPS+RNAi group. Then, ganglial cells were collected from the culture whereas the nodose ganglia and lungs were sampled from the animals, and PPT mRNA and substance P (SP) levels were analyzed. Also, airway reactivity to capsaicin was performed in vivo. LPS induced significant increases in PPT mRNA and SP levels in vitro and in vivo and an increase in airway reactivity to capsaicin in vivo. However, ds PPT RNA, but not scrambled RNA, prevented all LPS-induced alterations. The effect of ds PPT RNA was not enhanced by liposome in vitro. Therefore, we demonstrated that the local application of RNAi prevents effectively the activation of the noncholinergic system modulating the lungs/airways

Specific RNA Interference in Caenorhabditis elegans by Ingested dsRNA Expressed in Bacillus subtilis

NARCIS (Netherlands)

Lezzerini, M.; van de Ven, K.; Veerman, M.; Brul, S.; Budovskaya, Y.V.

2015-01-01

In nematodes, genome-wide RNAi-screening has been widely used as a rapid and efficient method to identify genes involved in the aging processes. By far the easiest way of inducing RNA interference (RNAi) in Caenorhabditis elegans is by feeding Escherichia coli that expresses specific double stranded

Antifibrotic effects of Smad4 small interfering RNAs in injured skeletal muscle after acute contusion.

Science.gov (United States)

Li, H; Chen, J; Chen, S; Zhang, Q; Chen, S

2011-10-01

Muscle injuries are common musculoskeletal problems encountered in sports medicine clinics. In this study, we examined the effect of lentivirus-mediated small interfering RNA (siRNA) targeting Smad4 on the suppression of the fibrosis in injured skeletal muscles. We found that Smad4-siRNA could efficiently knock down the expression of Smad4 in the C2C12 myoblast cells and in the contunded mice gastrocnemius muscle. The expression of mRNA level of Smad4 decreased to 11% and 49% compared to the control group, respectively, and the expression of protein level decreased to 13% and 57% respectively. Moreover, the lentivirus-mediated siRNA was stably transfected only into the skeletal muscle and not into the liver of the animals. In contunded mice gastrocnemius, the collagenous and vimentin-positive area in the Smad4 siRNA group reduced to 36% and 37% compared to the control group, respectively. Furthermore, compared to the scrambled Smad4 siRNA-injected mice and PBS control-injected mice, the muscle function of the mice injected with lentivirus-mediated Smad4 siRNA improved in terms of both fast-twitch and tetanic strength (P<0.05). The results suggest that the gene therapy of inhibiting Smad4 by lentivirus-mediated siRNA could be a useful approach to prevent scar tissue formation and improve the function of injured skeletal muscle. © Georg Thieme Verlag KG Stuttgart · New York.

Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation.

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Yang, Binxia; Janardhanan, Rajiv; Vohra, Pawan; Greene, Eddie L; Bhattacharya, Santanu; Withers, Sarah; Roy, Bhaskar; Nieves Torres, Evelyn C; Mandrekar, Jaywant; Leof, Edward B; Mukhopadhyay, Debabrata; Misra, Sanjay

2014-02-01

Venous neointimal hyperplasia (VNH) causes hemodialysis vascular access failure. Here we tested whether VNH formation occurs in part due to local vessel hypoxia caused by surgical trauma to the vasa vasorum of the outflow vein at the time of arteriovenous fistula placement. Selective targeting of the adventitia of the outflow vein at the time of fistula creation was performed using a lentivirus-delivered small-hairpin RNA that inhibits VEGF-A expression. This resulted in significant increase in mean lumen vessel area, decreased media/adventitia area, and decreased constrictive remodeling with a significant increase in apoptosis (increase in caspase 3 activity and TUNEL staining) accompanied with decreased cellular proliferation and hypoxia-inducible factor-1α at the outflow vein. There was significant decrease in cells staining positive for α-smooth muscle actin (a myofibroblast marker) and VEGFR-1 expression with a decrease in MMP-2 and MMP-9. These results were confirmed in animals that were treated with humanized monoclonal antibody to VEGF-A with similar results. Since hypoxia can cause fibroblast to differentiate into myofibroblasts, we silenced VEGF-A gene expression in fibroblasts and subjected them to hypoxia. This decreased myofibroblast production, cellular proliferation, cell invasion, MMP-2 activity, and increased caspase 3. Thus, VEGF-A reduction at the time of arteriovenous fistula placement results in increased positive vascular remodeling.

Immunoregulation by interference RNA (iRNA – mechanisms, role, perspective

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Emilia Sikora

2011-08-01

Full Text Available The functioning of an organism depends on the precise control mechanisms, constantly adjusted to the actual state. Therefore, there is a need for efficient communication between both adjacent and distant cells, which may be executed by proteins such as hormones, neurotransmitters and cytokines. Recently another means of regulation has emerged – short regulatory RNAs (srRNAs. Although discovered only a couple of years ago, the mechanism of RNA interference has already become a topic of thousands of publications, defining its roles in both physiological and pathological processes, such as cancerogenesis and autoimmunization.RNAs regulating cell function may be coded in its genome (both exons and introns or be introduced from the external environment. In mammals microRNAs (miRNAs cooperate with proteins from the Ago/PIWI family to form effector ribonucleoprotein complexes, and owing to their complementarity to the target mRNA, control genes’ expression at the posttranscriptional level, either through the suppression of mRNA translation or through mRNA degradation.SrRNAs are crucial regulators throughout the development of immune cells, starting from hematopoietic stem cells, up to the effector cells of the adaptive immune response. Moreover, some of the regulatory cells perform their function by releasing miRNAs, which are then transported to the target cells, possibly enclosed in the exosomes.

Production of high-amylose maize lines using RNA interference in ...

African Journals Online (AJOL)

amylose maize lines with a low T-DNA copy number, demonstrating that RNAi is an efficient method for the production of high-amylose maize lines. Key words: Maize, high-amylose, RNA interference, starch branching enzyme gene sbe2a.

Retention and loss of RNA interference pathways in trypanosomatid protozoans.

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Lon-Fye Lye

2010-10-01

Full Text Available RNA interference (RNAi pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance, and/or alterations in parasite virulence.

Prior Puma Lentivirus Infection Modifies Early Immune Responses and Attenuates Feline Immunodeficiency Virus Infection in Cats

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Wendy S. Sprague

2018-04-01

Full Text Available We previously showed that cats that were infected with non-pathogenic Puma lentivirus (PLV and then infected with pathogenic feline immunodeficiency virus (FIV (co-infection with the host adapted/pathogenic virus had delayed FIV proviral and RNA viral loads in blood, with viral set-points that were lower than cats infected solely with FIV. This difference was associated with global CD4+ T cell preservation, greater interferon gamma (IFN-γ mRNA expression, and no cytotoxic T lymphocyte responses in co-infected cats relative to cats with a single FIV infection. In this study, we reinforced previous observations that prior exposure to an apathogenic lentivirus infection can diminish the effects of acute infection with a second, more virulent, viral exposure. In addition, we investigated whether the viral load differences that were observed between PLV/FIV and FIV infected cats were associated with different immunocyte phenotypes and cytokines. We found that the immune landscape at the time of FIV infection influences the infection outcome. The novel findings in this study advance our knowledge about early immune correlates and documents an immune state that is associated with PLV/FIV co-infection that has positive outcomes for lentiviral diseases.

Steric restrictions of RISC in RNA interference identified with size-expanded RNA nucleobases.

Science.gov (United States)

Hernández, Armando R; Peterson, Larryn W; Kool, Eric T

2012-08-17

Understanding the interactions between small interfering RNAs (siRNAs) and the RNA-induced silencing complex (RISC), the key protein complex of RNA interference (RNAi), is of great importance to the development of siRNAs with improved biological and potentially therapeutic function. Although various chemically modified siRNAs have been reported, relatively few studies with modified nucleobases exist. Here we describe the synthesis and hybridization properties of siRNAs bearing size-expanded RNA (xRNA) nucleobases and their use as a novel and systematic set of steric probes in RNAi. xRNA nucleobases are expanded by 2.4 Å using benzo-homologation and retain canonical Watson-Crick base-pairing groups. Our data show that the modified siRNA duplexes display small changes in melting temperature (+1.4 to -5.0 °C); substitutions near the center are somewhat destabilizing to the RNA duplex, while substitutions near the ends are stabilizing. RNAi studies in a dual-reporter luciferase assay in HeLa cells revealed that xRNA nucleobases in the antisense strand reduce activity at some central positions near the seed region but are generally well tolerated near the ends. Most importantly, we observed that xRNA substitutions near the 3'-end increased activity over that of wild-type siRNAs. The data are analyzed in terms of site-dependent steric effects in RISC. Circular dichroism experiments show that single xRNA substitutions do not significantly distort the native A-form helical structure of the siRNA duplex, and serum stability studies demonstrated that xRNA substitutions protect siRNAs against nuclease degradation.

Safety assessment of food and feed from biotechnology-derived crops employing RNA-mediated gene regulation to achieve desired traits: a scientific review.

Science.gov (United States)

Petrick, Jay S; Brower-Toland, Brent; Jackson, Aimee L; Kier, Larry D

2013-07-01

Gene expression can be modulated in plants to produce desired traits through agricultural biotechnology. Currently, biotechnology-derived crops are compared to their conventional counterparts, with safety assessments conducted on the genetic modification and the intended and unintended differences. This review proposes that this comparative safety assessment paradigm is appropriate for plants modified to express mediators of RNA-mediated gene regulation, including RNA interference (RNAi), a gene suppression mechanism that naturally occurs in plants and animals. The molecular mediators of RNAi, including long double-stranded RNAs (dsRNA), small interfering RNAs (siRNA), and microRNAs (miRNA), occur naturally in foods; therefore, there is an extensive history of safe consumption. Systemic exposure following consumption of plants containing dsRNAs that mediate RNAi is limited in higher organisms by extensive degradation of ingested nucleic acids and by biological barriers to uptake and efficacy of exogenous nucleic acids. A number of mammalian RNAi studies support the concept that a large margin of safety will exist for any small fraction of RNAs that might be absorbed following consumption of foods from biotechnology-derived plants that employ RNA-mediated gene regulation. Food and feed derived from these crops utilizing RNA-based mechanisms is therefore expected to be as safe as food and feed derived through conventional plant breeding. Copyright © 2013 Elsevier Inc. All rights reserved.

RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei

NARCIS (Netherlands)

Vondrusková, Eva; van den Burg, Janny; Zíková, Alena; Ernst, Nancy Lewis; Stuart, Kenneth; Benne, Rob; Lukes, Julius

2005-01-01

Mitochondrial RNA-binding proteins MRP1 and MRP2 occur in a heteromeric complex that appears to play a role in U-insertion/deletion editing in trypanosomes. Reduction in the levels of MRP1 (gBP21) and/or MRP2 (gBP25) mRNA by RNA interference in procyclic Trypanosoma brucei resulted in severe growth

RNA Interference in Insect Vectors for Plant Viruses

OpenAIRE

Kanakala, Surapathrudu; Ghanim, Murad

2016-01-01

Insects and other arthropods are the most important vectors of plant pathogens. The majority of plant pathogens are disseminated by arthropod vectors such as aphids, beetles, leafhoppers, planthoppers, thrips and whiteflies. Transmission of plant pathogens and the challenges in managing insect vectors due to insecticide resistance are factors that contribute to major food losses in agriculture. RNA interference (RNAi) was recently suggested as a promising strategy for controlling insect pests...

The SMN1 common variant c.22 dupA in Chinese patients causes spinal muscular atrophy by nonsense-mediated mRNA decay in humans.

Science.gov (United States)

Bai, JinLi; Qu, YuJin; Cao, YanYan; Yang, Lan; Ge, Lin; Jin, YuWei; Wang, Hong; Song, Fang

2018-02-20

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is mostly caused by homozygous deletion of the SMN1 gene. Approximately 5%-10% of SMA patients are believed to have SMN1 variants. c.22 dupA (p.Ser8lysfs*23) has been identified as the most frequent variant in the Chinese SMA population and to be associated with a severe phenotype. However, the exact molecular mechanism of the variant on the pathogenesis of SMA is unclear. We observed that SMN1 mRNA and the SMN protein in the peripheral blood cells of a patient with c.22 dupA were lower than those of controls. The aim of this study is to investigate whether nonsense-mediated mRNA decay (NMD) plays a role in the mechanism of the c.22 dupA variant of the SMN1 gene as it causes SMA. Two lymphoblasts cell lines from two patients (patient 1 and 2) with the c.22 dupA, and one dermal fibroblasts cell line from patient 2 were included in our study. Two-stage validation of the NMD mechanism was supplied. We first measured the changes in the transcript levels of the SMN1 gene by real-time quantitative PCR after immortalized B-lymphoblasts and dermal fibroblasts cells of the SMA patients were treated with inhibitors of the NMD pathway, including puromycin and cyclohemide. Next, lentivirus-mediated knockdown of the key NMD factor-Up-frameshift protein 1 (UPF1)-was performed in the fibroblasts cell line to further clarify whether the variant led to NMD, as UPF1 recognizes abnormally terminated transcripts as NMD substrates during translation. SC35 1.7-kb transcripts, a physiological NMD substrate was determined to be a NMD positive gene in our experiments. The two inhibitors resulted in a dramatic escalation of the levels of the full-length SMN1 (fl-SMN1) transcripts. Additionally, the SC35 1.7-kb mRNA levels were also increased, suggesting that NMD pathway is suppressed by the two inhibitors. For the 3 cell lines, the fold increase of the SMN1 transcript levels of cycloheximide ranged

RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast

Energy Technology Data Exchange (ETDEWEB)

Iida, Tetsushi, E-mail: tiida@nig.ac.jp [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012 (Japan); Iida, Naoko [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Tsutsui, Yasuhiro [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuda-cho, Midori-ku, Yokohama 226-8501 (Japan); Yamao, Fumiaki [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Kobayashi, Takehiko [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan)

2012-10-12

Highlights: Black-Right-Pointing-Pointer RNAi is linked to the cell cycle checkpoint in fission yeast. Black-Right-Pointing-Pointer Ptr1 co-purifies with Ago1. Black-Right-Pointing-Pointer The ptr1-1 mutation impairs the checkpoint but does not affect gene silencing. Black-Right-Pointing-Pointer ago1{sup +} and ptr1{sup +} regulate the cell cycle checkpoint via the same pathway. Black-Right-Pointing-Pointer Mutations in ago1{sup +} and ptr1{sup +} lead to the nuclear accumulation of poly(A){sup +} RNAs. -- Abstract: Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1{sup +}, the overexpression of ago1{sup +} alleviated the cell cycle defect in dcr1{Delta}. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1{sup +} is dependent on ptr1{sup +}. Nuclear accumulation of poly(A){sup +} RNAs was detected in mutants of ago1{sup +} and ptr1{sup +}, suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

Transgenic Sugarcane Resistant to Sorghum mosaic virus Based on Coat Protein Gene Silencing by RNA Interference

Directory of Open Access Journals (Sweden)

Jinlong Guo

2015-01-01

Full Text Available As one of the critical diseases of sugarcane, sugarcane mosaic disease can lead to serious decline in stalk yield and sucrose content. It is mainly caused by Potyvirus sugarcane mosaic virus (SCMV and/or Sorghum mosaic virus (SrMV, with additional differences in viral strains. RNA interference (RNAi is a novel strategy for producing viral resistant plants. In this study, based on multiple sequence alignment conducted on genomic sequences of different strains and isolates of SrMV, the conserved region of coat protein (CP genes was selected as the target gene and the interference sequence with size of 423 bp in length was obtained through PCR amplification. The RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence and cp4-epsps herbicide-tolerant gene was transferred to sugarcane cultivar ROC22 via Agrobacterium-mediated transformation. After herbicide screening, PCR molecular identification, and artificial inoculation challenge, anti-SrMV positive transgenic lines were successfully obtained. SrMV resistance rate of the transgenic lines with the interference sequence was 87.5% based on SrMV challenge by artificial inoculation. The genetically modified SrMV-resistant lines of cultivar ROC22 provide resistant germplasm for breeding lines and can also serve as resistant lines having the same genetic background for study of resistance mechanisms.

Interspecific RNA interference of SHOOT MERISTEMLESS-like disrupts Cuscuta pentagona plant parasitism.

Science.gov (United States)

Alakonya, Amos; Kumar, Ravi; Koenig, Daniel; Kimura, Seisuke; Townsley, Brad; Runo, Steven; Garces, Helena M; Kang, Julie; Yanez, Andrea; David-Schwartz, Rakefet; Machuka, Jesse; Sinha, Neelima

2012-07-01

Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA-mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection.

Short hairpin RNA targeting 2B gene of coxsackievirus B3 exhibits potential antiviral effects both in vitro and in vivo

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Yao Hailan

2012-08-01

Full Text Available Abstract Background Coxsackievirus B3 is an important infectious agent of viral myocarditis, pancreatitis and aseptic meningitis, but there are no specific antiviral therapeutic reagents in clinical use. RNA interference-based technology has been developed to prevent the viral infection. Methods To evaluate the impact of RNA interference on viral replication, cytopathogenicity and animal survival, short hairpin RNAs targeting the viral 2B region (shRNA-2B expressed by a recombinant vector (pGCL-2B or a recombinant lentivirus (Lenti-2B were tansfected in HeLa cells or transduced in mice infected with CVB3. Results ShRNA-2B exhibited a significant effect on inhibition of viral production in HeLa cells. Furthermore, shRNA-2B improved mouse survival rate, reduced the viral tissues titers and attenuated tissue damage compared with those of the shRNA-NC treated control group. Lenti-2B displayed more effective role in inhibition of viral replication than pGCL-2B in vivo. Conclusions Coxsackievirus B3 2B is an effective target of gene silencing against coxsackievirus B3 infection, suggesting that shRNA-2B is a potential agent for further development into a treatment for enterviral diseases.

Creating Transgenic shRNA Mice by Recombinase-Mediated Cassette Exchange

Science.gov (United States)

Premsrirut, Prem K.; Dow, Lukas E.; Park, Youngkyu; Hannon, Gregory J.; Lowe, Scott W.

2014-01-01

RNA interference (RNAi) enables sequence-specific, experimentally induced silencing of virtually any gene by tapping into innate regulatory mechanisms that are conserved among most eukaryotes. The principles that enable transgenic RNAi in cell lines can also be used to create transgenic animals, which express short-hairpin RNAs (shRNAs) in a regulated or tissue-specific fashion. However, RNAi in transgenic animals is somewhat more challenging than RNAi in cultured cells. The activities of promoters that are commonly used for shRNA expression in cell culture can vary enormously in different tissues, and founder lines also typically vary in transgene expression due to the effects of their single integration sites. There are many ways to produce mice carrying shRNA transgenes and the method described here uses recombinase-mediated cassette exchange (RMCE). RMCE permits insertion of the shRNA transgene into a well-characterized locus that gives reproducible and predictable expression in each founder and enhances the probability of potent expression in many cell types. This procedure is more involved and complex than simple pronuclear injection, but if even a few shRNA mice are envisioned, for example, to probe the functions of several genes, the effort of setting up the processes outlined below are well worthwhile. Note that when creating a transgenic mouse, one should take care to use the most potent shRNA possible. As a rule of thumb, the sequence chosen should provide >90% knockdown when introduced into cultured cells at single copy (e.g., on retroviral infection at a multiplicity of ≤0.3). PMID:24003198

Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo

International Nuclear Information System (INIS)

Anesti, Anna-Maria; Simpson, Guy R; Price, Toby; Pandha, Hardev S; Coffin, Robert S

2010-01-01

Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEX GM-CSF , we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and β-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials

Lentivirus-mediated delivery of sonic hedgehog into the striatum stimulates neuroregeneration in a rat model of Parkinson disease.

Science.gov (United States)

Zhang, Yi; Dong, Weiren; Guo, Suiqun; Zhao, Shu; He, Suifen; Zhang, Lihua; Tang, Yinjuan; Wang, Haihong

2014-12-01

Parkinson disease (PD) is a progressive neurodegenerative disorder in which the nigrostriatal pathway, consisting of dopaminergic neuronal projections from the substantia nigra to the striatum, degenerates. Viral transduction is currently the most promising in vivo strategy for delivery of therapeutic proteins into the brain for treatment of PD. Sonic hedgehog (Shh) is necessary for cell proliferation, differentiation and neuroprotection in the central nervous system. In this study, we investigated the effects of overexpressed N-terminal product of SHH (SHH-N) in a PD model rat. A lentiviral vector containing SHH-N was stereotactically injected into the striatum 24 h after a striatal 6-OHDA lesion. We found that overexpressed SHH-N attenuated behavioral deficits and reduced the loss of dopamine neurons in the substantia nigra and the loss of dopamine fibers in the striatum. In addition, fluoro-ruby-labeled nigrostriatal projections were also repaired. Together, our results demonstrate the feasibility and efficacy of using the strategy of lentivirus-mediated Shh-N delivery to delay nigrostriatal pathway degeneration. This strategy holds the potential for therapeutic application in the treatment of PD.

Bioreducible poly(amido amine)s for siRNA delivery

NARCIS (Netherlands)

van der Aa, L.J.

2011-01-01

Successes in RNA interference based therapies are still limited due to the lack of efficient delivery of the mediator, small interfering RNA (siRNA), to the targeted site. The key to success can be the delivery of the siRNA molecules by polymer-based carrier systems, since they can be chemically

RNA interference: ready to silence cancer?

Science.gov (United States)

Mocellin, Simone; Costa, Rodolfo; Nitti, Donato

2006-01-01

RNA interference (RNAi) is considered the most promising functional genomics tool recently developed. As in other medical fields, this biotechnology might revolutionize the approach to dissecting the biology of cancer, ultimately speeding up the discovery pace of novel targets suitable for molecularly tailored antitumor therapies. In addition, preclinical results suggest that RNAi itself might be used as a therapeutic weapon. With the aim of illustrating not only the potentials but also the current limitations of RNAi as a tool in the fight against cancer, here we summarize the physiology of RNAi, discuss the main technical issues of RNAi-based gene silencing, and review some of the most interesting preclinical results obtained so far with its implementation in the field of oncology.

Theoretical studies on sRNA-mediated regulation in bacteria

Science.gov (United States)

Chang, Xiao-Xue; Xu, Liu-Fang; Shi, Hua-Lin

2015-12-01

Small RNA(sRNA)-mediated post-transcriptional regulation differs from protein-mediated regulation. Through base-pairing, sRNA can regulate the target mRNA in a catalytic or stoichiometric manner. Some theoretical models were built for comparison of the protein-mediated and sRNA-mediated modes in the steady-state behaviors and noise properties. Many experiments demonstrated that a single sRNA can regulate several mRNAs, which causes crosstalk between the targets. Here, we focus on some models in which two target mRNAs are silenced by the same sRNA to discuss their crosstalk features. Additionally, the sequence-function relationship of sRNA and its role in the kinetic process of base-pairing have been highlighted in model building. Project supported by the National Basic Research Program of China (Grant No. 2013CB834100), the National Natural Science Foundation of China (Grant Nos. 11121403 and 11274320), the Open Project Program of State Key Laboratory of Theoretical Physics, Institute of Theoretical Physics, Chinese Academy of Sciences, China (Grant No. Y4KF171CJ1), the National Natural Science Foundation for Young Scholar of China (Grant No. 11304115), and the China Postdoctoral Science Foundation (Grant No. 2013M541282).

(AAV)-mediated expression of small interfering RNA

African Journals Online (AJOL)

Effective inhibition of specific gene by adenoassociated virus (AAV)-mediated expression of small interfering RNA. ... To perform functional tests on siRNA, which was expressed by the viral vector, recombinant AAVs, coding for siRNA against exogenous gene, EGFP, and endogenous gene, p53, were established and ...

Stable RNA interference of ErbB-2 gene synergistic with epirubicin suppresses breast cancer growth in vitro and in vivo

International Nuclear Information System (INIS)

Hu Xiaoqu; Su Fengxi; Qin Li; Jia Weijuan; Gong Chang; Yu Fengyan; Guo Jujiang; Song Erwei

2006-01-01

Overexpression of human epidermal growth factor receptor-2 (Her2, ErbB-2) contributes to the progression and metastasis of breast cancer, implying that Her2 gene is a suitable target of RNA interference (RNAi) for breast cancer therapy. Here, we employed plasmid-mediated expression of 2 different Her2-shRNAs (pU6-Her2shRNAs) efficiently silenced the target gene expression on Her2 expressing SKBR-3 breast cancer cells in both mRNA and protein levels. Consequently, pU6-Her2shRNA increased apoptosis and reduced proliferation of SKBR-3 cells assayed by TUNEL and MTT, respectively. In vivo, intra-tumor injection of pU6-Her2shRNA inhibited the growth of SKBR-3 tumors inoculated subcutaneously in nude mice. Furthermore, pU6-Her2shRNA synergized the tumor suppression effect of epirubicin to SKBR-3 cells in vitro and implanted subcutaneously in nude mice. Therefore, we concluded that stable silencing of Her2 gene expression with plasmid expressing shRNA may hold great promise as a novel therapy for Her2 expressing breast cancers alone or in combination with anthracycline chemotherapy

PsOr1, a potential target for RNA interference-based pest management.

Science.gov (United States)

Zhao, Y Y; Liu, F; Yang, G; You, M S

2011-02-01

Insect pests cause billions of dollars in agricultural losses, and attempts to kill them have resulted in growing threats from insecticide resistance, dietary pesticide pollution and environmental destruction. New approaches to control refractory insect pests are therefore needed. The host-plant preferences of insect pests rely on olfaction and are mediated via a seven transmembrane-domain odorant receptor (Or) family. The present study reports the cloning and characterization of PsOr1, the first candidate member of the Or gene family from Phyllotreta striolata, a devastating beetle pest that causes damage worldwide. PsOr1 is remarkably well conserved with respect to other insect orthologues, including DmOr83b from Drosophila melanogaster. These insect orthologues form an essential non-conventional Or sub-family and may play an important and generalized role in insect olfaction. We designed double-stranded (ds) RNA directly against the PsOr1 gene and exploited RNA interference (RNAi) to control P. striolata. The chemotactic behavioural measurements showed that adult beetles were unable to sense the attractant or repellent odour stimulus after microinjection of dsRNA against PsOr1. Reverse Transcription (RT)-PCR analysis showed specific down-regulation of mRNA transcript levels for this gene. Furthermore, host-plant preference experiments confirmed that silencing PsOr1 by RNAi treatment impaired the host-plant preferences of P. striolata for cruciferous vegetables. These results demonstrate that this insect control approach of using RNAi to target PsOr1 and its orthologues might be effective in blocking host-plant-seeking behaviours in diverse insect pests. The results also support the theory that this unique receptor type plays an essential general role in insect olfaction. © 2010 Fujian Agriculture and Forestry University. Insect Molecular Biology © 2010 The Royal Entomological Society.

RNA interference targets arbovirus replication in Culicoides cells.

Science.gov (United States)

Schnettler, Esther; Ratinier, Maxime; Watson, Mick; Shaw, Andrew E; McFarlane, Melanie; Varela, Mariana; Elliott, Richard M; Palmarini, Massimo; Kohl, Alain

2013-03-01

Arboviruses are transmitted to vertebrate hosts by biting arthropod vectors such as mosquitoes, ticks, and midges. These viruses replicate in both arthropods and vertebrates and are thus exposed to different antiviral responses in these organisms. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism that has been shown to play a major role in the antiviral response against arboviruses in mosquitoes. Culicoides midges are important vectors of arboviruses, known to transmit pathogens of humans and livestock such as bluetongue virus (BTV) (Reoviridae), Oropouche virus (Bunyaviridae), and likely the recently discovered Schmallenberg virus (Bunyaviridae). In this study, we investigated whether Culicoides cells possess an antiviral RNAi response and whether this is effective against arboviruses, including those with double-stranded RNA (dsRNA) genomes, such as BTV. Using reporter gene-based assays, we established the presence of a functional RNAi response in Culicoides sonorensis-derived KC cells which is effective in inhibiting BTV infection. Sequencing of small RNAs from KC and Aedes aegypti-derived Aag2 cells infected with BTV or the unrelated Schmallenberg virus resulted in the production of virus-derived small interfering RNAs (viRNAs) of 21 nucleotides, similar to the viRNAs produced during arbovirus infections of mosquitoes. In addition, viRNA profiles strongly suggest that the BTV dsRNA genome is accessible to a Dicer-type nuclease. Thus, we show for the first time that midge cells target arbovirus replication by mounting an antiviral RNAi response mainly resembling that of other insect vectors of arboviruses.

Inhibition of osteoclastogenesis by RNA interference targeting RANK

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Ma Ruofan

2012-08-01

Full Text Available Abstract Background Osteoclasts and osteoblasts regulate bone resorption and formation to allow bone remodeling and homeostasis. The balance between bone resorption and formation is disturbed by abnormal recruitment of osteoclasts. Osteoclast differentiation is dependent on the receptor activator of nuclear factor NF-kappa B (RANK ligand (RANKL as well as the macrophage colony-stimulating factor (M-CSF. The RANKL/RANK system and RANK signaling induce osteoclast formation mediated by various cytokines. The RANK/RANKL pathway has been primarily implicated in metabolic, degenerative and neoplastic bone disorders or osteolysis. The central role of RANK/RANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies in treatment of osteolysis. The purpose of this study was to assess the effect of inhibition of RANK expression in mouse bone marrow macrophages on osteoclast differentiation and bone resorption. Methods Three pairs of short hairpin RNAs (shRNA targeting RANK were designed and synthesized. The optimal shRNA was selected among three pairs of shRNAs by RANK expression analyzed by Western blot and Real-time PCR. We investigated suppression of osteoclastogenesis of mouse bone marrow macrophages (BMMs using the optimal shRNA by targeting RANK. Results Among the three shRANKs examined, shRANK-3 significantly suppressed [88.3%] the RANK expression (p Conclusions These findings suggest that retrovirus-mediated shRNA targeting RANK inhibits osteoclast differentiation and osteolysis. It may appear an attractive target for preventing osteolysis in humans with a potential clinical application.

Apoptotic block in colon cancer cells may be rectified by lentivirus mediated overexpression of caspase-9.

Science.gov (United States)

Xu, D; Wang, C; Shen, X; Yu, Y; Rui, Y; Zhang, D; Zhou, Z

2013-12-01

At present, the inhibition of apoptosis during pathogenesis of colorectal cancer is widely recognized while the role of caspase-9 in this process remains controversial. We aimed to investigate the differential expression of caspase-9 and evaluate the therapeutic potential of expression intervention in this study. We first examined the different expression of caspase-9 in normal colon mucosa, adenoma and cancer, investigating the relationship between its expression and clinico-pathological characteristics. Secondly, overexpression of caspase-9 was established in colon cancer cell lines by lentivirus infection to study the changes in growth, proliferation and apoptosis. Compared with normal colon mucosa, the expression of caspase-9 was higher in adenoma while lower in cancer both at mRNA and protein level (P expression is more common in poorly differentiated cancers (P expression of caspase-9, poorer colony formation and slower cell proliferation. In terms of apoptosis related indicators, caspase-9 overexpression leads to higher apoptosis rate and GO/G1 arrest, while up-regulating the expression of caspase-3 (P expression from colon mucosa, adenoma to cancer suggested it may be involved in the carcinogenesis of colon cancer. The overexpression of caspase-9 exhibits an inhibitory role in cancer growth and proliferation while promoting apoptosis. However, a non-apoptotic role of caspase-9 facilitating differentiation was also implied.

Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

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El Far Mohamed

2009-05-01

Full Text Available Abstract Background Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC. While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain. Results We show that the TRBP binding site in Dicer is a 165 amino acid (aa region located between the ATPase and the helicase domains. The binding site in TRBP is a 69 aa domain, called C4, located at the C-terminal end of TRBP. The TRBP1 and TRBP2 isoforms, but not TRBPs lacking the C4 site (TRBPsΔC4, co-immunoprecipitated with Dicer. The C4 domain is therefore necessary to bind Dicer, irrespective of the presence of RNA. Immunofluorescence shows that while full-length TRBPs colocalize with Dicer, TRBPsΔC4 do not. tarbp2-/- cells, which do not express TRBP, do not support RNA interference (RNAi mediated by short hairpin or micro RNAs against EGFP. Both TRBPs, but not TRBPsΔC4, were able to rescue RNAi function. In human cells with low RNAi activity, addition of TRBP1 or 2, but not TRBPsΔC4, rescued RNAi function. Conclusion The mapping of the interaction sites between TRBP and Dicer show unique domains that are required for their binding. Since TRBPsΔC4 do not interact or colocalize with Dicer, we suggest that TRBP and Dicer, both dsRBPs, do not interact through bound dsRNA. TRBPs, but not TRBPsΔC4, rescue RNAi activity in RNAi-compromised cells, indicating that the binding of Dicer to TRBP is critical for RNAi function.

Isolation and identification of a South African lentivirus from jaagsiekte lungs

International Nuclear Information System (INIS)

Payne, A.; York, D.F.; De Villiers, E-M.; Verwoerd, D.W.; Querat, G.; Barban, V.; Sauze, N.; Vigne, R.

1986-01-01

In the course of attempts to grow the jaagsiekte retrovirus in cell culture, a typical lentivirus was isolated for the first time in South Africa from adenomatous lungs. Morphologically the virus could not be distinguished from other lentiviruses, but serologically it was shown to be more closely related to visna virus than to caprine arthritis-encephalitis virus. However, a preliminary restriction enzyme analysis of the linear proviral DNA of this new lentivirus (SA-DMVV) revealed that it is significantly district from visna virus and CAEV and therefore may represent a third type of lentivirus. Antibodies to the virus were demonstrated in a number of sheep in various parts of the country, but a direct link to a disease condition was not found. Attempts to produce lung lesions by intratracheal injection of the virus have been unsuccessful to date but a transient arthritis was produced by intra-articular inoculation. Viral replication seems to be enhanced in jaagsiekte lungs

The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved nucleotide analog interference probing (trNAIP).

Science.gov (United States)

Soares, Emilie; Schwartz, Annie; Nollmann, Marcello; Margeat, Emmanuel; Boudvillain, Marc

2014-08-01

Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2'-hydroxyl group of the incoming 3'-RNA nucleotide by a Rho subunit. We propose that this step arises from the intrinsic weakness of one of the subunit interfaces caused by asymmetric, split-ring arrangement of primary RNA tethers around the Rho hexamer. Translocation is at highest stake every seventh nucleotide when the weak interface engages the incoming 3'-RNA nucleotide or breaks, depending on RNA threading constraints in the Rho pore. This substrate-governed, 'test to run' iterative mechanism offers a new perspective on how a ring-translocase may function or be regulated. It also illustrates the interest and versatility of the new trNAIP methodology to unveil the molecular mechanisms of complex RNA-based systems. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Efficient Inhibition of wear debris-induced inflammation by locally delivered siRNA

International Nuclear Information System (INIS)

Peng Xiaochun; Tao Kun; Cheng Tao; Zhu Junfeng; Zhang Xianlong

2008-01-01

Aseptic loosening is the most common long-term complication of total joint replacement, which is associated with the generation of wear debris. The purpose of this study was to investigate the inhibitory effect of small interfering RNA (siRNA) targeting tumor necrosis factor-α (TNF-α) on wear debris-induced inflammation. A local delivery of lentivirus-mediated TNF-α siRNA into the modified murine air pouch, which was stimulated by polymethylmethacrylate (PMMA) particles, resulted in significant blockage of TNF-α both in mRNA and protein levels for up to 4 weeks. In addition, significant down-regulation of interleukin-1 (IL-1) and interleukin-6 (IL-6) was observed in TNF-α siRNA-treated pouches. The safety profile of gene therapy was proven by Bioluminescent assay and quantitative fluorescent flux. Histological analysis revealed less inflammatory responses (thinner pouch membrane and decreased cellular infiltration) in TNF-α siRNA-treated pouches. These findings suggest that local delivery of TNF-α siRNA might be an excellent therapeutic candidate to inhibit particle-induced inflammation.

RNA-Interference Components Are Dispensable for Transcriptional Silencing of the Drosophila Bithorax-Complex

KAUST Repository

Cernilogar, Filippo M.

2013-06-13

Background:Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.Principal Findings:Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (BX-C). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the BX-C. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins.Conclusions:We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in Drosophila. © 2013 Cernilogar et al.

In vivo targeting of ADAM9 gene expression using lentivirus-delivered shRNA suppresses prostate cancer growth by regulating REG4 dependent cell cycle progression.

Directory of Open Access Journals (Sweden)

Che-Ming Liu

Full Text Available Cancer cells respond to stress by activating a variety of survival signaling pathways. A disintegrin and metalloproteinase (ADAM 9 is upregulated during cancer progression and hormone therapy, functioning in part through an increase in reactive oxygen species. Here, we present in vitro and in vivo evidence that therapeutic targeting of ADAM9 gene expression by lentivirus-delivered small hairpin RNA (shRNA significantly inhibited proliferation of human prostate cancer cell lines and blocked tumor growth in a murine model of prostate cancer bone metastasis. Cell cycle studies confirmed an increase in the G1-phase and decrease in the S-phase population of cancer cells under starvation stress conditions, which correlated with elevated intracellular superoxide levels. Microarray data showed significantly decreased levels of regenerating islet-derived family member 4 (REG4 expression in prostate cancer cells with knockdown of ADAM9 gene expression. This REG4 downregulation also resulted in induction of expression of p21(Cip1/WAF1, which negatively regulates cyclin D1 and blocks the G1/S transition. Our data reveal a novel molecular mechanism of ADAM9 in the regulation of prostate cancer cell proliferation, and suggests a combined modality of ADAM9 shRNA gene therapy and cytotoxic agents for hormone refractory and bone metastatic prostate cancer.

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

Science.gov (United States)

Parrish, S; Fire, A

2001-10-01

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.

Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes.

Science.gov (United States)

Göertz, G P; Fros, J J; Miesen, P; Vogels, C B F; van der Bent, M L; Geertsema, C; Koenraadt, C J M; van Rij, R P; van Oers, M M; Pijlman, G P

2016-11-15

Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5'-3' exoribonuclease XRN1/Pacman on conserved RNA structures in the 3' untranslated region (UTR) of the viral genomic RNA. sfRNA production is conserved in insect-specific, mosquito-borne, and tick-borne flaviviruses and flaviviruses with no known vector, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here, we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed growth kinetics similar to those of wild-type WNV in both RNA interference (RNAi)-competent and -compromised mosquito cell lines. Small-RNA deep sequencing of WNV-infected mosquitoes indicated an active small interfering RNA (siRNA)-based antiviral response for both the wild-type and sfRNA1-deficient viruses. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo Two reproducible small-RNA hot spots within the 3' UTR/sfRNA of the wild-type virus mapped to RNA stem-loops SL-III and 3' SL, which stick out of the three-dimensional (3D) sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administered via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production. Understanding the flavivirus transmission

Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

Directory of Open Access Journals (Sweden)

Alexey Berezhnoy

2012-01-01

Full Text Available Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

RNA Interference in Moths: Mechanisms, Applications, and Progress

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Jin Xu

2016-10-01

Full Text Available The vast majority of lepidopterans, about 90%, are moths. Some moths, particularly their caterpillars, are major agricultural and forestry pests in many parts of the world. However, some other members of moths, such as the silkworm Bombyx mori, are famous for their economic value. Fire et al. in 1998 initially found that exogenous double-stranded RNA (dsRNA can silence the homolog endogenous mRNA in organisms, which is called RNA interference (RNAi. Soon after, the RNAi technique proved to be very promising not only in gene function determination but also in pest control. However, later studies demonstrate that performing RNAi in moths is not as straightforward as shown in other insect taxa. Nevertheless, since 2007, especially after 2010, an increasing number of reports have been published that describe successful RNAi experiments in different moth species either on gene function analysis or on pest management exploration. So far, more than 100 peer-reviewed papers have reported successful RNAi experiments in moths, covering 10 families and 25 species. By using classic and novel dsRNA delivery methods, these studies effectively silence the expression of various target genes and determine their function in larval development, reproduction, immunology, resistance against chemicals, and other biological processes. In addition, a number of laboratory and field trials have demonstrated that RNAi is also a potential strategy for moth pest management. In this review, therefore, we summarize and discuss the mechanisms and applications of the RNAi technique in moths by focusing on recent progresses.

RNA Interference in Insect Vectors for Plant Viruses

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Surapathrudu Kanakala

2016-12-01

Full Text Available Insects and other arthropods are the most important vectors of plant pathogens. The majority of plant pathogens are disseminated by arthropod vectors such as aphids, beetles, leafhoppers, planthoppers, thrips and whiteflies. Transmission of plant pathogens and the challenges in managing insect vectors due to insecticide resistance are factors that contribute to major food losses in agriculture. RNA interference (RNAi was recently suggested as a promising strategy for controlling insect pests, including those that serve as important vectors for plant pathogens. The last decade has witnessed a dramatic increase in the functional analysis of insect genes, especially those whose silencing results in mortality or interference with pathogen transmission. The identification of such candidates poses a major challenge for increasing the role of RNAi in pest control. Another challenge is to understand the RNAi machinery in insect cells and whether components that were identified in other organisms are also present in insect. This review will focus on summarizing success cases in which RNAi was used for silencing genes in insect vector for plant pathogens, and will be particularly helpful for vector biologists.

Eukaryotic interference with homoserine lactone mediated procaryotic signalling

DEFF Research Database (Denmark)

Givskov, Michael Christian; de Nys, Rocky; Gram, Lone

1996-01-01

Acylated homoserine lactones (AHLs) plays a widespread role in intercellular communication among bacteria. The Australian macroalga Delisea pulchra produces secondary metabolites which have structural similarities to AHL molecules. We report here that these metabolites inhibited AHL-controlled pr......-controlled processes in prokaryotes. Our results suggest that the interaction between higher organisms and their surface-associated bacteria may be mediated by interference with bacterial regulatory systems....

Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

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Jonathan A Kopechek

Full Text Available RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14. Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9 or control RNA (n = 8 during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3 confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively. Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.

Short-hairpin RNA-mediated stable silencing of Grb2 impairs cell growth and DNA synthesis

International Nuclear Information System (INIS)

Di Fulvio, Mauricio; Henkels, Karen M.; Gomez-Cambronero, Julian

2007-01-01

Grb2 is an SH2-SH3 protein adaptor responsible for linking growth factor receptors with intracellular signaling cascades. To study the role of Grb2 in cell growth, we have generated a new COS7 cell line (COS7 shGrb2 ), based on RNAi technology, as null mutations in mammalian Grb2 genes are lethal in early development. This novel cell line continuously expresses a short hairpin RNA that targets endogenous Grb2. Stable COS7 shGrb2 cells had the shGrb2 integrated into the genomic DNA and carried on SiL construct (made refractory to the shRNA-mediated interference), but not with an SH2-deficient mutant (R86K). Thus, a viable knock-down and rescue protocol has demonstrated that Grb2 is crucial for cell proliferation

CXCR4 Is Required by a Nonprimate Lentivirus: Heterologous Expression of Feline Immunodeficiency Virus in Human, Rodent, and Feline Cells

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Poeschla, Eric M.; Looney, David J.

1998-01-01

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors. PMID:9658135

Dysregulation of RNA Mediated Gene Expression in Motor Neuron Diseases.

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Gonçalves, Inês do Carmo G; Rehorst, Wiebke A; Kye, Min Jeong

2016-01-01

Recent findings indicate an important role for RNA-mediated gene expression in motor neuron diseases, including ALS (amyotrophic lateral sclerosis) and SMA (spinal muscular atrophy). ALS, also known as Lou Gehrig's disease, is an adult-onset progressive neurodegenerative disorder, whereby SMA or "children's Lou Gehrig's disease" is considered a pediatric neurodevelopmental disorder. Despite the difference in genetic causes, both ALS and SMA share common phenotypes; dysfunction/loss of motor neurons that eventually leads to muscle weakness and atrophy. With advanced techniques in molecular genetics and cell biology, current data suggest that these two distinct motor neuron diseases share more than phenotypes; ALS and SMA have similar cellular pathological mechanisms including mitochondrial dysfunction, oxidative stress and dysregulation in RNA-mediated gene expression. Here, we will discuss the current findings on these two diseases with specific focus on RNA-mediated gene regulation including miRNA expression, pre-mRNA processing and RNA binding proteins.

RNA interference for performance enhancement and detection in doping control.

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Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

2011-10-01

RNA interference represents a comparably new route of regulating and manipulating specific gene expression. Promising results were obtained in experimental therapies aim at the treatment of different kinds of diseases including cancer, diabetes mellitus or Dychenne muscular dystrophy. While studies on down-regulation efficiency are often performed by analyzing the regulated protein, the direct detection of small, interfering RNA molecules and antisense oligonucleotides is of great interest for the investigation of the metabolism and degradation and also for the detection of a putative misuse of these molecules in sports. Myostatin down-regulation was shown to result in increased performance and muscle growth and the regulation of several other proteins could be relevant for performance enhancement. This mini-review summarizes current approaches for the mass spectrometric analysis of siRNA and antisense oligonucleotides from biological matrices and the available data on biodistribution, metabolism, and half-life of relevant substances are discussed. Copyright © 2011 John Wiley & Sons, Ltd.

A lion lentivirus related to feline immunodeficiency virus: epidemiologic and phylogenetic aspects.

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Brown, E W; Yuhki, N; Packer, C; O'Brien, S J

1994-09-01

Feline immunodeficiency virus (FIV) is a novel lentivirus that is genetically homologous and functionally analogous to the human AIDS viruses, human immunodeficiency virus types 1 and 2. FIV causes immunosuppression in domestic cats by destroying the CD4 T-lymphocyte subsets in infected hosts. A serological survey of over 400 free-ranging African and Asian lions (Panthera leo) for antibodies to FIV revealed endemic lentivirus prevalence with an incidence of seropositivity as high as 90%. A lion lentivirus (FIV-Ple) was isolated by infection of lion lymphocytes in vitro. Seroconversion was documented in two Serengeti lions, and discordance of mother-cub serological status argues against maternal transmission (in favor of horizontal spread) as a major route of infection among lions. A phylogenetic analysis of cloned FIV-Ple pol gene sequences from 27 lions from four African populations (from the Serengeti reserve, Ngorongoro Crater, Lake Manyara, and Kruger Park) revealed remarkably high intra- and interindividual genetic diversity at the sequence level. Three FIV-Ple phylogenetic clusters or clades were resolved with phenetic, parsimony, and likelihood analytical procedures. The three clades, which occurred not only together in the same population but throughout Africa, were as divergent from each other as were homologous pol sequences of lentivirus isolated from distinct feline species, i.e., puma and domestic cat. The FIV-Ple clades, however, were more closely related to each other than to other feline lentiviruses (monophyletic for lion species), suggesting that the ancestors of FIV-Ple evolved in allopatric (geographically isolated) lion populations that converged recently. To date, there is no clear evidence of FIV-Ple-associated pathology, raising the possibility of a historic genetic accommodation of the lion lentivirus and its host leading to a coevolved host-parasite symbiosis (or commensalism) in the population similar to that hypothesized for endemic

Role of receptor-mediated endocytosis in the antiangiogenic effects of human T lymphoblastic cell-derived microparticles.

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Yang, Chun; Xiong, Wei; Qiu, Qian; Shao, Zhuo; Shao, Zuo; Hamel, David; Tahiri, Houda; Leclair, Grégoire; Lachapelle, Pierre; Chemtob, Sylvain; Hardy, Pierre

2012-04-15

Microparticles possess therapeutic potential regarding angiogenesis. We have demonstrated the contribution of apoptotic human CEM T lymphocyte-derived microparticles (LMPs) as inhibitors of angiogenic responses in animal models of inflammation and tumor growth. In the present study, we characterized the antivascular endothelial growth factor (VEGF) effects of LMPs on pathological angiogenesis in an animal model of oxygen-induced retinopathy and explored the role of receptor-mediated endocytosis in the effects of LMPs on human retinal endothelial cells (HRECs). LMPs dramatically inhibited cell growth of HRECs, suppressed VEGF-induced cell migration in vitro experiments, and attenuated VEGF-induced retinal vascular leakage in vivo. Intravitreal injections of fluorescently labeled LMPs revealed accumulation of LMPs in retinal tissue, with more than 60% reductions of the vascular density in retinas of rats with oxygen-induced neovascularization. LMP uptake experiments demonstrated that the interaction between LMPs and HRECs is dependent on temperature. In addition, endocytosis is partially dependent on extracellular calcium. RNAi-mediated knockdown of low-density lipoprotein receptor (LDLR) reduced the uptake of LMPs and attenuated the inhibitory effects of LMPs on VEGF-A protein expression and HRECs cell growth. Intravitreal injection of lentivirus-mediated RNA interference reduced LDLR protein expression in retina by 53% and significantly blocked the antiangiogenic effects of LMPs on pathological vascularization. In summary, the potent antiangiogenic LMPs lead to a significant reduction of pathological retinal angiogenesis through modulation of VEGF signaling, whereas LDLR-mediated endocytosis plays a partial, but pivotal, role in the uptake of LMPs in HRECs.

Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo

NARCIS (Netherlands)

Mook, Olaf R.; Baas, Frank; de Wissel, Marit B.; Fluiter, Kees

2007-01-01

RNA interference has become widely used as an experimental tool to study gene function. In addition, small interfering RNA (siRNA) may have great potential for the treatment of diseases. Recently, it was shown that siRNA can be used to mediate gene silencing in mouse models. Locally administered

Modelling Toehold-Mediated RNA Strand Displacement

OpenAIRE

Šulc, Petr; Ouldridge, Thomas E.; Romano, Flavio; Doye, Jonathan P.K.; Louis, Ard A.

2015-01-01

We study the thermodynamics and kinetics of an RNA toehold-mediated strand displacement reaction with a recently developed coarse-grained model of RNA. Strand displacement, during which a single strand displaces a different strand previously bound to a complementary substrate strand, is an essential mechanism in active nucleic acid nanotechnology and has also been hypothesized to occur in vivo. We study the rate of displacement reactions as a function of the length of the toehold and temperat...

DMPD: Negative regulation of cytoplasmic RNA-mediated antiviral signaling. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 18703349 Negative regulation of cytoplasmic RNA-mediated antiviral signaling. Komur...Show Negative regulation of cytoplasmic RNA-mediated antiviral signaling. PubmedID 18703349 Title Negative r...egulation of cytoplasmic RNA-mediated antiviral signaling. Authors Komuro A, Bamm

Role of RNA interference in plant improvement.

Science.gov (United States)

Jagtap, Umesh Balkrishna; Gurav, Ranjit Gajanan; Bapat, Vishwas Anant

2011-06-01

Research to alter crops for their better performance involving modern technology is underway in numerous plants, and achievements in transgenic plants are impacting crop improvements in unparalleled ways. Striking progress has been made using genetic engineering technology over the past two decades in manipulating genes from diverse and exotic sources, and inserting them into crop plants for inducing desirable characteristics. RNA interference (RNAi) has recently been identified as a natural mechanism for regulation of gene expression in all higher organisms from plants to humans and promises greater accuracy and precision to plant improvement. The expression of any gene can be down-regulated in a highly explicit manner exclusive of affecting the expression of any other gene by using RNAi technologies. Additional research in this field has been focused on a number of other areas including microRNAs, hairpin RNA, and promoter methylation. Manipulating new RNAi pathways, which generate small RNA molecules to amend gene expression in crops, can produce new quality traits and having better potentiality of protection against abiotic and biotic stresses. Nutritional improvement, change in morphology, or enhanced secondary metabolite synthesis are some of the other advantages of RNAi technology. In addition to its roles in regulating gene expression, RNAi is also used as a natural defense mechanism against molecular parasites such as jumping genes and viral genetic elements that affect genome stability. Even though much advancement has been made on the field of RNAi over the preceding few years, the full prospective of RNAi for crop improvement remains to be fully realized. The intricacy of RNAi pathway, the molecular machineries, and how it relates to plant development are still to be explained.

Role of RNA interference in plant improvement

Science.gov (United States)

Jagtap, Umesh Balkrishna; Gurav, Ranjit Gajanan; Bapat, Vishwas Anant

2011-06-01

Research to alter crops for their better performance involving modern technology is underway in numerous plants, and achievements in transgenic plants are impacting crop improvements in unparalleled ways. Striking progress has been made using genetic engineering technology over the past two decades in manipulating genes from diverse and exotic sources, and inserting them into crop plants for inducing desirable characteristics. RNA interference (RNAi) has recently been identified as a natural mechanism for regulation of gene expression in all higher organisms from plants to humans and promises greater accuracy and precision to plant improvement. The expression of any gene can be down-regulated in a highly explicit manner exclusive of affecting the expression of any other gene by using RNAi technologies. Additional research in this field has been focused on a number of other areas including microRNAs, hairpin RNA, and promoter methylation. Manipulating new RNAi pathways, which generate small RNA molecules to amend gene expression in crops, can produce new quality traits and having better potentiality of protection against abiotic and biotic stresses. Nutritional improvement, change in morphology, or enhanced secondary metabolite synthesis are some of the other advantages of RNAi technology. In addition to its roles in regulating gene expression, RNAi is also used as a natural defense mechanism against molecular parasites such as jumping genes and viral genetic elements that affect genome stability. Even though much advancement has been made on the field of RNAi over the preceding few years, the full prospective of RNAi for crop improvement remains to be fully realized. The intricacy of RNAi pathway, the molecular machineries, and how it relates to plant development are still to be explained.

Identification of chemosensory receptor genes in Manduca sexta and knockdown by RNA interference

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Howlett Natalie

2012-05-01

Full Text Available Abstract Background Insects detect environmental chemicals via a large and rapidly evolving family of chemosensory receptor proteins. Although our understanding of the molecular genetic basis for Drosophila chemoreception has increased enormously in the last decade, similar understanding in other insects remains limited. The tobacco hornworm, Manduca sexta, has long been an important model for insect chemosensation, particularly from ecological, behavioral, and physiological standpoints. It is also a major agricultural pest on solanaceous crops. However, little sequence information and lack of genetic tools has prevented molecular genetic analysis in this species. The ability to connect molecular genetic mechanisms, including potential lineage-specific changes in chemosensory genes, to ecologically relevant behaviors and specializations in M. sexta would be greatly beneficial. Results Here, we sequenced transcriptomes from adult and larval chemosensory tissues and identified chemosensory genes based on sequence homology. We also used dsRNA feeding as a method to induce RNA interference in larval chemosensory tissues. Conclusions We report identification of new chemosensory receptor genes including 17 novel odorant receptors and one novel gustatory receptor. Further, we demonstrate that systemic RNA interference can be used in larval olfactory neurons to reduce expression of chemosensory receptor transcripts. Together, our results further the development of M. sexta as a model for functional analysis of insect chemosensation.

Theranostic Niosomes for Efficient siRNA/microRNA Delivery and Activatable Near-Infrared Fluorescent Tracking of Stem Cells

DEFF Research Database (Denmark)

Yang, Chuanxu; Shan, Gao; Song, Ping

2018-01-01

RNA interference (RNAi) mediated gene regulation in stem cells offers great potential in regenerative medicine. In this study, we developed a theranostic platform for efficient delivery of small RNAs (siRNA/miRNA) to human mesenchymal stem cells (hMSCs) to promote differentiation, and meanwhile...... OFF/ON activatable fluorescence upon cellular internalization, resulting in efficient NIR labeling and the capability to dynamically monitor stem cells in mice. In addition, iSPN/siRNA achieved simultaneous long-term cell tracking and in vivo gene silencing after implantation in mice. These results...

RNA-mediated epigenetic heredity requires the cytosine methyltransferase Dnmt2.

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Jafar Kiani

2013-05-01

Full Text Available RNA-mediated transmission of phenotypes is an important way to explain non-Mendelian heredity. We have previously shown that small non-coding RNAs can induce hereditary epigenetic variations in mice and act as the transgenerational signalling molecules. Two prominent examples for these paramutations include the epigenetic modulation of the Kit gene, resulting in altered fur coloration, and the modulation of the Sox9 gene, resulting in an overgrowth phenotype. We now report that expression of the Dnmt2 RNA methyltransferase is required for the establishment and hereditary maintenance of both paramutations. Our data show that the Kit paramutant phenotype was not transmitted to the progeny of Dnmt2(-/- mice and that the Sox9 paramutation was also not established in Dnmt2(-/- embryos. Similarly, RNA from Dnmt2-negative Kit heterozygotes did not induce the paramutant phenotype when microinjected into Dnmt2-deficient fertilized eggs and microinjection of the miR-124 microRNA failed to induce the characteristic giant phenotype. In agreement with an RNA-mediated mechanism of inheritance, no change was observed in the DNA methylation profiles of the Kit locus between the wild-type and paramutant mice. RNA bisulfite sequencing confirmed Dnmt2-dependent tRNA methylation in mouse sperm and also indicated Dnmt2-dependent cytosine methylation in Kit RNA in paramutant embryos. Together, these findings uncover a novel function of Dnmt2 in RNA-mediated epigenetic heredity.

Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

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Delphine Trochet

2016-01-01

Full Text Available Dynamin 2 (DNM2 is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.

RNA interference gene therapy in dominant retinitis pigmentosa and cone-rod dystrophy mouse models caused by GCAP1 mutations

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Li eJiang

2014-04-01

Full Text Available RNA interference (RNAi knockdown is an efficacious therapeutic strategy for silencing genes causative for dominant retinal dystrophies. To test this, we used self-complementary (sc AAV2/8 vector to develop an RNAi-based therapy in two dominant retinal degeneration mouse models. The allele-specific model expresses transgenic bovine GCAP1(Y99C establishing a rapid RP-like phenotype, whereas the nonallele-specific model expresses mouse GCAP1(L151F producing a slowly progressing cone/rod dystrophy (CORD. The late onset GCAP1(L151F-CORD mimics the dystrophy observed in human GCAP1-CORD patients. Subretinal injection of scAAV2/8 carrying shRNA expression cassettes specific for bovine or mouse GCAP1 showed strong expression at one week post-injection. In both allele-specific (GCAP1(Y99C-RP and nonallele-specific (GCAP1(L151F-CORD models of dominant retinal dystrophy, RNAi-mediated gene silencing enhanced photoreceptor survival, delayed onset of degeneration and improved visual function. Such results provide a proof of concept toward effective RNAi-based gene therapy mediated by scAAV2/8 for dominant retinal disease based on GCAP1 mutation. Further, nonallele-specific RNAi knockdown of GCAP1 may prove generally applicable toward the rescue of any human GCAP1-based dominant cone-rod dystrophy.

Small Interference RNA Targeting TLR4 Gene Effectively Attenuates Pulmonary Inflammation in a Rat Model

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Feixiang Wu

2012-01-01

Full Text Available Objective. The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA targeting Toll-like receptor 4 (TLR4 gene in ameliorating lipopolysaccharide- (LPS- induced acute lung injury (ALI. Methods. In vitro, alveolar macrophages (AMs were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL for 2 h, and the function and expression of TLR4 were evaluated. In vivo, rats received intratracheal injection of 300 μL of normal saline (control group, 300 μL of Ad-EGFP (Ad-EGFP group, or 300 μL of Ad-siTLR4 (Ad-siTLR4 group and then were intravenously treated with LPS (50 mg/kg to induce ALI. Results. Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals. Conclusion. TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

Effective mRNA Inhibition in PANC-1 Cells in Vitro Mediated via an mPEG-SeSe-PEI Delivery System.

Science.gov (United States)

Zhang, Yuefeng; Yang, Bin; Liu, Yajie; Qin, Wenjie; Li, Chao; Wang, Lantian; Zheng, Wen; Wu, Yulian

2016-05-01

RNA interference (RNAi)-mediated gene therapy is a promising approach to cure various diseases. However, developing an effective, safe, specific RNAi delivery system remains a major challenge. In this study, a novel redox-responsive polyetherimide (PEI)-based nanovector, mPEG-SeSe-PEI, was developed and its efficacy evaluated. We prepared three mPEG-SeSe-PEI vector candidates for small interfering glyceraldehyde-3-phosphate dehydrogenase (siGADPH) and determined their physiochemical properties and transfection efficiency using flow cytometry and PEG11.6-SeSe-PEI polymer. We investigated the silencing efficacy of GADPH mRNA expression in PANC-1 cells and observed that PEG11.6-SeSe-PEI/siGADPH (N/P ratio=10) polyplexes possessed the appropriate size and zeta-potential and exhibited excellent in vitro gene silencing effects with the least cytotoxicity in PANC-1 cells. In conclusion, we present PEG11.6-SeSe-PEI as a potential therapeutic gene delivery system for small interfering RNA (siRNA).

RNA interference: learning gene knock-down from cell physiology

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Provenzano Maurizio

2004-11-01

Full Text Available Summary Over the past decade RNA interference (RNAi has emerged as a natural mechanism for silencing gene expression. This ancient cellular antiviral response can be exploited to allow specific inhibition of the function of any chosen target gene. RNAi is proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, RNAi technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes. This review briefly describes the molecular principles underlying the biology of RNAi phenomenon and discuss the main technical issues regarding optimization of RNAi experimental design.

RNA interference: learning gene knock-down from cell physiology

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Mocellin, Simone; Provenzano, Maurizio

2004-01-01

Over the past decade RNA interference (RNAi) has emerged as a natural mechanism for silencing gene expression. This ancient cellular antiviral response can be exploited to allow specific inhibition of the function of any chosen target gene. RNAi is proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, RNAi technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes. This review briefly describes the molecular principles underlying the biology of RNAi phenomenon and discuss the main technical issues regarding optimization of RNAi experimental design. PMID:15555080

A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines.

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Wang, Fei; Guo, Tao; Jiang, Hongmei; Li, Ruobi; Wang, Ting; Zeng, Ni; Dong, Guanghui; Zeng, Xiaowen; Li, Daochuan; Xiao, Yongmei; Hu, Qiansheng; Chen, Wen; Xing, Xiumei; Wang, Qing

2018-06-01

Gene knockdown and knockout using RNAi and CRISPR/Cas9 allow for efficient evaluation of gene function, but it is unclear how the choice of technology can influence the results. To compare the phenotypes obtained using siRNA and CRISPR/Cas9 technologies, aldehyde dehydrogenase 2 (ALDH2) was selected as an example. In this study, we constructed one HepG2 cell line with a homozygous mutation in the fifth exon of ALDH2 (ALDH2-KO1 cell) using the eukaryotic CRISPR/Cas9 expression system followed by the limited dilution method and one HepG2 cell line with different mutations in the ALDH2 gene (ALDH2-KO2 cell) using the lentivirus CRISPR/Cas9 system. Additionally, one ALDH2-knockdown (KD) HepG2 cell line was created using siRNA. The reproducibility of these methods was further verified in the HEK293FT cell line. We found that the mRNA expression level of ALDH2 was significantly decreased and the protein expression level of ALDH2 was completely abolished in the ALDH2-KO cell lines, but not in ALDH2-KD cells. Furthermore, the functional activity of ALDH2 was also markedly disrupted in the two ALDH2-KO cell lines compared with ALDH2-KD and wild-type cells. The lack of ALDH2 expression mediated by CRIPSR/Cas9 resulted in a more dramatic increase in the cellular susceptibility to chemical-induced reactive oxygen species generation, cytotoxicity, apoptosis, and inflammation, especially at low concentrations compared with ALDH2-KD and WT cells. Therefore, we consider the gene knockout cell line created by CRISPR/Cas9 to be a more useful tool for identifying the function of a gene.

Thermodynamic control of small RNA-mediated gene silencing

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Kumiko eUi-Tei

2012-06-01

Full Text Available Small interfering RNAs (siRNAs and microRNAs (miRNAs are crucial regulators of posttranscriptional gene silencing, which is referred to as RNA interference (RNAi or RNA silencing. In RNAi, siRNA loaded onto the RNA-induced silencing complex (RISC downregulates target gene expression by cleaving mRNA whose sequence is perfectly complementary to the siRNA guide strand. We previously showed that highly functional siRNAs possessed the following characteristics: A or U residues at nucleotide position 1 measured from the 5’ terminal, four to seven A/Us in positions 1–7, and G or C residues at position 19. This finding indicated that an RNA strand with a thermodynamically unstable 5’ terminal is easily retained in the RISC and functions as a guide strand. In addition, it is clear that unintended genes with complementarities only in the seed region (positions 2–8 are also downregulated by off-target effects. siRNA efficiency is mainly determined by the Watson-Crick base-pairing stability formed between the siRNA seed region and target mRNA. siRNAs with a low seed-target duplex melting temperature (Tm have little or no seed-dependent off-target activity. Thus, important parts of the RNA silencing machinery may be regulated by nucleotide base-pairing thermodynamic stability. A mechanistic understanding of thermodynamic control may enable an efficient target gene-specific RNAi for functional genomics and safe therapeutic applications.

SAF-A forms a complex with BRG1 and both components are required for RNA polymerase II mediated transcription.

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Dzeneta Vizlin-Hodzic

Full Text Available BACKGROUND: Scaffold attachment factor A (SAF-A participates in the regulation of gene expression by organizing chromatin into transcriptionally active domains and by interacting directly with RNA polymerase II. METHODOLOGY: Here we use co-localization, co-immunoprecipitation (co-IP and in situ proximity ligation assay (PLA to identify Brahma Related Gene 1 (BRG1, the ATP-driven motor of the human SWI-SNF chromatin remodeling complex, as another SAF-A interaction partner in mouse embryonic stem (mES cells. We also employ RNA interference to investigate functional aspects of the SAF-A/BRG1 interaction. PRINCIPAL FINDINGS: We find that endogenous SAF-A protein interacts with endogenous BRG1 protein in mES cells, and that the interaction does not solely depend on the presence of mRNA. Moreover the interaction remains intact when cells are induced to differentiate. Functional analyses reveal that dual depletion of SAF-A and BRG1 abolishes global transcription by RNA polymerase II, while the nucleolar RNA polymerase I transcription machinery remains unaffected. CONCLUSIONS: We demonstrate that SAF-A interacts with BRG1 and that both components are required for RNA Polymerase II Mediated Transcription.

Neuroprotection by biodegradable PAMAM ester (e-PAM-R)-mediated HMGB1 siRNA delivery in primary cortical cultures and in the postischemic brain.

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Kim, Il-Doo; Lim, Chae-Moon; Kim, Jung-Bin; Nam, Hye Yeong; Nam, Kihoon; Kim, Seung-Woo; Park, Jong-Sang; Lee, Ja-Kyeong

2010-03-19

Although RNA interference (RNAi)-mediated gene silencing provides a powerful strategy for modulating specific gene functions, difficulties associated with siRNA delivery have impeded the development of efficient therapeutic applications. In particular, the efficacy of siRNA delivery into neurons has been limited by extremely low transfection efficiencies. e-PAM-R is a biodegradable arginine ester of PAMAM dendrimer, which is readily degradable under physiological conditions (pH 7.4, 37 degrees C). In the present study, we investigated the efficiency of siRNA delivery by e-PAM-R in primary cortical cultures and in rat brain. e-PAM-R/siRNA complexes showed high transfection efficiencies and low cytotoxicities in primary cortical cultures. Localization of fluorescence-tagged siRNA revealed that siRNA was delivered not only into the nucleus and cytoplasm, but also along the processes of the neuron. e-PAM-R/siRNA complex-mediated target gene reduction was observed in over 40% of cells and it was persistent for over 48 h. The potential use of e-PAM-R was demonstrated by gene knockdown after transfecting High mobility group box-1 (HMGB1, a novel cytokine-like molecule) siRNA into H(2)O(2)- or NMDA-treated primary cortical cultures. In these cells, HMGB1 siRNA delivery successfully reduced both basal and H(2)O(2)- or NMDA-induced HMGB1 levels, and as a result of that, neuronal cell death was significantly suppressed in both cases. Furthermore, we showed that e-PAM-R successfully delivered HMGB1 siRNA into the rat brain, wherein HMGB1 expression was depleted in over 40% of neurons and astrocytes of the normal brain. Moreover, e-PAM-R-mediated HMGB1 siRNA delivery notably reduced infarct volume in the postischemic rat brain, which is generated by occluding the middle cerebral artery for 60 min. These results indicate that e-PAM-R, a novel biodegradable nonviral gene carrier, offers an efficient means of transfecting siRNA into primary neuronal cells and in the brain and of

Evaluation of carrier-mediated siRNA delivery

DEFF Research Database (Denmark)

Colombo, Stefano; Nielsen, Hanne Mørck; Foged, Camilla

2013-01-01

RNA delivery. An in vitro cell culture model system expressing enhanced green fluorescent protein (EGFP) was used to develop the assay, which was based on the intracellular quantification of a full-length double-stranded Dicer substrate siRNA by stem-loop RT qPCR. The result is a well-documented protocol......RNA delivered by use of carriers remains an analytical challenge. The purpose of the present study was to optimize and validate an analytical protocol based on stem-loop reverse transcription quantitative polymerase chain reaction (RT qPCR) to quantitatively monitor the carrier-mediated intracellular si...

RNA interference: Applications and advances in insect toxicology and insect pest management.

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Kim, Young Ho; Soumaila Issa, Moustapha; Cooper, Anastasia M W; Zhu, Kun Yan

2015-05-01

Since its discovery, RNA interference (RNAi) has revolutionized functional genomic studies due to its sequence-specific nature of post-transcriptional gene silencing. In this paper, we provide a comprehensive review of the recent literature and summarize the current knowledge and advances in the applications of RNAi technologies in the field of insect toxicology and insect pest management. Many recent studies have focused on identification and validation of the genes encoding insecticide target proteins, such as acetylcholinesterases, ion channels, Bacillus thuringiensis receptors, and other receptors in the nervous system. RNAi technologies have also been widely applied to reveal the role of genes encoding cytochrome P450 monooxygenases, carboxylesterases, and glutathione S-transferases in insecticide detoxification and resistance. More recently, studies have focused on understanding the mechanism of insecticide-mediated up-regulation of detoxification genes in insects. As RNAi has already shown great potentials for insect pest management, many recent studies have also focused on host-induced gene silencing, in which several RNAi-based transgenic plants have been developed and tested as proof of concept for insect pest management. These studies indicate that RNAi is a valuable tool to address various fundamental questions in insect toxicology and may soon become an effective strategy for insect pest management. Copyright © 2015 Elsevier Inc. All rights reserved.

Non-Target Effects of Green Fluorescent Protein (GFP-Derived Double-Stranded RNA (dsRNA-GFP Used in Honey Bee RNA Interference (RNAi Assays

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Francis M. F. Nunes

2013-01-01

Full Text Available RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP-derived double-stranded RNA (dsRNA-GFP is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays.

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Nunes, Francis M F; Aleixo, Aline C; Barchuk, Angel R; Bomtorin, Ana D; Grozinger, Christina M; Simões, Zilá L P

2013-01-04

RNA interference has been frequently applied to modulate gene function in organisms where the production and maintenance of mutants is challenging, as in our model of study, the honey bee, Apis mellifera. A green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP) is currently commonly used as control in honey bee RNAi experiments, since its gene does not exist in the A. mellifera genome. Although dsRNA-GFP is not expected to trigger RNAi responses in treated bees, undesirable effects on gene expression, pigmentation or developmental timing are often observed. Here, we performed three independent experiments using microarrays to examine the effect of dsRNA-GFP treatment (introduced by feeding) on global gene expression patterns in developing worker bees. Our data revealed that the expression of nearly 1,400 genes was altered in response to dsRNA-GFP, representing around 10% of known honey bee genes. Expression changes appear to be the result of both direct off-target effects and indirect downstream secondary effects; indeed, there were several instances of sequence similarity between putative siRNAs generated from the dsRNA-GFP construct and genes whose expression levels were altered. In general, the affected genes are involved in important developmental and metabolic processes associated with RNA processing and transport, hormone metabolism, immunity, response to external stimulus and to stress. These results suggest that multiple dsRNA controls should be employed in RNAi studies in honey bees. Furthermore, any RNAi studies involving these genes affected by dsRNA-GFP in our studies should use a different dsRNA control.

Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

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Cecere, Germano; Hoersch, Sebastian; O'Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

2014-04-01

Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, its role in gene expression regulation remains controversial. Here we used genome-wide profiling of nascent RNA transcripts and found that the CSR-1 RNA interference pathway promoted sense-oriented RNA polymerase II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. On the basis of these findings, we propose that the CSR-1 pathway helps maintain the directionality of active transcription, thereby propagating the distinction between transcriptionally active and silent genomic regions.

Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription.

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Hicks, Jessica A; Li, Liande; Matsui, Masayuki; Chu, Yongjun; Volkov, Oleg; Johnson, Krystal C; Corey, David R

2017-08-15

In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Branched RNA: A New Architecture for RNA Interference

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Anna Aviñó

2011-01-01

Full Text Available Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

[VEGF165 transfected endothelial progenitor cells mediated by lentivirus alleviated ALI in rats].

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He, Zhaohui; He, Huiwei; Lu, Yuanhua; Chen, Zhi; Xu, Fanghua; Wang, Rongsheng; Yang, Chunli

2017-11-01

To investigate the protective effects of vascular endothelial growth factor-165 (VEGF165) transfected the endothelial progenitor cells (EPCs) mediated by lentivirus on acute lung injury (ALI) in rats. The mononuclear cells from the male Sprague-Dawley (SD) rats were isolated and cultured to get the EPCs for study. The lentivirus vector carrying the human VEGF165 gene was constructed. According to the random number table method, 90 male SD rats were divided into ALI model group, phosphate buffer solution (PBS) group, EPCs treatment group, none transfected EPCs treatment group and VEGF165 transfected EPCs treatment group, and the rats in each group were subdivided into 4, 12 and 48 hours subgroups, with 6 rats in each subgroup. The rat model of ALI was reproduced by intravenous injection of oleic acid (0.15 μL/g). Then each treatment group was given PBS, EPCs, none transfected EPCs and VEGF165 transfected EPCs respectively with the same volume of 0.2 mL. For the groups with cells, about 1×10 6 cells were contained. Abdominal aortic blood and lung tissue were harvested at 4, 12 and 48 hours. Arterial blood gas analysis was performed. The lung wet/dry weight ratio (W/D) was calculated. The expressions of induced nitric oxide synthase (iNOS), endothelin-1 (ET-1) and VEGF165 were determined by enzyme-linked immunosorbent assay (ELISA). After dyed with hematoxylin-eosin (HE), the lung tissue pathology was observed and the lung injury score was performed. Compared with the ALI model group, the arterial partial pressure of oxygen (PaO 2 ) in EPCs, none transfected EPCs and VEGF165 transfected EPCs treatment groups was significantly increased from 4 hours, and lung W/D, expressions of iNOS and ET-1 were significantly decreased, and VEGF165 expression was significantly increased. Compared with the EPCs treatment group, the increase in PaO 2 , the decrease in lung W/D and expressions of iNOS and ET-1, and the increase in VEGF165 expression in VEGF165 transfected EPCs

Stars and Symbiosis: MicroRNA- and MicroRNA*-Mediated Transcript Cleavage Involved in Arbuscular Mycorrhizal Symbiosis1[W][OA

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Devers, Emanuel A.; Branscheid, Anja; May, Patrick; Krajinski, Franziska

2011-01-01

The majority of plants are able to form the arbuscular mycorrhizal (AM) symbiosis in association with AM fungi. During symbiosis development, plant cells undergo a complex reprogramming resulting in profound morphological and physiological changes. MicroRNAs (miRNAs) are important components of the regulatory network of plant cells. To unravel the impact of miRNAs and miRNA-mediated mRNA cleavage on root cell reprogramming during AM symbiosis, we carried out high-throughput (Illumina) sequencing of small RNAs and degradome tags of Medicago truncatula roots. This led to the annotation of 243 novel miRNAs. An increased accumulation of several novel and conserved miRNAs in mycorrhizal roots suggest a role of these miRNAs during AM symbiosis. The degradome analysis led to the identification of 185 root transcripts as mature miRNA and also miRNA*-mediated mRNA cleavage targets. Several of the identified miRNA targets are known to be involved in root symbioses. In summary, the increased accumulation of specific miRNAs and the miRNA-mediated cleavage of symbiosis-relevant genes indicate that miRNAs are an important part of the regulatory network leading to symbiosis development. PMID:21571671

Improvement of Morphine-Mediated Analgesia by Inhibition of β-Arrestin 2 Expression in Mice Periaqueductal Gray Matter

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Li, Yuting; Liu, Xing; Liu, Chang; Kang, Jiuhong; Yang, Jingyu; Pei, Gang; Wu, Chunfu

2009-01-01

Morphine is a well-known μ-opioid receptor (MOR) agonist and an efficient analgesic, but its long-term use inevitably leads to drug addiction and tolerance. Here, we show that specific inhibition of β-arrestin2 with its siRNA lentivirus microinjected in mice periaqueductal gray matter (PAG) significantly improved both acute and chronic morphine analgesia and delayed the tolerance in the hotplate test. The specific effect of β-arrestin2 was proven by overexpression or knockdown of its homology β-arrestin1 in PAG, which showed no significant effects on morphine analgesia. These findings suggest that specific siRNA targeting β-arrestin2 may constitute a new approach to morphine therapy and other MOR agonist-mediated analgesia and tolerance. PMID:19399231

Systemic delivery of microRNA-101 potently inhibits hepatocellular carcinoma in vivo by repressing multiple targets.

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Fang Zheng

2015-02-01

Full Text Available Targeted therapy based on adjustment of microRNA (miRNAs activity takes great promise due to the ability of these small RNAs to modulate cellular behavior. However, the efficacy of miR-101 replacement therapy to hepatocellular carcinoma (HCC remains unclear. In the current study, we first observed that plasma levels of miR-101 were significantly lower in distant metastatic HCC patients than in HCCs without distant metastasis, and down-regulation of plasma miR-101 predicted a worse disease-free survival (DFS, P<0.05. In an animal model of HCC, we demonstrated that systemic delivery of lentivirus-mediated miR-101 abrogated HCC growth in the liver, intrahepatic metastasis and distant metastasis to the lung and to the mediastinum, resulting in a dramatic suppression of HCC development and metastasis in mice without toxicity and extending life expectancy. Furthermore, enforced overexpression of miR-101 in HCC cells not only decreased EZH2, COX2 and STMN1, but also directly down-regulated a novel target ROCK2, inhibited Rho/Rac GTPase activation, and blocked HCC cells epithelial-mesenchymal transition (EMT and angiogenesis, inducing a strong abrogation of HCC tumorigenesis and aggressiveness both in vitro and in vivo. These results provide proof-of-concept support for systemic delivery of lentivirus-mediated miR-101 as a powerful anti-HCC therapeutic modality by repressing multiple molecular targets.

Inhibition of CD147 expression by RNA interference reduces proliferation, invasion and increases chemosensitivity in cancer stem cell-like HT-29 cells.

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Chen, Jie; Pan, Yuqin; He, Bangshun; Ying, Houqun; Wang, Feng; Sun, Huiling; Deng, Qiwen; Liu, Xian; Lin, Kang; Peng, Hongxin; Cho, William C; Wang, Shukui

2015-10-01

The association between CD147 and cancer stem cells (CSCs) provides a new angle for cancer treatments. The aim of this study was to investigate the biological roles of CD147 in colorectal CSCs. The Oct4-green fluorescent protein (GFP) vector was used to isolate CSCs and pYr-mir30-shRNA was used to generate short hairpin RNA (shRNA) specifically for CD147. After RNA interference (RNAi), CD147 was evaluated by reverse transcription‑quantitative PCR and western blot analysis, and its biological functions were assessed by MTT and invasion assays. The results showed that the differentiation of isolated CSC-like HT-29 cells was blocked and these cells were highly positive for CD44 and CD147. RNAi-mediated CD147 silencing reduced the expression of CD147 at both mRNA and protein levels. Moreover, the activities of proliferation and invasion were decreased obviously in CSCs. Knockdown of CD147 increased the chemosensitivity of CSC-like cells to gemcitabine, cisplatin, docetaxel at 0.1, 1 and 10 µM respectively, however, there was no significant difference among the three groups to paclitaxel at 10 µM. In conclusion, these results suggest that CD147 plays an important role in colorectal CSCs and might be regarded as a novel CSC-specific targeted strategy against colorectal cancer.

Hybrid Lentivirus-transposon Vectors With a Random Integration Profile in Human Cells

DEFF Research Database (Denmark)

Staunstrup, Nicklas H; Moldt, Brian; Mátés, Lajos

2009-01-01

Gene delivery by human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) is efficient, but genomic integration of the viral DNA is strongly biased toward transcriptionally active loci resulting in an increased risk of insertional mutagenesis in gene therapy protocols. Nonviral...... Sleeping Beauty (SB) transposon vectors have a significantly safer insertion profile, but efficient delivery into relevant cell/tissue types is a limitation. In an attempt to combine the favorable features of the two vector systems we established a novel hybrid vector technology based on SB transposase......-mediated insertion of lentiviral DNA circles generated during transduction of target cells with integrase (IN)-defective LVs (IDLVs). By construction of a lentivirus-transposon hybrid vector allowing transposition exclusively from circular viral DNA substrates, we demonstrate that SB transposase added in trans...

The Mediator Complex: At the Nexus of RNA Polymerase II Transcription.

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Jeronimo, Célia; Robert, François

2017-10-01

Mediator is an essential, large, multisubunit, transcriptional co-activator highly conserved across eukaryotes. Mediator interacts with gene-specific transcription factors at enhancers as well as with the RNA polymerase II (RNAPII) transcription machinery bound at promoters. It also interacts with several other factors involved in various aspects of transcription, chromatin regulation, and mRNA processing. Hence, Mediator is at the nexus of RNAPII transcription, regulating its many steps and connecting transcription with co-transcriptional events. To achieve this flexible role, Mediator, which is divided into several functional modules, reorganizes its conformation and composition while making transient contacts with other components. Here, we review the mechanisms of action of Mediator and propose a unifying model for its function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sensitivity and specificity of a nested polymerase chain reaction for detection of lentivirus infection in lions (Panthera leo).

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Adams, Hayley; van Vuuren, Moritz; Kania, Stephen; Bosman, Anna-Mari; Keet, Dewald; New, John; Kennedy, Melissa

2010-12-01

Feline immunodeficiency virus (FIV) is a lentivirus in the Retroviridae family that causes lifelong infection in domestic cats. The lentivirus of African lions (Panthera leo), referred to as FIVple, is endemic in certain lion populations in eastern and southern Africa. Lentivirus infection leads to immunologic dysfunction and immunosuppressive disease in domestic cats; however, little is known about the pathogenic effects of infection in lions, nor about the epidemiologic impact on free-ranging and captive populations. Whole blood and serum samples were collected opportunistically from free-ranging lions in Kruger National Park, Republic of South Africa (RSA). Whole blood and serum samples were also collected from captive wild lions in the RSA. A nested polymerase chain reaction (PCR) assay for detection of FIV was performed on all whole blood samples. In addition, serum samples were tested for cross-reactive antibodies to domestic feline lentivirus antigens and puma lentivirus synthetic envelope peptide antigen. The PCR assay successfully amplified the lion lentivirus from African lions. The relative sensitivity and relative specificity were 79% and 100%, respectively, and the positive and negative predictive values were 100% and 67%, respectively. This research represents the first study to compare genetic material with antibody-based methods of lentivirus detection on lions in RSA. Using PCR as an additional diagnostic test for FIV in lions will increase screening sensitivity and will allow viral characterization among circulating isolates and monitoring of changes in the viral epidemiology within geographic regions and populations over time.

Does Work-Home Interference mediate the relationship between workload and well-being?

NARCIS (Netherlands)

Geurts, S.A.E.; Kompier, M.A.J.; Roxburgh, S.; Houtman, I.L.D.

2003-01-01

Drawing on the Effort-Recovery (E-R) model, the current study investigated to what extent Work-Home Interference (WHI) mediated the relationship between workload and two indicators of well-being, that is, (a) affective well-being (i.e., work-related negative affect and depressive mood) and (b)

Immobilized Lentivirus Vector on Chondroitin Sulfate-Hyaluronate Acid-Silk Fibroin Hybrid Scaffold for Tissue-Engineered Ligament-Bone Junction

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Liguo Sun

2014-01-01

Full Text Available The lack of a fibrocartilage layer between graft and bone remains the leading cause of graft failure after anterior cruciate ligament (ACL reconstruction. The objective of this study was to develop a gene-modified silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS hybrid scaffold for reconstructing the fibrocartilage layer. The scaffold was fabricated by lyophilizing the CHS mixture with braided silk cables. The scanning electronic microscopy (SEM showed that microporous CHS sponges were formed around silk cables. Each end of scaffold was modified with lentiviral-mediated transforming growth factor-β3 (TGF-β3 gene. The cells on scaffold were transfected by bonded lentivirus. In vitro culture demonstrated that mesenchymal stem cells (MSCs on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of cartilage-specific genes also increased with culture time. After 2 weeks, the MSCs were distributed uniformly throughout scaffold. Deposited collagen was also found to increase. The chondral differentiation of MSCs was verified by expressions of collagen II and TGF-β3 genes in mRNA and protein level. Histology also confirmed the production of cartilage extracellular matrix (ECM components. The results demonstrated that gene-modified silk cable-reinforced CHS scaffold was capable of supporting cell proliferation and differentiation to reconstruct the cartilage layer of interface.

A discontinuous RNA platform mediates RNA virus replication: building an integrated model for RNA-based regulation of viral processes.

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Baodong Wu

2009-03-01

Full Text Available Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

[Construction of BAD Lentivirus Vector and Its Effect on Proliferation in A549 Cell Lines].

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Huang, Na; He, Yan-qi; Zhu, Jing; Li, Wei-min

2015-05-01

To construct the recombinant lentivirus expressing vector BAD (Bcl-2-associated death protein) gene and to study its effect on A549 cell proliferation. The BAD gene was amplified from plasmid pAV-MCMV-BAD-GFP by PCR. The purified BAD gene fragment was inserted into a lentivirus vector (pLVX-IRES-ZsGreen 1), and the insertion was identified by PCR, restriction endonuclease analysis and DNA sequencing. A549 cells were then transfected with the packaged recombinant lentivirus, and resistant cell clones were selected with flow cytometry. The expression of BAD in A549 cell lines stably transduction with a lentivirus was examined using Western blot. The effect of BAD overexpression on proliferation of A549 cells was evaluated by using CCK-8 kit. Restriction enzyme digestion and DNA sequencing showed that the full-length BAD gene (507 bp) had been successfully subcloned into the lentiviral vector to result in the recombinant vector pLVX-IRES-ZsGreen 1. Monoclonal cell lines BAD-A549 was produced after transfection with the recombinant lentivirus and selected with flow cytometry. Stable expression of BAD protein was verified by Western blot. In vitro, the OD value in BAD group was significantly lower than that of control groups from 120-144 h (PBAD gene had been successfully generated. In vitro, BAD overexpression significantly inhibited A549 cells proliferation.

The interferon-induced antiviral protein PML (TRIM19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion.

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Masroori, Nasser; Merindol, Natacha; Berthoux, Lionel

2016-03-22

The promyelocytic leukemia (PML) protein, a type I interferon (IFN-I)-induced gene product and a member of the tripartite motif (TRIM) family, modulates the transcriptional activity of viruses belonging to various families. Whether PML has an impact on the replication of HIV-1 has not been fully addressed, but recent studies point to its possible involvement in the restriction of HIV-1 in human cells and in the maintenance of transcriptional latency in human cell lines in which HIV-1 is constitutively repressed. We investigated further the restriction of HIV-1 and a related lentivirus, SIVmac, by PML in murine cells and in a lymphocytic human cell line. In particular, we studied the relevance of PML to IFN-I-mediated inhibition and the role of individual human isoforms. We demonstrate that both human PML (hPML) and murine PML (mPML) inhibit the early post-entry stages of the replication of HIV-1 and a related lentivirus, SIVmac. In addition, HIV-1 was transcriptionally silenced by mPML and by hPML isoforms I, II, IV and VI in MEFs. This PML-mediated transcriptional repression was attenuated in presence of the histone deacetylase inhibitor SAHA. In contrast, depletion of PML had no effect on HIV-1 gene expression in a human T cell line. PML was found to contribute to the inhibition of HIV-1 by IFN-I. Specifically, IFN-α and IFN-β treatments of MEFs enhanced the PML-dependent inhibition of HIV-1 early replication stages. We show that PML can inhibit HIV-1 and other lentiviruses as part of the IFN-I-mediated response. The restriction takes place at two distinct steps, i.e. reverse transcription and transcription, and in an isoform-specific, cellular context-specific fashion. Our results support a model in which PML activates innate immune antilentiviral effectors. These data are relevant to the development of latency reversal-inducing pharmacological agents, since PML was previously proposed as a pharmacological target for such inhibitors. This study also has

Lentiviral Nef Proteins Utilize PAK2-Mediated Deregulation of Cofilin as a General Strategy To Interfere with Actin Remodeling▿ †

Science.gov (United States)

Stolp, Bettina; Abraham, Libin; Rudolph, Jochen M.; Fackler, Oliver T.

2010-01-01

Nef is an accessory protein and pathogenicity factor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which elevates virus replication in vivo. We recently described for HIV type 1SF2 (HIV-1SF2) the potent interference of Nef with T-lymphocyte chemotaxis via its association with the cellular kinase PAK2. Mechanistic analysis revealed that this interaction results in deregulation of the actin-severing factor cofilin and thus blocks the chemokine-mediated actin remodeling required for cell motility. However, the efficiency of PAK2 association is highly variable among Nef proteins from different lentiviruses, prompting us to evaluate the conservation of this actin-remodeling/cofilin-deregulating mechanism. Based on the analysis of a total of 17 HIV-1, HIV-2, and SIV Nef proteins, we report here that inhibition of chemokine-induced actin remodeling as well as inactivation of cofilin are strongly conserved activities of lentiviral Nef proteins. Of note, even for Nef variants that display only marginal PAK2 association in vitro, these activities require the integrity of a PAK2 recruitment motif and the presence of endogenous PAK2. Thus, reduced in vitro affinity to PAK2 does not indicate limited functionality of Nef-PAK2 complexes in intact HIV-1 host cells. These results establish hijacking of PAK2 for deregulation of cofilin and inhibition of triggered actin remodeling as a highly conserved function of lentiviral Nef proteins, supporting the notion that PAK2 association may be critical for Nef's activity in vivo. PMID:20147394

Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus

Directory of Open Access Journals (Sweden)

Chu Justin

2010-02-01

Full Text Available Abstract Background Dengue virus (DENV is the causative agent of Dengue fever and the life-threatening Dengue Haemorrhagic fever or Dengue shock syndrome. In the absence of anti-viral agents or vaccine, there is an urgent need to develop an effective anti-viral strategy against this medically important viral pathogen. The initial interplay between DENV and the host cells may represent one of the potential anti-viral targeting sites. Currently the involvements of human membrane trafficking host genes or factors that mediate the infectious cellular entry of dengue virus are not well defined. Results In this study, we have used a targeted small interfering RNA (siRNA library to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics and endosome trafficking that are important and essential for DENV infection. The infectious entry of DENV into Huh7 cells was shown to be potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. Furthermore, DENV infection was shown to be sensitive to the disruption of human genes in regulating the early to late endosomal trafficking as well as the endosomal acidic pH. The importance and involvement of both actin and microtubule dynamics in mediating the infectious entry of DENV was also revealed in this study. Conclusions Together, the findings from this study have provided a detail profiling of the human membrane trafficking cellular genes and the mechanistic insight into the interplay of these host genes with DENV to initiate an infection, hence broadening our understanding on the entry pathway of this medically important viral pathogen. These data may also provide a new potential avenue for development of anti

Construction of lentiviral shRNA expression vector targeting ...

African Journals Online (AJOL)

DNA oligo was cloned into lentiviral expression vector, and then polymerase chain reaction (PCR) and sequencing analyses were conducted to verify the constructs. The verified vectors were co-transfected into 293FT cells that could produce lentiviral. shRNA lentiviruses from the selected constructs were propagated and ...

RNA interference mediated pten knock-down inhibit the formation of polycystic ovary.

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Ouyang, Jie-Xiu; Luo, Tao; Sun, Hui-Yun; Huang, Jian; Tang, Dan-Feng; Wu, Lei; Zheng, Yue-Hui; Zheng, Li-Ping

2013-08-01

Pten (phosphatase and tensin homolog deleted on chromosome 10), a kind of tumor suppressor gene, plays important roles in female reproductive system. But its expression and roles in the formation of polycystic ovaries are yet to be known. In this study, we constructed a rat model of PCOS using norethindrone and HCG injections and found the expressions of pten mRNA and PTEN protein increased significantly in the polycystic ovary tissue by immunohistochemistry, RT-PCR, and western blot. Furthermore, the results showed that in vivo ovaries could be effectively transfected by lentiviral vectors through the ovarian microinjection method and indicated that pten shRNA may inhibit the formation of polycystic ovaries by pten down-regulation. Our study provides new information regarding the role of PTEN in female reproductive disorders, such as polycystic ovary syndrome.

RNA interference: concept to reality in crop improvement.

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Saurabh, Satyajit; Vidyarthi, Ambarish S; Prasad, Dinesh

2014-03-01

The phenomenon of RNA interference (RNAi) is involved in sequence-specific gene regulation driven by the introduction of dsRNA resulting in inhibition of translation or transcriptional repression. Since the discovery of RNAi and its regulatory potentials, it has become evident that RNAi has immense potential in opening a new vista for crop improvement. RNAi technology is precise, efficient, stable and better than antisense technology. It has been employed successfully to alter the gene expression in plants for better quality traits. The impact of RNAi to improve the crop plants has proved to be a novel approach in combating the biotic and abiotic stresses and the nutritional improvement in terms of bio-fortification and bio-elimination. It has been employed successfully to bring about modifications of several desired traits in different plants. These modifications include nutritional improvements, reduced content of food allergens and toxic compounds, enhanced defence against biotic and abiotic stresses, alteration in morphology, crafting male sterility, enhanced secondary metabolite synthesis and seedless plant varieties. However, crop plants developed by RNAi strategy may create biosafety risks. So, there is a need for risk assessment of GM crops in order to make RNAi a better tool to develop crops with biosafety measures. This article is an attempt to review the RNAi, its biochemistry, and the achievements attributed to the application of RNAi in crop improvement.

Selection Demands and Working Memory Mediate Interference during Naming

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Julie W Hughes

2015-05-01

Analyzing RTs, we replicated previous SI effects in BCN in healthy subjects and patients (reported effects’ p’s < .05. Subjects responded more slowly to items in semantically related vs. unrelated groups, and this interference increased across cycles (evidence of increasing selection demands; Figures 1a/c. Indicative of WM, healthy subjects were faster to name items as the number of unrelated items to be named (i.e., remembered decreased. However, this was attenuated by selection demand, as there was no decrease in RTs across repeated related items (Figure 1b. Converging evidence from patients demonstrated more impaired selection capacity produced increasingly slower RTs across item positions within the related condition (high selection demand (Figure 1d. Larger WM capacity (rhyme probe but not category probe, p = .54 produced increasingly faster RTs across items positions in the unrelated condition (low selection demand (Figure 1e. Together, these findings suggest that both selection demand and phonological WM processes mediate interference during naming.

Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR-Cas interference in Sulfolobus

DEFF Research Database (Denmark)

Mousaei, Marzieh; Deng, Ling; She, Qunxin

2016-01-01

The stringency of crRNA-protospacer DNA base pair matching required for effective CRISPR-Cas interference is relatively low in crenarchaeal Sulfolobus species in contrast to that required in some bacteria. To understand its biological significance we studied crRNA-protospacer interactions...... in Sulfolobus islandicus REY15A which carries multiple, and functionally diverse, interference complexes. A range of mismatches were introduced into a vector-borne protospacer that was identical to spacer 1 of CRISPR locus 2, with a cognate CCN PAM sequence. Two important crRNA annealing regions were identified...

RNA Interference - Towards RNA becoming a Medicine -42 ...

Indian Academy of Sciences (India)

research. A brief history of the development ofRNAi is shown in. Box 2. Mechanism of ... new RNA strand using target RNA as the template and thereby converting it ... thought to excise precursor stRNA from their -70 nt stem loop precursor to ...

Enhancement of antiproliferative activity of interferons by RNA interference-mediated silencing of SOCS gene expression in tumor cells.

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Takahashi, Yuki; Kaneda, Haruka; Takasuka, Nana; Hattori, Kayoko; Nishikawa, Makiya; Watanabe, Yoshihiko; Takakura, Yoshinobu

2008-08-01

The suppressor of cytokine signaling (SOCS) proteins, negative regulators of interferon (IFN)-induced signaling pathways, is involved in IFN resistance of tumor cells. To improve the growth inhibitory effect of IFN-beta and IFN-gamma on a murine melanoma cell line, B16-BL6, and a murine colon carcinoma cell line, Colon26 cells, SOCS-1 and SOCS-3 gene expression in tumor cells was downregulated by transfection of plasmid DNA expressing short hairpin RNA targeting one of these genes (pshSOCS-1 and pshSOCS-3, respectively). Transfection of pshSOCS-1 significantly increased the antiproliferative effect of IFN-gamma on B16-BL6 cells. However, any other combinations of plasmids and IFN had little effect on the growth of B16-BL6 cells. In addition, transfection of pshSOCS-1 and pshSOCS-3 produced little improvement in the effect of IFN on Colon26 cells. To understand the mechanism underlining these findings, the level of SOCS gene expression was measured by real time polymerase chain reaction. Addition of IFN-gamma greatly increased the SOCS-1 mRNA expression in B16-BL6 cells. Taking into account the synergistic effect of pshSOCS-1 and IFN-gamma on the growth of B16-BL6 cells, these findings suggest that IFN-gamma-induced high SOCS-1 gene expression in B16-BL6 cells significantly interferes with the antiproliferative effect of IFN-gamma. These results indicate that silencing SOCS gene expression can be an effective strategy to enhance the antitumor effect of IFN under conditions in which the SOCS gene expression is upregulated by IFN.

miRNA-mediated functional changes through co-regulating function related genes.

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Jie He

Full Text Available BACKGROUND: MicroRNAs play important roles in various biological processes involving fairly complex mechanism. Analysis of genome-wide miRNA microarray demonstrate that a single miRNA can regulate hundreds of genes, but the regulative extent on most individual genes is surprisingly mild so that it is difficult to understand how a miRNA provokes detectable functional changes with such mild regulation. RESULTS: To explore the internal mechanism of miRNA-mediated regulation, we re-analyzed the data collected from genome-wide miRNA microarray with bioinformatics assay, and found that the transfection of miR-181b and miR-34a in Hela and HCT-116 tumor cells regulated large numbers of genes, among which, the genes related to cell growth and cell death demonstrated high Enrichment scores, suggesting that these miRNAs may be important in cell growth and cell death. MiR-181b induced changes in protein expression of most genes that were seemingly related to enhancing cell growth and decreasing cell death, while miR-34a mediated contrary changes of gene expression. Cell growth assays further confirmed this finding. In further study on miR-20b-mediated osteogenesis in hMSCs, miR-20b was found to enhance osteogenesis by activating BMPs/Runx2 signaling pathway in several stages by co-repressing of PPARγ, Bambi and Crim1. CONCLUSIONS: With its multi-target characteristics, miR-181b, miR-34a and miR-20b provoked detectable functional changes by co-regulating functionally-related gene groups or several genes in the same signaling pathway, and thus mild regulation from individual miRNA targeting genes could have contributed to an additive effect. This might also be one of the modes of miRNA-mediated gene regulation.

Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

International Nuclear Information System (INIS)

Zuo, Keqiang; Li, Dan; Pulli, Benjamin; Yu, Fei; Cai, Haidong; Yuan, Xueyu; Zhang, Xiaoping; Lv, Zhongwei

2012-01-01

Highlights: ► Hsp90 is over-expressed in human breast cancer. ► The shRNA-mediated gene silencing of Hsp90 resulted in inhibition of cell growth. ► Akt and NF-kB were down-regulation after transfection due to Hsp90 silencing. ► The tumor growth ratio was decline due to Hsp90 silencing. ► The PCNA expression was down-regulation due to Hsp90 silencing. -- Abstract: Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.

RNA interference silences Microplitis demolitor bracovirus genes and implicates glc1.8 in disruption of adhesion in infected host cells

International Nuclear Information System (INIS)

Beck, Markus; Strand, Michael R.

2003-01-01

The family Polydnaviridae consists of ds-DNA viruses that are symbiotically associated with certain parasitoid wasps. PDVs are transmitted vertically but also are injected by wasps into hosts where they cause several physiological alterations including immunosuppression. The PDV genes responsible for mediating immunosuppression and other host alterations remain poorly characterized in large measure because viral mutants cannot be produced to study gene function. Here we report the use of RNA interference (RNAi) to specifically silence the glc1.8 and egf1.0 genes from Microplitis demolitor bracovirus (MdBV) in High Five cells derived from the lepidopteran Trichoplusia ni. Dose-response studies indicated that MdBV infects High Five cells and blocks the ability of these cells to adhere to culture plates. This response was very similar to what occurs in two classes of hemocytes, granular cells, and plasmatocytes, after infection by MdBV. Screening of monoclonal antibody (mAb) markers that distinguish different classes of lepidopteran hemocytes indicated that High Five cells cross-react with three mAbs that recognize granular cells from T. ni. Double-stranded RNA (dsRNA) complementary to glc1.8 specifically silenced glc1.8 expression and rescued the adhesive phenotype of High Five cells. Reciprocally, dsRNA complementary to egf1.0 silenced egf1.0 expression but had no effect on adhesion. The simplicity and potency of RNAi could be extremely useful for analysis of other PDV genes

Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

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Schnettler, Esther; Hemmes, Hans; Huismann, Rik; Goldbach, Rob; Prins, Marcel; Kormelink, Richard

2010-11-01

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

Short hairpin RNA interference therapy for ischemic heart disease

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Huang, Mei; Chan, Denise; Jia, Fangjun; Xie, Xiaoyan; Li, Zongjin; Hoyt, Grant; Robbins, Robert C.; Chen, Xiaoyuan; Giaccia, Amato; Wu, Joseph C.

2013-01-01

Background During hypoxia, upregulation of hypoxia inducible factor-1 alpha (HIF-1α) transcriptional factor can activate several downstream angiogenic genes. However, HIF-1α is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Here we hypothesize that short hairpin RNA (shRNA) interference therapy targeting PHD2 can be used for treatment of myocardial ischemia and this process can be followed noninvasively by molecular imaging. Methods and Results PHD2 was cloned from mouse embryonic stem (ES) cells by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted into the pSuper vector driven by the H1 promoter, followed by a separate hypoxia response element (HRE)-incorporated promoter driving a firefly luciferase (Fluc) reporter gene. This construct was used to transfect mouse C2C12 myoblast cell line for in vitro confirmation. Compared to the control short hairpin scramble (shScramble) as control, inhibition of PHD2 increased levels of HIF-1α protein and several downstream angiogenic genes by >30% (P<0.01). Afterwards, shRNA targeting PHD2 (shPHD2) plasmid was injected intramyocardially following ligation of left anterior descending (LAD) artery in mice. Animals were randomized into shPHD2 group (n=20) versus shScramble sequence as control (n=20). Bioluminescence imaging detected transgene expression for 4–5 weeks. Echocardiographic study showed the shPHD2 group had improved fractional shortening compared with the shScramble group at week 4 (33.7%±1.9% vs. 28.4%±2.8%; P<0.05). Postmortem analysis showed increased presence of small capillaries and venules in the infarcted zones by CD31 staining. Finally, Western blot anlaysis of explanted hearts also confirm that animals treated with shPHD2 had significantly higher levels of HIF-1α protein. Conclusions This is the first study to image the biological role of shRNA therapy for improving cardiac function. Inhibition of PHD2 by shRNA led to

Vector-based RNA interference against vascular endothelial growth factor-A significantly limits vascularization and growth of prostate cancer in vivo.

Science.gov (United States)

Wannenes, Francesca; Ciafré, Silvia Anna; Niola, Francesco; Frajese, Gaetano; Farace, Maria Giulia

2005-12-01

RNA interference technology is emerging as a very potent tool to obtain a cellular knockdown of a desired gene. In this work we used vector-based RNA interference to inhibit vascular endothelial growth factor (VEGF) expression in prostate cancer in vitro and in vivo. We demonstrated that transduction with a plasmid carrying a small interfering RNA targeting all isoforms of VEGF, dramatically impairs the expression of this growth factor in the human prostate cancer cell line PC3. As a consequence, PC3 cells loose their ability to induce one of the fundamental steps of angiogenesis, namely the formation of a tube-like network in vitro. Most importantly, our "therapeutic" vector is able to impair tumor growth rate and vascularization in vivo. We show that a single injection of naked plasmid in developing neoplastic mass significantly decreases microvessel density in an androgen-refractory prostate xenograft and is able to sustain a long-term slowing down of tumor growth. In conclusion, our results confirm the basic role of VEGF in the angiogenic development of prostate carcinoma, and suggest that the use of our vector-based RNA interference approach to inhibit angiogenesis could be an effective tool in view of future gene therapy applications for prostate cancer.

Defective RNA particles derived from Tomato black ring virus genome interfere with the replication of parental virus.

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Hasiów-Jaroszewska, Beata; Minicka, Julia; Zarzyńska-Nowak, Aleksandra; Budzyńska, Daria; Elena, Santiago F

2018-05-02

Tomato black ring virus (TBRV) is the only member of the Nepovirus genus that is known to form defective RNA particles (D RNAs) during replication. Here, de novo generation of D RNAs was observed during prolonged passages of TBRV isolates originated from Solanum lycopersicum and Lactuca sativa in Chenopodium quinoa plants. D RNAs of about 500 nt derived by a single deletion in the RNA1 molecule and contained a portion of the 5' untranslated region and viral replicase, and almost the entire 3' non-coding region. Short regions of sequence complementarity were found at the 5' and 3' junction borders, which can facilitate formation of the D RNAs. Moreover, in this study we analyzed the effects of D RNAs on TBRV replication and symptoms development of infected plants. C. quinoa, S. lycopersicum, Nicotiana tabacum, and L. sativa were infected with the original TBRV isolates (TBRV-D RNA) and those containing additional D RNA particles (TBRV + D RNA). The viral accumulation in particular hosts was measured up to 28 days post inoculation by RT-qPCR. Statistical analyses revealed that D RNAs interfere with TBRV replication and thus should be referred to as defective interfering particles. The magnitude of the interference effect depends on the interplay between TBRV isolate and host species. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of nonviable genes affecting touch sensitivity in Caenorhabditis elegans using neuronally enhanced feeding RNA interference.

Science.gov (United States)

Chen, Xiaoyin; Cuadros, Margarete Diaz; Chalfie, Martin

2015-01-09

Caenorhabditis elegans senses gentle touch along the body via six touch receptor neurons. Although genetic screens and microarray analyses have identified several genes needed for touch sensitivity, these methods miss pleiotropic genes that are essential for the viability, movement, or fertility of the animals. We used neuronally enhanced feeding RNA interference to screen genes that cause lethality or paralysis when mutated, and we identified 61 such genes affecting touch sensitivity, including five positive controls. We confirmed 18 genes by using available alleles, and further studied one of them, tag-170, now renamed txdc-9. txdc-9 preferentially affects anterior touch response but is needed for tubulin acetylation and microtubule formation in both the anterior and posterior touch receptor neurons. Our results indicate that neuronally enhanced feeding RNA interference screens complement traditional mutageneses by identifying additional nonviable genes needed for specific neuronal functions. Copyright © 2015 Chen et al.

Integrative analyses reveal a long noncoding RNA-mediated sponge regulatory network in prostate cancer.

Science.gov (United States)

Du, Zhou; Sun, Tong; Hacisuleyman, Ezgi; Fei, Teng; Wang, Xiaodong; Brown, Myles; Rinn, John L; Lee, Mary Gwo-Shu; Chen, Yiwen; Kantoff, Philip W; Liu, X Shirley

2016-03-15

Mounting evidence suggests that long noncoding RNAs (lncRNAs) can function as microRNA sponges and compete for microRNA binding to protein-coding transcripts. However, the prevalence, functional significance and targets of lncRNA-mediated sponge regulation of cancer are mostly unknown. Here we identify a lncRNA-mediated sponge regulatory network that affects the expression of many protein-coding prostate cancer driver genes, by integrating analysis of sequence features and gene expression profiles of both lncRNAs and protein-coding genes in tumours. We confirm the tumour-suppressive function of two lncRNAs (TUG1 and CTB-89H12.4) and their regulation of PTEN expression in prostate cancer. Surprisingly, one of the two lncRNAs, TUG1, was previously known for its function in polycomb repressive complex 2 (PRC2)-mediated transcriptional regulation, suggesting its sub-cellular localization-dependent function. Our findings not only suggest an important role of lncRNA-mediated sponge regulation in cancer, but also underscore the critical influence of cytoplasmic localization on the efficacy of a sponge lncRNA.

Early secreted antigen ESAT-6 of Mycobacterium Tuberculosis promotes apoptosis of macrophages via targeting the microRNA155-SOCS1 interaction.

Science.gov (United States)

Yang, Shaojun; Li, Fake; Jia, Shuangrong; Zhang, Kejun; Jiang, Wenbing; Shang, Ya; Chang, Kai; Deng, Shaoli; Chen, Ming

2015-01-01

The early secreted antigenic target 6-kDa protein (ESAT-6) of Mycobacterium tuberculosis (Mtb) not only acts as a key player for virulence but also exhibits a strong immunotherapeutic potential against Mtb. However, little is known about the molecular basis for its potential in immunotherapy. The present study was designed to unravel the role of miRNA-155 in ESAT-6-mediated enhancement of host immunity and apoptosis in macrophages. Lentivirus-mediated miR-155 sponge and miR-155 and SOCS1 overexpression vectors were developed in macrophages. TLR2- or p65-specific siRNA knockdown was employed to silence TLR2 or p65. Quantitative polymerase chain reaction and western blotting analyses were performed to determine mRNA and protein expression levels, respectively. Macrophage apoptosis was analyzed by flow cytometry. ESAT-6 significantly increased miR-155 expression, which was dependent on TLR2/NF-κB activation in macrophages. Induced expression of miRNA-155 was required for the ESAT-6-mediated protective immune response and macrophage apoptosis. ESAT-6 promoted macrophage apoptosis by targeting the miR-155-SOCS1 pathway. The differential expression levels of TLR2, BIC, and SOCS1 were involved in regulating the immune response in human peripheral blood mononuclear cells of patients with active tuberculosis (TB) and latent TB (LTB). ESAT-6 promotes apoptosis of macrophages via targeting the miRNA155-SOCS1 interaction. © 2015 S. Karger AG, Basel.

Early Secreted Antigen ESAT-6 of Mycobacterium Tuberculosis Promotes Apoptosis of Macrophages via Targeting the MicroRNA155-SOCS1 Interaction

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Shaojun Yang

2015-02-01

Full Text Available Background: The early secreted antigenic target 6-kDa protein (ESAT-6 of Mycobacterium tuberculosis (Mtb not only acts as a key player for virulence but also exhibits a strong immunotherapeutic potential against Mtb. However, little is known about the molecular basis for its potential in immunotherapy. The present study was designed to unravel the role of miRNA-155 in ESAT-6-mediated enhancement of host immunity and apoptosis in macrophages. Methods: Lentivirus-mediated miR-155 sponge and miR-155 and SOCS1 overexpression vectors were developed in macrophages. TLR2- or p65-specific siRNA knockdown was employed to silence TLR2 or p65. Quantitative polymerase chain reaction and western blotting analyses were performed to determine mRNA and protein expression levels, respectively. Macrophage apoptosis was analyzed by flow cytometry. Results: ESAT-6 significantly increased miR-155 expression, which was dependent on TLR2/NF-κB activation in macrophages. Induced expression of miRNA-155 was required for the ESAT-6-mediated protective immune response and macrophage apoptosis. ESAT-6 promoted macrophage apoptosis by targeting the miR-155-SOCS1 pathway. The differential expression levels of TLR2, BIC, and SOCS1 were involved in regulating the immune response in human peripheral blood mononuclear cells of patients with active tuberculosis (TB and latent TB (LTB. Conclusion: ESAT-6 promotes apoptosis of macrophages via targeting the miRNA155-SOCS1 interaction.

C to U RNA editing mediated by APOBEC1 requires RNA-binding protein RBM47.

Science.gov (United States)

Fossat, Nicolas; Tourle, Karin; Radziewic, Tania; Barratt, Kristen; Liebhold, Doreen; Studdert, Joshua B; Power, Melinda; Jones, Vanessa; Loebel, David A F; Tam, Patrick P L

2014-08-01

Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is accomplished by the deaminase APOBEC1 and its partnership with the RNA-binding protein A1CF. We identify and characterise here a novel RNA-binding protein, RBM47, that interacts with APOBEC1 and A1CF and is expressed in tissues where C to U RNA editing occurs. RBM47 can substitute for A1CF and is necessary and sufficient for APOBEC1-mediated editing in vitro. Editing is further impaired in Rbm47-deficient mutant mice. These findings suggest that RBM47 and APOBEC1 constitute the basic machinery for C to U RNA editing. © 2014 The Authors.

A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi.

Science.gov (United States)

Tsai, Hsin-Yue; Chen, Chun-Chieh G; Conte, Darryl; Moresco, James J; Chaves, Daniel A; Mitani, Shohei; Yates, John R; Tsai, Ming-Daw; Mello, Craig C

2015-01-29

Effective silencing by RNA-interference (RNAi) depends on mechanisms that amplify and propagate the silencing signal. In some organisms, small-interfering RNAs (siRNAs) are amplified from target mRNAs by RNA-dependent RNA polymerase (RdRP). Both RdRP recruitment and mRNA silencing require Argonaute proteins, which are generally thought to degrade RNAi targets by directly cleaving them. However, in C. elegans, the enzymatic activity of the primary Argonaute, RDE-1, is not required for silencing activity. We show that RDE-1 can instead recruit an endoribonuclease, RDE-8, to target RNA. RDE-8 can cleave RNA in vitro and is needed for the production of 3' uridylated fragments of target mRNA in vivo. We also find that RDE-8 promotes RdRP activity, thereby ensuring amplification of siRNAs. Together, our findings suggest a model in which RDE-8 cleaves target mRNAs to mediate silencing, while generating 3' uridylated mRNA fragments to serve as templates for the RdRP-directed amplification of the silencing signal. Copyright © 2015 Elsevier Inc. All rights reserved.

Oligopeptide-based enzyme immunoassay for ovine lentivirus antibody detection.

OpenAIRE

Kwang, J; Torres, J V

1994-01-01

Ovine progressive pneumonia virus (OPPV) is a lentivirus which causes a progressive disease in sheep. Immunodominant epitopes have been identified in the envelope gp40 glycoprotein. Synthetic peptides representing these regions are able to detect the presence of OPPV antibodies in 96% of infected sheep.

Functional Crosstalk between Human Papillomaviruses and Lentiviruses

OpenAIRE

Pryszlak, Anna Marta

2016-01-01

Human papillomaviruses (HPVs) and human immunodeficiency virus‐1 (HIV‐1) are human pathogens of high biomedical significance worldwide. Interestingly, increasing epidemiological evidence indicates that individuals with active HPV infections possess an enhanced risk of being infected by HIV‐1. These findings raise the possibility that HPVs may directly or indirectly increase the pathogenicity of lentiviruses, such as HIV‐1. Using a Vesicular Stomatitis Virus‐G‐(VSV‐G)‐pseudotype...

Adenovirus Vector-Derived VA-RNA-Mediated Innate Immune Responses

Directory of Open Access Journals (Sweden)

Hiroyuki Mizuguchi

2011-07-01

Full Text Available The major limitation of the clinical use of replication-incompetent adenovirus (Ad vectors is the interference by innate immune responses, including induction of inflammatory cytokines and interferons (IFN, following in vivo application of Ad vectors. Ad vector-induced production of inflammatory cytokines and IFNs also results in severe organ damage and efficient induction of acquired immune responses against Ad proteins and transgene products. Ad vector-induced innate immune responses are triggered by the recognition of Ad components by pattern recognition receptors (PRRs. In order to reduce the side effects by Ad vector-induced innate immune responses and to develop safer Ad vectors, it is crucial to clarify which PRRs and which Ad components are involved in Ad vector-induced innate immune responses. Our group previously demonstrated that myeloid differentiating factor 88 (MyD88 and toll-like receptor 9 (TLR9 play crucial roles in the Ad vector-induced inflammatory cytokine production in mouse bone marrow-derived dendritic cells. Furthermore, our group recently found that virus associated-RNAs (VA-RNAs, which are about 160 nucleotide-long non-coding small RNAs encoded in the Ad genome, are involved in IFN production through the IFN-β promoter stimulator-1 (IPS-1-mediated signaling pathway following Ad vector transduction. The aim of this review is to highlight the Ad vector-induced innate immune responses following transduction, especially VA-RNA-mediated innate immune responses. Our findings on the mechanism of Ad vector-induced innate immune responses should make an important contribution to the development of safer Ad vectors, such as an Ad vector lacking expression of VA-RNAs.

Interference of transcription across H-NS binding sites and repression by H-NS.

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Rangarajan, Aathmaja Anandhi; Schnetz, Karin

2018-05-01

Nucleoid-associated protein H-NS represses transcription by forming extended DNA-H-NS complexes. Repression by H-NS operates mostly at the level of transcription initiation. Less is known about how DNA-H-NS complexes interfere with transcription elongation. In vitro H-NS has been shown to enhance RNA polymerase pausing and to promote Rho-dependent termination, while in vivo inhibition of Rho resulted in a decrease of the genome occupancy by H-NS. Here we show that transcription directed across H-NS binding regions relieves H-NS (and H-NS/StpA) mediated repression of promoters in these regions. Further, we observed a correlation of transcription across the H-NS-bound region and de-repression. The data suggest that the transcribing RNA polymerase is able to remodel the H-NS complex and/or dislodge H-NS from the DNA and thus relieve repression. Such an interference of transcription and H-NS mediated repression may imply that poorly transcribed AT-rich loci are prone to be repressed by H-NS, while efficiently transcribed loci escape repression. © 2018 John Wiley & Sons Ltd.

Modelling toehold-mediated RNA strand displacement.

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Šulc, Petr; Ouldridge, Thomas E; Romano, Flavio; Doye, Jonathan P K; Louis, Ard A

2015-03-10

We study the thermodynamics and kinetics of an RNA toehold-mediated strand displacement reaction with a recently developed coarse-grained model of RNA. Strand displacement, during which a single strand displaces a different strand previously bound to a complementary substrate strand, is an essential mechanism in active nucleic acid nanotechnology and has also been hypothesized to occur in vivo. We study the rate of displacement reactions as a function of the length of the toehold and temperature and make two experimentally testable predictions: that the displacement is faster if the toehold is placed at the 5' end of the substrate; and that the displacement slows down with increasing temperature for longer toeholds. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Long-term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy.

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Tschernutter, M; Schlichtenbrede, F C; Howe, S; Balaggan, K S; Munro, P M; Bainbridge, J W B; Thrasher, A J; Smith, A J; Ali, R R

2005-04-01

The Royal College of Surgeons (RCS) rat is a well-characterized model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding , a receptor tyrosine kinase found in RPE cells, that is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. In order to evaluate the efficacy of lentiviral-mediated gene replacement therapy in the RCS rat, we produced recombinant VSV-G pseudotyped HIV-1-based lentiviruses containing a murine Mertk cDNA driven by a spleen focus forming virus (SFFV) promoter. The vector was subretinally injected into the right eye of 10-day-old RCS rats; the left eye was left untreated as an internal control. Here, we present a detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits. We examined animals at various time points over a period of 7 months by light and electron microscopy, and electroretinography. We observed correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function for up to 7 months. This study demonstrates the potential of gene therapy approaches for the treatment of retinal degenerations caused by defects specific to the RPE and supports the use of lentiviral vectors for the treatment of such disorders.

The epidemiology of lion lentivirus infection among a population of free-ranging lions (Panthera leo) in the Kruger National Park, South Africa.

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Adams, H; van Vuuren, M; Bosman, A-M; Keet, D; New, J; Kennedy, M

2009-09-01

Feline immunodeficiency virus is a lentivirus of domestic cats that causes significant lifelong infection. Infection with this or similar lentiviruses has been detected in several nondomestic feline species, including African lions (Panthera leo). Although lion lentivirus (FIVple) infection is endemic in certain lion populations in eastern and southern Africa, little is known about its pathogenic effects or its epidemiological impact in free-ranging lions. This report describes the epidemiological investigation of lentivirus positivity of free-ranging lions in the Kruger National Park, South Africa. A nested polymerase chain reaction assay for virus detection was performed on all whole blood samples collected. In addition, serum samples were tested for cross-reactive antibodies to domestic feline lentivirus antigens and to puma lentivirus synthetic envelope peptide antigen. The results were analysed in conjunction with epidemiological data to provide a descriptive epidemiological study on lion lentivirus infection in a free-ranging population of lions. The overall prevalence of lentivirus infection was 69%, with a prevalence of 41% in the north of the park, and 80% in the south. Adult males had the highest prevalence when combining the factors of sex and age: 94%. The lowest prevalences were found among juveniles, with male juveniles at 29%. Adults were 5.58 times more likely to test positive for FIVple than juveniles, with adult males being 35 times more likely to be test positive for FIVple compared with juvenile males. This research represents the 1st epidemiological study of the lion lentivirus among free-ranging lions in the Kruger National Park.

The epidemiology of lion lentivirus infection among a population of free-ranging lions (Panthera leo in the Kruger National Park, South Africa

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H. Adams

2009-05-01

Full Text Available Feline immunodeficiency virus is a lentivirus of domestic cats that causes significant lifelong infection. Infection with this or similar lentiviruses has been detected in several non-domestic feline species, including African lions (Panthera leo. Although lion lentivirus (FIVple infection is endemic in certain lion populations in eastern and southern Africa, little is known about its pathogenic effects or its epidemiological impact in free-ranging lions. This report describes the epidemiological investigation of lentivirus positivity of free-ranging lions in the Kruger National Park, South Africa. A nested polymerase chain reaction assay for virus detection was performed on all whole blood samples collected. In addition, serum samples were tested for cross-reactive antibodies to domestic feline lentivirus antigens and to puma lentivirus synthetic envelope peptide antigen. The results were analysed in conjunction with epidemiological data to provide a descriptive epidemiological study on lion lentivirus infection in a free-ranging population of lions. The overall prevalence of lentivirus infection was 69 %, with a prevalence of 41 % in the north of the park, and 80 %in the south. Adult males had the highest prevalence when combining the factors of sex and age: 94 %. The lowest prevalences were found among juveniles, with male juveniles at 29 %. Adults were 5.58 times more likely to test positive for FIVple than juveniles, with adult males being 35 times more likely to be test positive for FIVple compared with juvenile males. This research represents the 1st epidemiological study of the lion lentivirus among free-ranging lions in the Kruger National Park.

Delivery of dsRNA through topical feeding for RNA interference in the citrus sap piercing-sucking hemipteran, Diaphorina citri.

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Killiny, Nabil; Kishk, Abdelaziz

2017-06-01

RNA interference (RNAi) is a powerful means to study functional genomics in insects. The delivery of dsRNA is a challenging step in the development of RNAi assay. Here, we describe a new delivery method to increase the effectiveness of RNAi in the Asian citrus psyllid Diaphorina citri. Bromophenol blue droplets were topically applied to fifth instar nymphs and adults on the ventral side of the thorax between the three pairs of legs. In addition to video recordings that showed sucking of the bromophenol blue by the stylets, dissected guts turned blue indicating that the uptake was through feeding. Thus, we called the method topical feeding. We targeted the abnormal wing disc gene (awd), also called nucleoside diphosphate kinase (NDPK), as a reporter gene to prove the uptake of dsRNA via this method of delivery. Our results showed that dsRNA-awd caused reduction of awd expression and nymph mortality. Survival and lifespan of adults emerged from treated nymphs and treated adults were affected. Silencing awd caused wing malformation in the adults emerged from treated nymphs. Topical feeding as a delivery of dsRNA is highly efficient for both nymphs and adults. The described method could be used to increase the efficiency of RNAi in D. citri and other sap piercing-sucking hemipterans. © 2017 Wiley Periodicals, Inc.

Over-expression of brain-derived neurotrophic factor in mesenchymal stem cells transfected with recombinant lentivirus BDNF gene.

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Zhang, X; Zhu, J; Zhang, K; Liu, T; Zhang, Z

2016-12-30

This study was aimed at investigating the expression of brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells (MSCs) modified with recombinant lentivirus bearing BDNF gene. Lentivirus vectors bearing BDNF gene were constructed. MSCs were isolated from rats and cultured. The lentiviral vectors containing BDNF gene were transfected into the MSCs, and BDNF gene and protein expressions were monitored with enhanced green fluorescent protein (EGFP). RT-PCR and Western blot were used to measure gene and protein expressions, respectibvely in MSCs, MSCs-EGFP and MSCs-EGFP-BDNF groups. Green fluorescence assay confirmed successful transfection of BDNF gene recombinant lentivirus into MSCs. RT-PCR and Western blot revealed that BDNF gene and protein expressions in the MSCs-EGFP-BDNF group were significantly higher than that in MSCs group and MSCs-EGFP group. There were no statistically significant differences in gene expression between MSCs and MSCs-EGFP groups. MSCs can over-express BDNF when transfected with recombinant lentivirus bearing BDNF gene.

Establishment and Evaluation of Stable Cell Lines Inhibiting Foot-and-Mouth Disease Virus by RNA Interference

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Yuan-xing Gu

2014-01-01

Full Text Available RNA interference (RNAi has been proved to be a powerful tool for foot-and-mouth disease virus FMDV inhibition in vitro and in vivo. We established five stable baby hamster kidney 21 cell lines (BHK-21 containing five short hairpin RNAs (shRNAs expression plasmids (p3D1shRNA, p3D2shRNA, p3D3shRNA, p3D4shRNA, and p3D5shRNA targeting 3D gene of FMDV. Immunofluorescent assay, virus titration, and real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR were conducted to detect the effect of shRNAs on FMDV replication. After challenged with FMDV of O/CHA/99, two cell lines (p3D1shRNA and p3D4shRNA showed a significant reduction in the synthesis of viral protein and RNA, accompanied by a sharp decrease in viral yield, and the inhibition could last for at least thirty passages. We developed an efficient procedure for the establishment and evaluation of stable cell lines for anti-FMDV research based on RNAi technology, which can be a candidate method for anti-FMDV research.

RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

International Nuclear Information System (INIS)

Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

2006-01-01

RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling.

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Asha, Srinivasan; Sreekumar, Sweda; Soniya, E V

2016-01-01

Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.). Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

RNA Interference: A Novel Source of Resistance to Combat Plant Parasitic Nematodes

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Sagar Banerjee

2017-05-01

Full Text Available Plant parasitic nematodes cause severe damage and yield loss in major crops all over the world. Available control strategies include use of insecticides/nematicides but these have proved detrimental to the environment, while other strategies like crop rotation and resistant cultivars have serious limitations. This scenario provides an opportunity for the utilization of technological advances like RNA interference (RNAi to engineer resistance against these devastating parasites. First demonstrated in the model free living nematode, Caenorhabtidis elegans; the phenomenon of RNAi has been successfully used to suppress essential genes of plant parasitic nematodes involved in parasitism, nematode development and mRNA metabolism. Synthetic neurotransmitants mixed with dsRNA solutions are used for in vitro RNAi in plant parasitic nematodes with significant success. However, host delivered in planta RNAi has proved to be a pioneering phenomenon to deliver dsRNAs to feeding nematodes and silence the target genes to achieve resistance. Highly enriched genomic databases are exploited to limit off target effects and ensure sequence specific silencing. Technological advances like gene stacking and use of nematode inducible and tissue specific promoters can further enhance the utility of RNAi based transgenics against plant parasitic nematodes.

A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

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Pompey, Justine M; Foda, Bardees; Singh, Upinder

2015-01-01

Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

A Single RNaseIII Domain Protein from Entamoeba histolytica Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System.

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Justine M Pompey

Full Text Available Dicer enzymes process double-stranded RNA (dsRNA into small RNAs that target gene silencing through the RNA interference (RNAi pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, Saccharomyces castellii Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite Entamoeba histolytica 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage in vitro, it can process dsRNA in the RNAi-negative background of Saccharomyces cerevisiae, and in conjunction with S. castellii Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in Entamoeba as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.

Optimizing sgRNA position markedly improves the efficiency of CRISPR/dCas9-mediated transcriptional repression

DEFF Research Database (Denmark)

Radzisheuskaya, Aliaksandra; Shlyueva, Daria; Müller, Iris

2016-01-01

CRISPR interference (CRISPRi) represents a newly developed tool for targeted gene repression. It has great application potential for studying gene function and mapping gene regulatory elements. However, the optimal parameters for efficient single guide RNA (sgRNA) design for CRISPRi are not fully...

Efficient inhibition of the formation of joint adhesions by ERK2 small interfering RNAs

International Nuclear Information System (INIS)

Li, Fengfeng; Ruan, Hongjiang; Fan, Cunyi; Zeng, Bingfang; Wang, Chunyang; Wang, Xiang

2010-01-01

Transforming growth factor-β1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which extracellular signal-regulated kinase (ERK)2 is considered to be crucial. Based on these theories, we examined the effects of a lentivirus-mediated small interfering RNA (siRNA) targeting ERK2 on the suppression of joint adhesion formation in vivo. The effects were assessed in vivo from different aspects including the adhesion score, histology and joint contracture angle. We found that the adhesions in the ERK2 siRNA group became soft and weak, and were easily stretched. Accordingly, the flexion contracture angles in the ERK2 siRNA group were also reduced (P < 0.05 compared with the control group). The animals appeared healthy, with no signs of impaired wound healing. In conclusion, local delivery of a lentivirus-mediated siRNA targeting ERK2 can ameliorate joint adhesion formation effectively and safely.

Long Noncoding RNA HOXC-AS1 Suppresses Ox-LDL-Induced Cholesterol Accumulation Through Promoting HOXC6 Expression in THP-1 Macrophages.

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Huang, Chuan; Hu, Yan-Wei; Zhao, Jing-Jing; Ma, Xin; Zhang, Yuan; Guo, Feng-Xia; Kang, Chun-Min; Lu, Jing-Bo; Xiu, Jian-Cheng; Sha, Yan-Hua; Gao, Ji-Juan; Wang, Yan-Chao; Li, Pan; Xu, Bang-Ming; Zheng, Lei; Wang, Qian

2016-11-01

Atherosclerosis is a common pathological basis of cardiovascular disease, which remains the leading cause of mortality. Long noncoding RNAs (lncRNAs) are newly studied non-protein-coding RNAs involved in gene regulation, but how lncRNAs exert regulatory effect on atherosclerosis remains unclear. In this study, we found that lncRNA HOXC cluster antisense RNA 1 (HOXC-AS1) and homeobox C6 (HOXC6) were downregulated in carotid atherosclerosis by performing microarray analysis. The results were verified in atherosclerotic plaques and normal arterial intima tissues by quantitative reverse transcription PCR and western blot analysis. Lentivirus-mediated overexpression of HOXC-AS1 induced HOXC6 expression at mRNA and protein levels in THP-1 macrophages. Besides, oxidized low-density lipoprotein (Ox-LDL) decreased expression of HOXC-AS1 and HOXC6 in a time-dependent manner. Induction of cholesterol accumulation by Ox-LDL could be partly suppressed by overexpression of HOXC-AS1.

Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi

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Manev Hari

2003-08-01

Full Text Available Abstract Background RNA interference (RNAi is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly gene (corresponding to a putative gene CG5652/GM06434, that we named beltless based on an embryonic loss-of-function phenotype. Results Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts. Conclusions We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should

RNA surveillance via nonsense-mediated mRNA decay is crucial for longevity in daf-2/insulin/IGF-1 mutant C. elegans.

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Son, Heehwa G; Seo, Mihwa; Ham, Seokjin; Hwang, Wooseon; Lee, Dongyeop; An, Seon Woo A; Artan, Murat; Seo, Keunhee; Kaletsky, Rachel; Arey, Rachel N; Ryu, Youngjae; Ha, Chang Man; Kim, Yoon Ki; Murphy, Coleen T; Roh, Tae-Young; Nam, Hong Gil; Lee, Seung-Jae V

2017-03-09

Long-lived organisms often feature more stringent protein and DNA quality control. However, whether RNA quality control mechanisms, such as nonsense-mediated mRNA decay (NMD), which degrades both abnormal as well as some normal transcripts, have a role in organismal aging remains unexplored. Here we show that NMD mediates longevity in C. elegans strains with mutations in daf-2/insulin/insulin-like growth factor 1 receptor. We find that daf-2 mutants display enhanced NMD activity and reduced levels of potentially aberrant transcripts. NMD components, including smg-2/UPF1, are required to achieve the longevity of several long-lived mutants, including daf-2 mutant worms. NMD in the nervous system of the animals is particularly important for RNA quality control to promote longevity. Furthermore, we find that downregulation of yars-2/tyrosyl-tRNA synthetase, an NMD target transcript, by daf-2 mutations contributes to longevity. We propose that NMD-mediated RNA surveillance is a crucial quality control process that contributes to longevity conferred by daf-2 mutations.

Double-stranded RNA interferes in a sequence-specific manner with the infection of representative members of the two viroid families

International Nuclear Information System (INIS)

Carbonell, Alberto; Martinez de Alba, Angel-Emilio; Flores, Ricardo; Gago, Selma

2008-01-01

Infection by viroids, non-protein-coding circular RNAs, occurs with the accumulation of 21-24 nt viroid-derived small RNAs (vd-sRNAs) with characteristic properties of small interfering RNAs (siRNAs) associated to RNA silencing. The vd-sRNAs most likely derive from dicer-like (DCL) enzymes acting on viroid-specific dsRNA, the key elicitor of RNA silencing, or on the highly structured genomic RNA. Previously, viral dsRNAs delivered mechanically or agroinoculated have been shown to interfere with virus infection in a sequence-specific manner. Here, we report similar results with members of the two families of nuclear- and chloroplast-replicating viroids. Moreover, homologous vd-sRNAs co-delivered mechanically also interfered with one of the viroids examined. The interference was sequence-specific, temperature-dependent and, in some cases, also dependent on the dose of the co-inoculated dsRNA or vd-sRNAs. The sequence-specific nature of these effects suggests the involvement of the RNA induced silencing complex (RISC), which provides sequence specificity to RNA silencing machinery. Therefore, viroid titer in natural infections might be regulated by the concerted action of DCL and RISC. Viroids could have evolved their secondary structure as a compromise between resistance to DCL and RISC, which act preferentially against RNAs with compact and relaxed secondary structures, respectively. In addition, compartmentation, association with proteins or active replication might also help viroids to elude their host RNA silencing machinery

Genetic Tools for Self-Organizing Culture of Mouse Embryonic Stem Cells via Small Regulatory RNA-Mediated Technologies, CRISPR/Cas9, and Inducible RNAi.

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Takata, Nozomu; Sakakura, Eriko; Sakuma, Tetsushi; Yamamoto, Takashi

2017-01-01

Approaches to investigate gene functions in experimental biology are becoming more diverse and reliable. Furthermore, several kinds of tissues and organs that possess their original identities can be generated in petri dishes from stem cells including embryonic, adult and induced pluripotent stem cells. Researchers now have several choices of experimental methods and their combinations to analyze gene functions in various biological systems. Here, as an example we describe one of the better protocols, which combines three-dimensional embryonic stem cell culture with small regulatory RNA-mediated technologies, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), and inducible RNA interference (RNAi). This protocol allows investigation of genes of interest to better understand gene functions in target tissues (or organs) during in vitro development.

Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

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St-Laurent Georges

2006-11-01

Full Text Available Abstract Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

miR-380-5p-mediated repression of TEP1 and TSPYL5 interferes with telomerase activity and favours the emergence of an “ALT-like” phenotype in diffuse malignant peritoneal mesothelioma cells

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Graziella Cimino-Reale

2017-07-01

Full Text Available Abstract Background Understanding the molecular/cellular underpinnings of diffuse malignant peritoneal mesothelioma (DMPM, a fatal malignancy with limited therapeutic options, is of utmost importance for the fruitful management of the disease. In this context, we previously found that telomerase activity (TA, which accounts for the limitless proliferative potential of cancer cells, is prognostic for disease relapse and cancer-related death in DMPM patients. Consequently, the identification of factors involved in telomerase activation/regulation may pave the way towards the development of novel therapeutic interventions for the disease. Here, the capability of miR-380-5p, a microRNA negligibly expressed in telomerase-positive DMPM clinical specimens, to interfere with telomerase-mediated telomere maintenance and, hence, with cancer cell growth was assessed on preclinical models of DMPM. Methods DMPM cells were transfected with a miR-380-5p synthetic precursor, and the effects of miRNA replacement were evaluated in terms of growing capability, induction of apoptosis and interference with TA. Reiterated weekly transfections were also performed in order to analyse the phenotype arising upon prolonged miR-380-5p reconstitution in DMPM cells. Results The ectopic expression of miR-380-5p elicited a remarkable inhibition of TA and resulted in DMPM cell growth impairment and apoptosis induction. In particular, we demonstrated for the first time that these effects were the result of a molecular circuitry converging on telomerase associated protein 1 (TEP1, where the miRNA was able to target the gene both directly in unconventional targeting modality and indirectly via p53 accumulation consequent to miRNA-mediated downregulation of testis-specific protein, Y-encoded-like 5 gene. Moreover, miR-380-5p did not cause telomere attrition and cell growth arrest in long-term DMPM transfectants, which in turn showed slightly elongated telomeres and molecular

Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes.

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Aizawa, Shu; Contu, Viorica Raluca; Fujiwara, Yuuki; Hase, Katsunori; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

2017-01-02

Lysosomes degrade macromolecules such as proteins and nucleic acids. We previously identified 2 novel types of autophagy, RNautophagy and DNautophagy, where lysosomes directly take up RNA and DNA, in an ATP-dependent manner, for degradation. We have also reported that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference defective-1), mediates RNA translocation during RNautophagy. In this addendum, we report that SIDT2 also mediates DNA translocation in the process of DNautophagy. These findings help elucidate the mechanisms underlying the direct uptake of nucleic acids by lysosomes and the physiological functions of DNautophagy.

RNA Interference Technology to Control Pest Sea Lampreys - A Proof-of-Concept

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Heath, George; Childs, Darcy; Docker, Margaret F.; McCauley, David W.; Whyard, Steven

2014-01-01

The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0–fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species. PMID:24505485

RNA interference technology to control pest sea lampreys--a proof-of-concept.

Directory of Open Access Journals (Sweden)

George Heath

Full Text Available The parasitic sea lamprey (Petromyzon marinus has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin reduced transcript levels 2.5, 3.6, and 5.0-fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species.

RNA interference technology to control pest sea lampreys--a proof-of-concept.

Science.gov (United States)

Heath, George; Childs, Darcy; Docker, Margaret F; McCauley, David W; Whyard, Steven

2014-01-01

The parasitic sea lamprey (Petromyzon marinus) has caused extensive losses to commercial fish stocks of the upper Great Lakes of North America. Methods of controlling the sea lamprey include trapping, barriers to prevent migration, and use of a chemical lampricide (3-trifluoromethyl-4-nitrophenol) to kill the filter-feeding larvae. Concerns about the non-specificity of these methods have prompted continued development of species-specific methods to control lampreys outside their native range. In this study, we considered the utility of RNA interference to develop a sea lamprey-specific lampricide. Injection of six different short interfering, double-stranded RNAs (siRNAs) into lamprey embryos first confirmed that the siRNAs could reduce the targeted transcript levels by more than 50%. Two size classes of lamprey larvae were then fed the siRNAs complexed with liposomes, and three of the siRNAs (targeting elongation factor 1α, calmodulin, and α-actinin) reduced transcript levels 2.5, 3.6, and 5.0-fold, respectively, within the lamprey midsections. This is not only the first demonstration of RNAi in lampreys, but it is also the first example of delivery of siRNAs to a non-mammalian vertebrate through feeding formulations. One of the siRNA treatments also caused increased mortality of the larvae following a single feeding of siRNAs, which suggests that prolonged or multiple feedings of siRNAs could be used to kill filter-feeding larvae within streams, following development of a slow-release formulation. The genes targeted in this study are highly conserved across many species, and only serve as a proof-of-concept demonstration that siRNAs can be used in lampreys. Given that RNA interference is a sequence-specific phenomenon, it should be possible to design siRNAs that selectively target gene sequences that are unique to sea lampreys, and thus develop a technology to control these pests without adversely affecting non-target species.

MicroRNA-mediated down-regulation of NKG2D ligands contributes to glioma immune escape.

Science.gov (United States)

Codo, Paula; Weller, Michael; Meister, Gunter; Szabo, Emese; Steinle, Alexander; Wolter, Marietta; Reifenberger, Guido; Roth, Patrick

2014-09-15

Malignant gliomas are intrinsic brain tumors with a dismal prognosis. They are well-adapted to hypoxic conditions and poorly immunogenic. NKG2D is one of the major activating receptors of natural killer (NK) cells and binds to several ligands (NKG2DL). Here we evaluated the impact of miRNA on the expression of NKG2DL in glioma cells including stem-like glioma cells. Three of the candidate miRNA predicted to target NKG2DL were expressed in various glioma cell lines as well as in glioblastomas in vivo: miR-20a, miR-93 and miR-106b. LNA inhibitor-mediated miRNA silencing up-regulated cell surface NKG2DL expression, which translated into increased susceptibility to NK cell-mediated lysis. This effect was reversed by neutralizing NKG2D antibodies, confirming that enhanced lysis upon miRNA silencing was mediated through the NKG2D system. Hypoxia, a hallmark of glioblastomas in vivo, down-regulated the expression of NKG2DL on glioma cells, associated with reduced susceptibility to NK cell-mediated lysis. This process, however, was not mediated through any of the examined miRNA. Accordingly, both hypoxia and the expression of miRNA targeting NKG2DL may contribute to the immune evasion of glioma cells at the level of the NKG2D recognition pathway. Targeting miRNA may therefore represent a novel approach to increase the immunogenicity of glioblastoma.

Ubiquitous expression of CUG or CAG trinucleotide repeat RNA causes common morphological defects in a Drosophila model of RNA-mediated pathology.

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Kynan T Lawlor

Full Text Available Expanded DNA repeat sequences are known to cause over 20 diseases, including Huntington's disease, several types of spinocerebellar ataxia and myotonic dystrophy type 1 and 2. A shared genetic basis, and overlapping clinical features for some of these diseases, indicate that common pathways may contribute to pathology. Multiple mechanisms, mediated by both expanded homopolymeric proteins and expanded repeat RNA, have been identified by the use of model systems, that may account for shared pathology. The use of such animal models enables identification of distinct pathways and their 'molecular hallmarks' that can be used to determine the contribution of each pathway in human pathology. Here we characterise a tergite disruption phenotype in adult flies, caused by ubiquitous expression of either untranslated CUG or CAG expanded repeat RNA. Using the tergite phenotype as a quantitative trait we define a new genetic system in which to examine 'hairpin' repeat RNA-mediated cellular perturbation. Further experiments use this system to examine whether pathways involving Muscleblind sequestration or Dicer processing, which have been shown to mediate repeat RNA-mediated pathology in other model systems, contribute to cellular perturbation in this model.

A fast, simple method for screening radiation susceptibility genes by RNA interference

International Nuclear Information System (INIS)

Tsuji, Atsushi B.; Sudo, Hitomi; Sugyo, Aya; Otsuki, Marika; Miyagishi, Makoto; Taira, Kazunari; Imai, Takashi; Harada, Yoshi-nobu

2005-01-01

Radiotherapy can cause unacceptable levels of damage to normal tissues in some cancer patients. To understand the molecular mechanisms underlying radiation-induced physiological responses, and to be able to predict the radiation susceptibility of normal tissues in individual patients, it is important to identify a comprehensive set of genes responsible for radiation susceptibility. We have developed a simple and rapid 96-well screening protocol using cell proliferation assays and RNA interference to identify genes associated with radiation susceptibility. We evaluated the performance of alamarBlue-, BrdU-, and sulforhodamine B-based cell proliferation assays using the 96-well format. Each proliferation assay detected the known radiation susceptibility gene, PRKDC. In a trial screen using 28 shRNA vectors, another known gene, CDKN1A, and one new radiation susceptibility gene, ATP5G3, were identified. Our results indicate that this method may be useful for large-scale screens designed to identify novel radiation susceptibility genes

Reduced lentivirus susceptibility in sheep with TMEM154 mutations.

Science.gov (United States)

Heaton, Michael P; Clawson, Michael L; Chitko-Mckown, Carol G; Leymaster, Kreg A; Smith, Timothy P L; Harhay, Gregory P; White, Stephen N; Herrmann-Hoesing, Lynn M; Mousel, Michelle R; Lewis, Gregory S; Kalbfleisch, Theodore S; Keen, James E; Laegreid, William W

2012-01-01

Visna/Maedi, or ovine progressive pneumonia (OPP) as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154) that exceeded genome-wide significance (unadjusted p-value 3×10(-9)). Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E) at position 35 were associated with infection while a third haplotype with lysine (K) at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-valuesheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-valuesheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.

Transcranial cavitation-mediated ultrasound therapy at sub-MHz frequency via temporal interference modulation

Science.gov (United States)

Sun, Tao; Sutton, Jonathan T.; Power, Chanikarn; Zhang, Yongzhi; Miller, Eric L.; McDannold, Nathan J.

2017-10-01

Sub-megahertz transmission is not usually adopted in pre-clinical small animal experiments for focused ultrasound (FUS) brain therapy due to the large focal size. However, low frequency FUS is vital for preclinical evaluations due to the frequency-dependence of cavitation behavior. To maximize clinical relevance, a dual-aperture FUS system was designed for low-frequency (274.3 kHz) cavitation-mediated FUS therapy. Combining two spherically curved transducers provides significantly improved focusing in the axial direction while yielding an interference pattern with strong side lobes, leading to inhomogeneously distributed cavitation activities. By operating the two transducers at slightly offset frequencies to modulate this interference pattern over the period of sonication, the acoustic energy was redistributed and resulted in a spatially homogenous treatment profile. Simulation and pressure field measurements in water were performed to assess the beam profiles. In addition, the system performance was demonstrated in vivo in rats via drug delivery through microbubble-mediated blood-brain barrier disruption. This design resulted in a homogenous treatment profile that was fully contained within the rat brain at a clinically relevant acoustic frequency.

Genomic Characterization of Variable Surface Antigens Reveals a Telomere Position Effect as a Prerequisite for RNA Interference-Mediated Silencing in Paramecium tetraurelia

Science.gov (United States)

Baranasic, Damir; Oppermann, Timo; Cheaib, Miriam; Cullum, John; Schmidt, Helmut

2014-01-01

ABSTRACT Antigenic or phenotypic variation is a widespread phenomenon of expression of variable surface protein coats on eukaryotic microbes. To clarify the mechanism behind mutually exclusive gene expression, we characterized the genetic properties of the surface antigen multigene family in the ciliate Paramecium tetraurelia and the epigenetic factors controlling expression and silencing. Genome analysis indicated that the multigene family consists of intrachromosomal and subtelomeric genes; both classes apparently derive from different gene duplication events: whole-genome and intrachromosomal duplication. Expression analysis provides evidence for telomere position effects, because only subtelomeric genes follow mutually exclusive transcription. Microarray analysis of cultures deficient in Rdr3, an RNA-dependent RNA polymerase, in comparison to serotype-pure wild-type cultures, shows cotranscription of a subset of subtelomeric genes, indicating that the telomere position effect is due to a selective occurrence of Rdr3-mediated silencing in subtelomeric regions. We present a model of surface antigen evolution by intrachromosomal gene duplication involving the maintenance of positive selection of structurally relevant regions. Further analysis of chromosome heterogeneity shows that alternative telomere addition regions clearly affect transcription of closely related genes. Consequently, chromosome fragmentation appears to be of crucial importance for surface antigen expression and evolution. Our data suggest that RNAi-mediated control of this genetic network by trans-acting RNAs allows rapid epigenetic adaptation by phenotypic variation in combination with long-term genetic adaptation by Darwinian evolution of antigen genes. PMID:25389173

New insights into the interplay between the translation machinery and nonsense-mediated mRNA decay factors.

Science.gov (United States)

Raimondeau, Etienne; Bufton, Joshua C; Schaffitzel, Christiane

2018-06-19

Faulty mRNAs with a premature stop codon (PTC) are recognized and degraded by nonsense-mediated mRNA decay (NMD). Recognition of a nonsense mRNA depends on translation and on the presence of NMD-enhancing or the absence of NMD-inhibiting factors in the 3'-untranslated region. Our review summarizes our current understanding of the molecular function of the conserved NMD factors UPF3B and UPF1, and of the anti-NMD factor Poly(A)-binding protein, and their interactions with ribosomes translating PTC-containing mRNAs. Our recent discovery that UPF3B interferes with human translation termination and enhances ribosome dissociation in vitro , whereas UPF1 is inactive in these assays, suggests a re-interpretation of previous experiments and modification of prevalent NMD models. Moreover, we discuss recent work suggesting new functions of the key NMD factor UPF1 in ribosome recycling, inhibition of translation re-initiation and nascent chain ubiquitylation. These new findings suggest that the interplay of UPF proteins with the translation machinery is more intricate than previously appreciated, and that this interplay quality-controls the efficiency of termination, ribosome recycling and translation re-initiation. © 2018 The Author(s).

Interspecific RNA Interference of SHOOT MERISTEMLESS-Like Disrupts Cuscuta pentagona Plant Parasitism[C][W][OA

Science.gov (United States)

Alakonya, Amos; Kumar, Ravi; Koenig, Daniel; Kimura, Seisuke; Townsley, Brad; Runo, Steven; Garces, Helena M.; Kang, Julie; Yanez, Andrea; David-Schwartz, Rakefet; Machuka, Jesse; Sinha, Neelima

2012-01-01

Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA–mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection. PMID:22822208

Gene interactions in the DNA damage-response pathway identified by genome-wide RNA-interference analysis of synthetic lethality

NARCIS (Netherlands)

van Haaften, Gijs; Vastenhouw, Nadine L; Nollen, Ellen A A; Plasterk, Ronald H A; Tijsterman, Marcel

2004-01-01

Here, we describe a systematic search for synthetic gene interactions in a multicellular organism, the nematode Caenorhabditis elegans. We established a high-throughput method to determine synthetic gene interactions by genome-wide RNA interference and identified genes that are required to protect

3' RNA ligase mediated rapid amplification of cDNA ends for validating viroid induced cleavage at the 3' extremity of the host mRNA.

Science.gov (United States)

Adkar-Purushothama, Charith Raj; Bru, Pierrick; Perreault, Jean-Pierre

2017-12-01

5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) is a widely-accepted method for the validation of direct cleavage of a target gene by a microRNA (miRNA) and viroid-derived small RNA (vd-sRNA). However, this method cannot be used if cleavage takes place in the 3' extremity of the target RNA, as this gives insufficient sequence length to design nested PCR primers for 5' RLM RACE. To overcome this hurdle, we have developed 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE). In this method, an oligonucleotide adapter having 5' adenylated and 3' blocked is ligated to the 3' end of the cleaved RNA followed by PCR amplification using gene specific primers. In other words, in 3' RLM RACE, 3' end is mapped using 5' fragment instead of small 3' fragment. The method developed here was verified by examining the bioinformatics predicted and parallel analysis of RNA ends (PARE) proved cleavage sites of chloride channel protein CLC-b-like mRNA in Potato spindle tuber viroid infected tomato plants. The 3' RLM RACE developed in this study has the potential to validate the miRNA and vd-sRNA mediated cleavage of mRNAs at its 3' untranslated region (3' UTR). Copyright © 2017 Elsevier B.V. All rights reserved.

Interferência por RNA: uma nova alternativa para terapia nas doenças reumáticas RNA interference: a new alternative for rheumatic diseases therapy

Directory of Open Access Journals (Sweden)

Natália Regine de França

2010-12-01

Full Text Available A interferência por RNA (RNAi é um mecanismo de silenciamento gênico pós-transcricional conservado durante a evolução. Esse mecanismo, recentemente descrito, é mediado por pequenos RNAs de fita dupla (dsRNAs capazes de reconhecer especificamente uma sequência de mRNA-alvo e mediar sua clivagem ou repressão traducional. O emprego da RNAi como uma ferramenta de terapia gênica tem sido muito estudado, especialmente em infecções virais, câncer, desordens genéticas herdadas, doenças cardiovasculares e mesmo em doenças reumáticas. Aliados aos dados do genoma humano, os conhecimentos do silenciamento gênico mediado por RNAi podem permitir a determinação funcional de praticamente qualquer gene expresso em uma célula e sua implicação para o funcionamento e homeostase celular. Vários estudos terapêuticos in vitro e in vivo em modelos de doenças autoimunes vêm sendo realizados com resultados encorajadores. As vias de quebra de tolerância e inflamação são alvos potenciais para terapia com RNAi em doenças inflamatórias e autoimunes. Nesta revisão vamos recordar os princípios básicos da RNAi e discutir os aspectos que levaram ao desenvolvimento de propostas terapêuticas baseadas em RNAi, começando pelos estudos in vitro de desenvolvimento de ferramentas e identificação de alvos, chegando até os estudos pré-clínicos de disponibilização da droga in vivo, e testes em células humanas e modelos animais de doenças autoimunes. Por fim, vamos revisar os últimos avanços da experiência clínica da terapia com RNAiRNA interference (RNAi is a post-transcriptional gene silencing mechanism preserved during evolution. This mechanism, recently described, is mediated by small double-stranded RNAs (dsRNAs that can specifically recognize a target mRNA sequence and mediate its cleavage or translational repression. The use of RNAi as a tool for gene therapy has been extensively studied, especially in viral infections, cancer

Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

Science.gov (United States)

Denise, Hubert; Moschos, Sterghios A; Sidders, Benjamin; Burden, Frances; Perkins, Hannah; Carter, Nikki; Stroud, Tim; Kennedy, Michael; Fancy, Sally-Ann; Lapthorn, Cris; Lavender, Helen; Kinloch, Ross; Suhy, David; Corbau, Romu

2014-02-04

TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

Emerging strategies for RNA interference (RNAi) applications in insects.

Science.gov (United States)

Nandety, Raja Sekhar; Kuo, Yen-Wen; Nouri, Shahideh; Falk, Bryce W

2015-01-01

RNA interference (RNAi) in insects is a gene regulatory process that also plays a vital role in the maintenance and in the regulation of host defenses against invading viruses. Small RNAs determine the specificity of the RNAi through precise recognition of their targets. These small RNAs in insects comprise small interfering RNAs (siRNAs), micro RNAs (miRNAs) and Piwi interacting RNAs (piRNAs) of various lengths. In this review, we have explored different forms of the RNAi inducers that are presently in use, and their applications for an effective and efficient fundamental and practical RNAi research with insects. Further, we reviewed trends in next generation sequencing (NGS) technologies and their importance for insect RNAi, including the identification of novel insect targets as well as insect viruses. Here we also describe a rapidly emerging trend of using plant viruses to deliver the RNAi inducer molecules into insects for an efficient RNAi response.

Aedes aegypti uses RNA interference in defense against Sindbis virus infection.

Science.gov (United States)

Campbell, Corey L; Keene, Kimberly M; Brackney, Douglas E; Olson, Ken E; Blair, Carol D; Wilusz, Jeffrey; Foy, Brian D

2008-03-17

RNA interference (RNAi) is an important anti-viral defense mechanism. The Aedes aegypti genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use Ae. aegypti as a model system to determine how the mosquito's anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family Togaviridae, genus Alphavirus). SINV (TR339-eGFP) (+) strand RNA, infectious virus titers and infection rates transiently increased in mosquitoes following dsRNA injection to cognate Ago2, Dcr2, or TSN mRNAs. Detection of SINV RNA-derived small RNAs at 2 and 7 days post-infection in non-silenced mosquitoes provided important confirmation of RNAi pathway activity. Two different recombinant SINV viruses (MRE16-eGFP and TR339-eGFP) with significant differences in infection kinetics were used to delineate vector/virus interactions in the midgut. We show virus-dependent effects on RNAi component transcript and protein levels during infection. Monitoring midgut Ago2, Dcr2, and TSN transcript levels during infection revealed that only TSN transcripts were significantly increased in midguts over blood-fed controls. Ago2 protein levels were depleted immediately following a non-infectious bloodmeal and varied during SINV infection in a virus-dependent manner. We show that silencing RNAi components in Ae. aegypti results in transient increases in SINV replication. Furthermore, Ae. aegypti RNAi is active during SINV infection as indicated by production of virus-specific siRNAs. Lastly, the RNAi response varies in a virus-dependent manner. These data define important features of RNAi anti-viral defense in Ae. aegypti.

Inhibition of KLF7-Targeting MicroRNA 146b Promotes Sciatic Nerve Regeneration.

Science.gov (United States)

Li, Wen-Yuan; Zhang, Wei-Ting; Cheng, Yong-Xia; Liu, Yan-Cui; Zhai, Feng-Guo; Sun, Ping; Li, Hui-Ting; Deng, Ling-Xiao; Zhu, Xiao-Feng; Wang, Ying

2018-06-01

A previous study has indicated that Krüppel-like factor 7 (KLF7), a transcription factor that stimulates Schwann cell (SC) proliferation and axonal regeneration after peripheral nerve injury, is a promising therapeutic transcription factor in nerve injury. We aimed to identify whether inhibition of microRNA-146b (miR-146b) affected SC proliferation, migration, and myelinated axon regeneration following sciatic nerve injury by regulating its direct target KLF7. SCs were transfected with miRNA lentivirus, miRNA inhibitor lentivirus, or KLF7 siRNA lentivirus in vitro. The expression of miR146b and KLF7, as well as SC proliferation and migration, were subsequently evaluated. In vivo, an acellular nerve allograft (ANA) followed by injection of GFP control vector or a lentiviral vector encoding an miR-146b inhibitor was used to assess the repair potential in a model of sciatic nerve gap. miR-146b directly targeted KLF7 by binding to the 3'-UTR, suppressing KLF7. Up-regulation of miR-146b and KLF7 knockdown significantly reduced the proliferation and migration of SCs, whereas silencing miR-146b resulted in increased proliferation and migration. KLF7 protein was localized in SCs in which miR-146b was expressed in vivo. Similarly, 4 weeks after the ANA, anti-miR-146b increased KLF7 and its target gene nerve growth factor cascade, promoting axonal outgrowth. Closer analysis revealed improved nerve conduction and sciatic function index score, and enhanced expression of neurofilaments, P0 (anti-peripheral myelin), and myelinated axon regeneration. Our findings provide new insight into the regulation of KLF7 by miR-146b during peripheral nerve regeneration and suggest a potential therapeutic strategy for peripheral nerve injury.

Neurogenesis-mediated forgetting minimizes proactive interference.

Science.gov (United States)

Epp, Jonathan R; Silva Mera, Rudy; Köhler, Stefan; Josselyn, Sheena A; Frankland, Paul W

2016-02-26

Established memories may interfere with the encoding of new memories, particularly when existing and new memories overlap in content. By manipulating levels of hippocampal neurogenesis, here we show that neurogenesis regulates this form of proactive interference. Increasing hippocampal neurogenesis weakens existing memories and, in doing so, facilitates the encoding of new, conflicting (but not non-conflicting) information in mice. Conversely, decreasing neurogenesis stabilizes existing memories, and impedes the encoding of new, conflicting information. These results suggest that reduced proactive interference is an adaptive benefit of neurogenesis-induced forgetting.

Immunization against Small Ruminant Lentiviruses

Directory of Open Access Journals (Sweden)

Beatriz Amorena

2013-08-01

Full Text Available Multisystemic disease caused by Small Ruminant Lentiviruses (SRLV in sheep and goats leads to production losses, to the detriment of animal health and welfare. This, together with the lack of treatments, has triggered interest in exploring different strategies of immunization to control the widely spread SRLV infection and, also, to provide a useful model for HIV vaccines. These strategies involve inactivated whole virus, subunit vaccines, DNA encoding viral proteins in the presence or absence of plasmids encoding immunological adjuvants and naturally or artificially attenuated viruses. In this review, we revisit, comprehensively, the immunization strategies against SRLV and analyze this double edged tool individually, as it may contribute to either controlling or enhancing virus replication and/or disease.

sRNA-Mediated Regulation of P-Fimbriae Phase Variation in Uropathogenic Escherichia coli

DEFF Research Database (Denmark)

Khandige, Surabhi; Kronborg, Tina; Uhlin, Bernt Eric

2015-01-01

Uropathogenic Escherichia coli (UPEC) are capable of occupying physiologically distinct intracellular and extracellular niches within the urinary tract. This feat requires the timely regulation of gene expression and small RNAs (sRNAs) are known to mediate such rapid adjustments in response to ch...... to changing environmental cues. This study aimed to uncover sRNA-mediated gene regulation in the UPEC strain UTI89, during infection of bladder epithelial cells. Hfq is an RNA chaperone known to facilitate and stabilize sRNA and target mRNA interactions with bacterial cells. The co...... to the discovery of a novel virulence-associated trans-acting sRNA-PapR. Deletion of papR was found to enhance adhesion of UTI89 to both bladder and kidney cell lines in a manner independent of type-1 fimbriae. We demonstrate PapR mediated posttranscriptional repression of the P-fimbriae phase regulator gene pap...

Development of functional genomic tools in trematodes: RNA interference and luciferase reporter gene activity in Fasciola hepatica.

Directory of Open Access Journals (Sweden)

Gabriel Rinaldi

2008-07-01

Full Text Available The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite-host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC. We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP, and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth

miRNA and mRNA Expression Profiles Reveal Insight into Chitosan-Mediated Regulation of Plant Growth.

Science.gov (United States)

Zhang, Xiaoqian; Li, Kecheng; Xing, Ronge; Liu, Song; Chen, Xiaolin; Yang, Haoyue; Li, Pengcheng

2018-04-18

Chitosan has been numerously studied as a plant growth regulator and stress tolerance inducer. To investigate the roles of chitosan as bioregulator on plant and unravel its possible metabolic responses mechanisms, we simultaneously investigated mRNAs and microRNAs (miRNAs) expression profiles of wheat seedlings in response to chitosan heptamer. We found 400 chitosan-responsive differentially expressed genes, including 268 up-regulated and 132 down-regulated mRNAs, many of which were related to photosynthesis, primary carbon and nitrogen metabolism, defense responses, and transcription factors. Moreover, miRNAs also participate in chitosan-mediated regulation on plant growth. We identified 87 known and 21 novel miRNAs, among which 56 miRNAs were induced or repressed by chitosan heptamer, such as miRNA156, miRNA159a, miRNA164, miRNA171a, miRNA319, and miRNA1127. The integrative analysis of miRNA and mRNA expression profiles in this case provides fundamental information for further investigation of regulation mechanisms of chitosan on plant growth and will facilitate its application in agriculture.

A Polytropic Caprine Arthritis Encephalitis Virus Promoter Isolated from Multiple Tissues from a Sheep with Multisystemic Lentivirus-Associated Inflammatory Disease

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Brian Murphy

2013-08-01

Full Text Available Caprine arthritis encephalitis virus (CAEV is a lentivirus that infects both goats and sheep and is closely related to maedi-visna virus that infects sheep; collectively, these viruses are known as small ruminant lentiviruses (SRLV. Infection of goats and sheep with SRLV typically results in discrete inflammatory diseases which include arthritis, mastitis, pneumonia or encephalomyelitis. SRLV-infected animals concurrently demonstrating lentivirus-associated lesions in tissues of lung, mammary gland, joint synovium and the central nervous system are either very rare or have not been reported. Here we describe a novel CAEV promoter isolated from a sheep with multisystemic lentivirus-associated inflammatory disease including interstitial pneumonia, mastitis, polyarthritis and leukomyelitis. A single, novel SRLV promoter was cloned and sequenced from five different anatomical locations (brain stem, spinal cord, lung, mammary gland and carpal joint synovium, all of which demonstrated lesions characteristic of lentivirus associated inflammation. This SRLV promoter isolate was found to be closely related to CAEV promoters isolated from goats in northern California and other parts of the world. The promoter was denoted CAEV-ovine-MS (multisystemic disease; the stability of the transcription factor binding sites within the U3 promoter sequence are discussed.

Lentivirus administration to rat muscle provides efficient sustained expression of erythropoietin

NARCIS (Netherlands)

Seppen, J.; Barry, S. C.; Harder, B.; Osborne, W. R.

2001-01-01

A lentivirus pseudotyped with vesicular stomatitis virus G protein (VSV-G) encoding rat erythropoietin (EPO) complementary DNA was administered to rat skeletal muscle and red blood cell production was serially monitored. After a single intramuscular injection hematocrit values increased and reached

Effects of Toll-like receptor 3 on herpes simplex virus type-1-infected mouse neural stem cells.

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Sun, Xiuning; Shi, Lihong; Zhang, Haoyun; Li, Ruifang; Liang, Ruiwen; Liu, Zhijun

2015-03-01

In this study, we aimed to investigate the effect of herpes simplex virus type-1 (HSV-1) infection on the phosphorylation of interferon regulatory factor 3 (IRF3) and the expression of interferon-β (IFN-β), as well as to clarify the functions of toll-like receptor 3 (TLR3) in mouse neural stem cells (NSCs) infected with HSV-1. In HSV-1-infected cultured NSCs, immunofluorescence, reverse transcription - polymerase chain reaction, Western blot, and ELISA were performed to reveal the expression patterns of TLR3, IRF3, and IFN-β. Then, lentivirus-mediated RNA interference (RNAi) was used to block the expression of TLR3, and its effect on host resistance to HSV-1 infection was investigated. Under uninfected conditions, NSCs expressed TLR3 and phosphorylated IRF3, but after infection, the expression level of TLR3 was upregulated and the phosphorylation level of IRF3 in the nucleus was significantly enhanced, while IFN-β was also expressed. After TLR3 expression was blocked by lentivirus-mediated RNAi, IRF3 phosphorylation and IFN-β expression were downregulated. Therefore, HSV-1 upregulated the expression of TLR3 in NSCs and promoted nuclear translocation after IRF3 was phosphorylated to induce IFN-β expression. TLR3 exhibited an anti-HSV-1 infection capacity via innate immune functions.

RNA interference: a promising technique for the improvement of traditional crops.

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Katoch, Rajan; Thakur, Neelam

2013-03-01

RNA interference (RNAi) is a homology-dependent gene-silencing technology that involves double-stranded RNA directed against a target gene. This technique has emerged as powerful tool in understanding the functions of a number of genes in recent years. For the improvement in the nutritional status of the plants and reduction in the level of antinutrients, the conventional breeding methods were not completely successful in achieving the tissue-specific regulation of some genes. RNAi has shown successful results in a number of plant species for nutritional improvement, change in morphology and alteration in metabolite synthesis. This technology has been applied mostly in genetic engineering of important crop plants, and till date there are no reports of its application for the improvement of traditional/underutilized crops. In this study, we discuss current knowledge of RNAi function and concept and strategies for the improvement of traditional crops. Practical application. Although RNAi has been extensively used for the improvement of popular crops, no attention has been given for the use of this technology for the improvement of underutilized crops. This study describes the importance of use of this technology for the improvement of underutilized crops.

RNA Interference: A Promising Tool in the Control of Important Vector Born Diseases Zika, Dengue Fever, and Malaria

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Jalil Nejati

2017-05-01

Full Text Available Background and Objectives: RNA interference is a process, in which a molecule of double-stranded RNA prevents the expression of a particular gene and leads to its silencing. Application of this technology in the control of disease-carrying insects is rising in agriculture and medical sciences. Also, its application in control of insect-borne diseases could be considered as a new, important, and effective approach. In this article, it was attempted to study the mechanisms of RNA interference, routs of its delivery to insects, as well as its application in genetic control of disease vector insects. Methods: In this study, 71 indexed articles in databases, such as Pubmed, SID, Scopus, Science direct, and Google scholar, were used. Results: dsRNA could be delivered to insect body through three routes of oral, injection, and Impregnation. The mechanism of dsRNA entrance into the cells has considerable effect on the success and applicability of this technique. Identification of host-parasite relationship in the insect body is one of the important applications of RNAi in medical entomology. Conclusion: Although, there is a considerable number of researches on RNAi in the agricultural pests field, studies on insect vectors of human diseases have been mostly in-vivo. However, application of RNAi is suggested as a new, safe and applicable approach, alone or along with other methods. Certainly, further researches in this field can pave the way for enforcement measures in the control of disease vectors, especially Zika, dengue fever, and malaria in the not so distant future.

Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site.

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Hatoum-Aslan, Asma; Maniv, Inbal; Marraffini, Luciano A

2011-12-27

Precise RNA processing is fundamental to all small RNA-mediated interference pathways. In prokaryotes, clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode small CRISPR RNAs (crRNAs) that protect against invasive genetic elements by antisense targeting. CRISPR loci are transcribed as a long precursor that is cleaved within repeat sequences by CRISPR-associated (Cas) proteins. In many organisms, this primary processing generates crRNA intermediates that are subject to additional nucleolytic trimming to render mature crRNAs of specific lengths. The molecular mechanisms underlying this maturation event remain poorly understood. Here, we defined the genetic requirements for crRNA primary processing and maturation in Staphylococcus epidermidis. We show that changes in the position of the primary processing site result in extended or diminished maturation to generate mature crRNAs of constant length. These results indicate that crRNA maturation occurs by a ruler mechanism anchored at the primary processing site. We also show that maturation is mediated by specific cas genes distinct from those genes involved in primary processing, showing that this event is directed by CRISPR/Cas loci.

RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells

International Nuclear Information System (INIS)

Bueren, André O von; Shalaby, Tarek; Oehler-Jänne, Christoph; Arnold, Lucia; Stearns, Duncan; Eberhart, Charles G; Arcaro, Alexandre; Pruschy, Martin; Grotzer, Michael A

2009-01-01

With current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis. To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA) and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR) and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425). siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT) expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector. In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly

RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells

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Arcaro Alexandre

2009-01-01

Full Text Available Abstract Background With current treatment strategies, nearly half of all medulloblastoma (MB patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis. Methods To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425. Results siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector. Conclusion In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly.

RNA interference suppression of A100A4 reduces the growth and metastatic phenotype of human renal cancer cells via NF-kB-dependent MMP-2 and bcl-2 pathway.

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Yang, X-C; Wang, X; Luo, L; Dong, D-H; Yu, Q-C; Wang, X-S; Zhao, K

2013-06-01

S100A4 is a well established marker and mediator of metastatic disease, but the exact mechanisms responsible for the metastasis promoting effects are less well defined. We tested a hypothesis that the S100A4 gene plays a role in the proliferation and invasiveness of human renal cancer cells (RCC) and may be associated with its metastatic spread. The small interference RNA vector pcDNA3.1-S100A4 siRNA was transfected in to the human renal cancer cell lines ACHN, Ketr-3, OS-RC-2, CaKi-2 and HTB-47, then treated with ABT-737 or BB94. Cell apoptosis and cell viability was detected by flow cytometry and MTT assay. Matrigel was used for cell motility and invasion assay. MMP-2, bcl-2 and S100A4 was detected by RT-PCR and western blot assay. NF-kB subunit p65 activity was detected by confocal microscopy assay. We then determine the effect S100A4 sliencing on tumor growth, lung metastasis development in vivo. Immunohistochemistry was used to detected the expression of S100A4, bcl-2, MMP-2, p65 and CD31. S100A4 silencing in ACHN cells by RNA interference significantly inhibited NF-kB and NF-kB-mediated MMP-2 and bcl-2 activation and cellular migration, proliferation, and promoted apoptosis. Furthermore, re-expression of S100A4 in S100A4-siRNA-transfected ACHN cells by transient S100A4 cDNA transfection restored the NF-kB and NF-kB-mediated MMP-2 and bcl-2 activation and their high migratory and cellular proliferative ability. An inhibitor ABT-737 (the Bcl-2 antagonist targets Bcl-2) against Bcl-2 suppressed cellular proliferation and promoted apoptosis induced by S100A4 re-expression in S100A4-siRNA-transfected ACHN cells. A inhibitor BB94 against MMPs to neutralize MMP-2 protein suppressed cellular invasion and migration induced by S100A4 re-expression in S100A4-siRNA-transfected ACHN cells. In the prevention model, S100A4 silencing inhibited primary tumor growth by (tumor weight) (76 ± 8%) and (tumor volum) (78 ± 4%) respectively and promoted apoptosis and the formation

The base excision repair pathway is required for efficient lentivirus integration.

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Kristine E Yoder

Full Text Available An siRNA screen has identified several proteins throughout the base excision repair (BER pathway of oxidative DNA damage as important for efficient HIV infection. The proteins identified included early repair factors such as the base damage recognition glycosylases OGG1 and MYH and the late repair factor POLß, implicating the entire BER pathway. Murine cells with deletions of the genes Ogg1, Myh, Neil1 and Polß recapitulate the defect of HIV infection in the absence of BER. Defective infection in the absence of BER proteins was also seen with the lentivirus FIV, but not the gammaretrovirus MMLV. BER proteins do not affect HIV infection through its accessory genes nor the central polypurine tract. HIV reverse transcription and nuclear entry appear unaffected by the absence of BER proteins. However, HIV integration to the host chromosome is reduced in the absence of BER proteins. Pre-integration complexes from BER deficient cell lines show reduced integration activity in vitro. Integration activity is restored by addition of recombinant BER protein POLß. Lentiviral infection and integration efficiency appears to depend on the presence of BER proteins.

Estudios estructurales de la proteína mayoritaria de la cápsida de lentivirus

OpenAIRE

Yélamos López, María Belén

1998-01-01

Los Lentivirus son un grupo de virus, subfamilia de los Retrovirus. Incluyen virus de gran importancia tanto veterinaria, como el virus de la anemia infecciosa equina (EIAV) o el virus de la inmunodeficiencia felina (FIV), como clínica como el virus de la inmunodeficiencia humana (HIV). Nuestro sistema de estudio es la proteína mayoritaria de la cápsida de lentivirus. Las proteínas de las cápsidas lentivirales tienen una gran homología de secuencia. Este alto grado de similitud, junto con la ...

RNA interference-based resistance against a legume mastrevirus

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Mansoor Shahid

2011-11-01

Full Text Available Abstract Background RNA interference (RNAi is a homology-dependant gene silencing mechanism and has been widely used to engineer resistance in plants against RNA viruses. However, its usefulness in delivering resistance against plant DNA viruses belonging to family Geminiviridae is still being debated. Although the RNAi approach has been shown, using a transient assay, to be useful in countering monocotyledonous plant-infecting geminiviruses of the genus Mastrevirus, it has yet to be investigated as a means of delivering resistance to dicot-infecting mastreviruses. Chickpea chlorotic dwarf Pakistan virus (CpCDPKV is a legume-infecting mastrevirus that affects chickpea and other leguminous crops in Pakistan. Results Here a hairpin (hpRNAi construct containing sequences encompassing part of replication-associated protein gene, intergenic region and part of the movement protein gene of CpCDPKV under the control of the Cauliflower mosaic virus 35S promoter has been produced and stably transformed into Nicotiana benthamiana. Plants harboring the hairpin construct were challenged with CpCDPKV. All non-transgenic N. benthamiana plants developed symptoms of CpCDPKV infection within two weeks post-inoculation. In contrast, none of the inoculated transgenic plants showed symptoms of infection and no viral DNA could be detected by Southern hybridization. A real-time quantitative PCR analysis identified very low-level accumulation of viral DNA in the inoculated transgenic plants. Conclusions The results presented show that the RNAi-based resistance strategy is useful in protecting plants from a dicot-infecting mastrevirus. The very low levels of virus detected in plant tissue of transgenic plants distal to the inoculation site suggest that virus movement and/or viral replication was impaired leading to plants that showed no discernible signs of virus infection.

RNA Interference and its therapeutic applications

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Srinivasa Rao T

2011-10-01

Full Text Available RNAi is a potent method, requiring only a few molecules of dsRNA per cell to silence the expression. Long molecules of double stranded RNA (dsRNA trigger the process. The dsRNA comes from virus and transposon activity in natural RNAi process, while it can be injected in the cells in experimental processes. The strand of the dsRNA that is identical in sequence to a region in target mRNA molecule is called the sense strand, and the other strand which is complimentary is termed the antisense strand. An enzyme complex called DICER thought to be similar to RNAase III then recognizes dsRNA, and cuts it into roughly 22- nucleotide long fragments. These fragments termed siRNAs for “small interfering RNAs” remain in double stranded duplexes with very short 3' overhangs. However, only one of the two strands, known as the guide strand or antisense strand binds the argonaute protein of RNA-induced silencing complex (RISC and target the complementary mRNA resulting gene silencing. The other anti-guide strand or passenger strand is degraded as a RISC substrate during the process of RISC activation. This form of RNAi is termed as post transcriptional gene silencing (PTGS; other forms are also thought to operate at the genomic or transcriptional level in some organisms. In mammals dsRNA longer than 30 base pairs induces a nonspecific antiviral response. This so-called interferon response results in a nonspecific arrest in translation and induction of apoptosis. This cascade induces a global non-specific suppression of translation, which in turn triggers apoptosis. Interestingly, dsRNAs less than 30 nt in length do not activate the antiviral response and specifically switched off genes in human cells without initiating the acute phase response. Thus these siRNAs are suitable for gene target validation and therapeutic applications in many species, including humans. [Vet. World 2011; 4(5.000: 225-229

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference

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Malgorzata Sierant

2011-01-01

Full Text Available RNA interference (RNAi technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G alleles of human Presenilin1 gene (PSEN1. This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimer's disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2-Thiouridine chemical modification introduced at the 3′-end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide.

Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions.

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Zhang, Feng-Lin; Shen, Guo-Min; Liu, Xiao-Ling; Wang, Fang; Zhao, Ying-Ze; Zhang, Jun-Wu

2012-08-01

Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type-specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34(+) haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Reduced lentivirus susceptibility in sheep with TMEM154 mutations.

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Michael P Heaton

2012-01-01

Full Text Available Visna/Maedi, or ovine progressive pneumonia (OPP as it is known in the United States, is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. This disease affects multiple tissues, including those of the respiratory and central nervous systems. Our aim was to identify ovine genetic risk factors for lentivirus infection. Sixty-nine matched pairs of infected cases and uninfected controls were identified among 736 naturally exposed sheep older than five years of age. These pairs were used in a genome-wide association study with 50,614 markers. A single SNP was identified in the ovine transmembrane protein (TMEM154 that exceeded genome-wide significance (unadjusted p-value 3×10(-9. Sanger sequencing of the ovine TMEM154 coding region identified six missense and two frameshift deletion mutations in the predicted signal peptide and extracellular domain. Two TMEM154 haplotypes encoding glutamate (E at position 35 were associated with infection while a third haplotype with lysine (K at position 35 was not. Haplotypes encoding full-length E35 isoforms were analyzed together as genetic risk factors in a multi-breed, matched case-control design, with 61 pairs of 4-year-old ewes. The odds of infection for ewes with one copy of a full-length TMEM154 E35 allele were 28 times greater than the odds for those without (p-value<0.0001, 95% CI 5-1,100. In a combined analysis of nine cohorts with 2,705 sheep from Nebraska, Idaho, and Iowa, the relative risk of infection was 2.85 times greater for sheep with a full-length TMEM154 E35 allele (p-value<0.0001, 95% CI 2.36-3.43. Although rare, some sheep were homozygous for TMEM154 deletion mutations and remained uninfected despite a lifetime of significant exposure. Together, these findings indicate that TMEM154 may play a central role in ovine lentivirus infection and removing sheep with the most susceptible genotypes may help eradicate OPP and protect flocks from reinfection.

Knock down of the myostatin gene by RNA interference increased body weight in chicken.

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Bhattacharya, T K; Shukla, R; Chatterjee, R N; Dushyanth, K

2017-01-10

Myostatin is a negative regulator of muscular growth in poultry and other animals. Of several approaches, knocking down the negative regulator is an important aspect to augment muscular growth in chicken. Knock down of myostatin gene has been performed by shRNA acting against the expression of gene in animals. Two methods of knock down of gene in chicken such as embryo manipulation and sperm mediated method have been performed. The hatching percentage in embryo manipulation and sperm mediated method of knock down was 58.0 and 41.5%, respectively. The shRNA in knock down chicken enhanced body weight at 6 weeks by 26.9%. The dressing percentage and serum biochemical parameters such as SGPT and alkaline phosphatase differed significantly (Pknock down and control birds. It is concluded that knocking down the myostatin gene successfully augmented growth in chicken. Copyright © 2016 Elsevier B.V. All rights reserved.

Involvement of activation of PKR in HBx-siRNA-mediated innate immune effects on HBV inhibition.

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Qiuju Han

Full Text Available RNA interference (RNAi of virus-specific genes offers the possibility of developing a new anti-hepatitis B virus (anti-HBV therapy. Recent studies have revealed that siRNAs can induce an innate immune response in vitro and in vivo. Here, HBVx (HBx mRNA expression and HBV replication were significantly inhibited, followed by the enhancement of expression of type I interferons (IFNs, IFN-stimulated genes (ISG15 and ISG56 and proinflammatory cytokines after HepG2.2.15 cells were transfected with chemically synthesized HBx-siRNAs. Transfection with HBx-siRNAs also significantly increased expression of dsRNA-dependent protein kinase R (PKR in HepG2.2.15 cells, followed by activation of downstream signaling events such as eukaryotic initiation factor 2α (eIF2-α. In PKR-over-expressing HepG2.2.15 cells, HBx-siRNAs exerted more potent inhibitory effects on HBV replication and greater production of type I IFNs. By contrast, the inhibitory effect of HBx-siRNAs on HBV replication was attenuated when PKR was inhibited or silenced, demonstrating that HBx-siRNAs greatly promoted PKR activation, leading to the higher production of type I IFN. Therefore, we concluded that PKR is involved in the innate immune effects mediated by HBx-siRNAs and further contributes to HBV inhibition. The bifunctional siRNAs with both gene silencing and innate immune activation properties may represent a new potential strategy for treatment of HBV.

Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity

NARCIS (Netherlands)

Charpentier, Emmanuelle; Richter, Hagen; Oost, van der John; White, Malcolm F.

2015-01-01

CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences

Silencing of RhoA and RhoC expression by RNA interference suppresses human colorectal carcinoma growth in vivo

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Wang Haibo

2010-09-01

Full Text Available Abstract Background RhoA and RhoC have been proved to be over-expressed in many solid cancers, including colorectal cancer. The reduction of RhoA and RhoC expression by RNA interference (RNAi resulted growth inhibition of cancer cells. The present study was to evaluate the effect of silencing of RhoA and RhoC expression by RNAi on growth of human colorectal carcinoma (CRC in tumor-bearing nude mice in vivo. Methods To establish HCT116 cell transplantable model, the nude mice were subcutaneously inoculated with 1.0 × 107 HCT116 cells and kept growing till the tumor xenografts reached 5-7 mm in diameter. Then the mice were randomly assigned to three groups(seven mice in each group: (1 normal saline(NS group, (2replication-defective recombinant adenovirus carrying the negative control shRNA (Ad-HK group and (3replication-defective recombinant adenovirus carrying the 4-tandem linked RhoA and RhoC shRNAs (Ad-RhoA-RhoC group. Ad-HK (4 × 108 pfu, 30 ul/mouse, Ad-RhoA-RhoC (4 × 108 pfu, 30 ul/mouse or PBS (30 ul/mouse was injected intratumorally four times once every other day. The weight and volumes of tumor xenografts were recorded. The levels of RhoA and RhoC mRNA transcripts and proteins in tumor xenografts were detected by reverse quantitative transcription polymerase chain reaction (QRT-PCR and immunohistochemical staining respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay was used to detect the death of cells. Results The xenografts in mice could be seen at 5th day from the implantation of HCT116 cells and all had reached 5-7 mm in size at 9th day. After injection intratumorally, the growth speed of tumor xenografts in Ad-RhoA-RhoC group was significantly delayed compared with those in NS and Ad-HK group(P RhoA and RhoC reduced more in Ad-RhoA-RhoC group than those in NS and Ad-HK group. The relative RhoA and RhoC mRNA transcripts were decreased to 48% and 43% respectively (P RhoA and Rho

[Expression of Jagged1 mRNA in human epithelial ovarian carcinoma tissues and effect of RNA interference of Jagged1 on growth of xenograft in nude mice].

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Liu, G Y; Gao, Z H; Li, L; Song, T T; Sheng, X G

2016-06-25

To investigate the expression of Jagged1 in human epithelial ovarian carcinoma tissues and the effect of Jagged1 on growth of xenograft in nude mice. (1) Forty-eight cases of ovarian cancer and 30 cases of patients with benign epithelial ovarian tumor in the Henan Province Xinxiang Central Hospital during Feb. 2011 to Mar. 2014 were enrolled in this study. The mRNA expression of Jagged1, Notch1 and the downstream target genes Hes1, Hey1 were analyzed by using realtime PCR method. (2) The ovarian cancer xenograft models in nude mice were constructed by injecting SKOV3 cells in axillary subcutaneouswere. The nude mice were randomly divided into Jagged1 interference group, blank plasmid group and control group. Each group had 10 mice. They were transfected with pcDNA3.1(+)-siRNA-Jagged1, blank plasmid pDC3.1 and phosphate buffer, respectively. The tumor volumes and tumor masses were measured 14 days after transfection and the inhibition rate was calculated. The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues after transfection in each group was detected by using realtime PCR technique and the relative protein expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues was detected by utilizing western blot method. (1) The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in ovarian cancer tissues were higher than benign ovarian tumor tissues, the differences were statistically significant (Ptissues of nude micein Jagged1 interference group were lower than that in the other two groups, the differences were statistically significant (Ptissues of nude mice among the three groups (P>0.05). Jagged1 is highly expressed in epithelial ovarian carcinoma. Jagged1 gene interference in xenograft tumor can inhibit ovarian cancer cell growth and improve tumor suppressor rate, which probably play roles by inhibiting Notch1 signaling pathway.

Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus

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Citrus paradisi Macf. cv. Duncan was transformed with constructs coding for the wild-type and mutant RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) for exploring replicase-mediated pathogen-derived resistance (RM-PDR). The RdRp gene was amplified from CTV genome and used to gener...

RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi?

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Unnikrishnan Unniyampurath

2016-02-01

Full Text Available The recent emergence of multiple technologies for modifying gene structure has revolutionized mammalian biomedical research and enhanced the promises of gene therapy. Over the past decade, RNA interference (RNAi based technologies widely dominated various research applications involving experimental modulation of gene expression at the post-transcriptional level. Recently, a new gene editing technology, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR and the CRISPR-associated protein 9 (Cas9 (CRISPR/Cas9 system, has received unprecedented acceptance in the scientific community for a variety of genetic applications. Unlike RNAi, the CRISPR/Cas9 system is bestowed with the ability to introduce heritable precision insertions and deletions in the eukaryotic genome. The combination of popularity and superior capabilities of CRISPR/Cas9 system raises the possibility that this technology may occupy the roles currently served by RNAi and may even make RNAi obsolete. We performed a comparative analysis of the technical aspects and applications of the CRISPR/Cas9 system and RNAi in mammalian systems, with the purpose of charting out a predictive picture on whether the CRISPR/Cas9 system will eclipse the existence and future of RNAi. The conclusion drawn from this analysis is that RNAi will still occupy specific domains of biomedical research and clinical applications, under the current state of development of these technologies. However, further improvements in CRISPR/Cas9 based technology may ultimately enable it to dominate RNAi in the long term.

Architecture of the RNA polymerase II-Mediator core initiation complex.

Science.gov (United States)

Plaschka, C; Larivière, L; Wenzeck, L; Seizl, M; Hemann, M; Tegunov, D; Petrotchenko, E V; Borchers, C H; Baumeister, W; Herzog, F; Villa, E; Cramer, P

2015-02-19

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

Understanding the core of RNA interference: The dynamic aspects of Argonaute-mediated processes

KAUST Repository

Zhu, Lizhe; Jiang, Hanlun; Sheong, Fu Kit; Cui, Xuefeng; Wang, Yanli; Gao, Xin; Huang, Xuhui

2016-01-01

and its interaction with the nucleic acids, considerable progress has been made to reveal the dynamic aspects of various Ago-mediated processes. Here we review these novel insights into the guide-strand loading, duplex unwinding, and effects of seed

RNA Interference Based Approach to Down Regulate Osmoregulators of Whitefly (Bemisia tabaci): Potential Technology for the Control of Whitefly

Science.gov (United States)

Over the past decade RNA interference (RNAi) technology has emerged as a successful tool not only for functional genomics, but in planta expression of short interfering RNAs (siRNAs) could offer potential for insect pest management. Insects feeding exclusively on plant sap depend on osmotic pressure...

Gold nanoparticle mediated laser transfection for efficient siRNA mediated gene knock down.

Directory of Open Access Journals (Sweden)

Dag Heinemann

Full Text Available Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types.

Gold nanoparticle mediated laser transfection for efficient siRNA mediated gene knock down.

Science.gov (United States)

Heinemann, Dag; Schomaker, Markus; Kalies, Stefan; Schieck, Maximilian; Carlson, Regina; Murua Escobar, Hugo; Ripken, Tammo; Meyer, Heiko; Heisterkamp, Alexander

2013-01-01

Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types.

Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

Science.gov (United States)

Heinemann, Dag; Schomaker, Markus; Kalies, Stefan; Schieck, Maximilian; Carlson, Regina; Escobar, Hugo Murua; Ripken, Tammo; Meyer, Heiko; Heisterkamp, Alexander

2013-01-01

Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types. PMID:23536802

RNA interference of carboxyesterases causes nymph mortality in the Asian citrus psyllid, Diaphorina citri.

Science.gov (United States)

Kishk, Abdelaziz; Anber, Helmy A I; AbdEl-Raof, Tsamoh K; El-Sherbeni, AbdEl-Hakeem D; Hamed, Sobhy; Gowda, Siddarame; Killiny, Nabil

2017-03-01

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important pest of citrus. In addition, D. citri is the vector of Huanglongbing, a destructive disease in citrus, also known as citrus greening disease caused by Candidatus Liberibacter asiaticus. Huanglongbing causes huge losses for citrus industries. Insecticide application for D. citri is the major strategy to prevent disease spread. The heavy use of insecticides causes development of insecticide resistance. We used RNA interference (RNAi) to silence genes implicated in pesticide resistance in order to increase the susceptibility. The activity of dsRNA to reduce the expression of carboxyesterases including esterases FE4 (EstFE4) and acetylcholinesterases (AChe) in D. citri was investigated. The dsRNA was applied topically to the fourth and fifth instars of nymphs. We targeted several EstFE4 and AChe genes using dsRNA against a consensus sequence for each of them. Five concentrations (25, 50, 75, 100, 125 ng/μl) from both dsRNAs were used. The treatments with the dsRNA caused concentration dependent nymph mortality. The highest gene expression levels of both AChe and EstFE4 were found in the fourth and fifth nymphal instars. Gene expression analysis showed that AChe genes were downregulated in emerged adults from dsRNA-AChe-treated nymphs compared to controls. However, EstFE4 genes were not affected. In the same manner, treatment with dsRNA-EstFE4 reduced expression level of EstFE4 genes in emerged adults from treated nymphs, but did not affect the expression of AChe genes. In the era of environmentally friendly control strategies, RNAi is a new promising venue to reduce pesticide applications. © 2017 Wiley Periodicals, Inc.

Resveratrol and pterostilbene as a microRNA-mediated chemopreventive and therapeutic strategy in prostate cancer

Science.gov (United States)

Growing evidence indicates deregulation of the epigenetic machinery comprising the microRNA (miRNA) network as a critical factor in the progression of various diseases including cancer. Concurrently, dietary phytochemicals are being intensively studied for their miRNA-mediated health beneficial prop...

CRISPR/Cas9-mediated noncoding RNA editing in human cancers.

Science.gov (United States)

Yang, Jie; Meng, Xiaodan; Pan, Jinchang; Jiang, Nan; Zhou, Chengwei; Wu, Zhenhua; Gong, Zhaohui

2018-01-02

Cancer is characterized by multiple genetic and epigenetic alterations, including a higher prevalence of mutations of oncogenes and/or tumor suppressors. Mounting evidences have shown that noncoding RNAs (ncRNAs) are involved in the epigenetic regulation of cancer genes and their associated pathways. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) system, a revolutionary genome-editing technology, has shed light on ncRNA-based cancer therapy. Here, we briefly introduce the classifications and mechanisms of CRISPR/Cas9 system. Importantly, we mainly focused on the applications of CRISPR/Cas9 system as a molecular tool for ncRNA (microRNA, long noncoding RNA and circular RNA, etc.) editing in human cancers, and the novel techniques that are based on CRISPR/Cas9 system. Additionally, the off-target effects and the corresponding solutions as well as the challenges toward CRISPR/Cas9 were also evaluated and discussed. Long- and short-ncRNAs have been employed as targets in precision oncology, and CRISPR/Cas9-mediated ncRNA editing may provide an excellent way to cure cancer.

Vaccination delays maedi-visna lentivirus infection in a naturally-infected sheep flock

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Gudnadóttir Margrét

2013-01-01

Full Text Available Abstract Background The Maedi-Visna (MV lentivirus causes two slowly progressive eventually fatal diseases of sheep, Maedi, a progressive interstitial pneumonia, and Visna, a progressive demyelinating disease of the central nervous system. Other lentiviruses also cause fatal slow infections in their natural hosts, e.g. the HIV virus in humans. Results of experimental vaccination against any lentivirus where vaccinees are challenged by natural routes, may therefore be of general interest. From 1991–1998 experiments with formalin-inactivated whole Maedi-Visna virus vaccine were carried out in the Department of Microbiology at the University of Iceland. Western Blot tests showed good immune response to all major proteins of the virus. When aluminium hydroxide was added to the vaccine all vaccinees developed neutralizing antibodies to the vaccine strain at titers 1/8 – 1/256. After housing 5 twin pairs, one twin in each pair vaccinated, the other unvaccinated, with infected sheep for 4 years, all the unvaccinated twins became infected, but only 2 of their vaccinated siblings as confirmed by virus cultivation experiments on tissues from their lungs spleens lymph nodes and choroid plexuses. Results One twin in each of 40 female twin pairs, born into a Maedi-Visna-infected sheep flock and kept under natural farming conditions in Cyprus, was vaccinated at birth, 3 weeks and 3 months, with formalin-inactivated whole Maedi-Visna lentivirus vaccine adjuvanted with aluminium hydroxide. 17 mothers of the twins were seronegative, 13 seroconverting and 10 had old infection. Of 17 vaccinees born to seronegative mothers 9 were uninfected at 28 months, but only 2 of their unvaccinated siblings. Of 13 unvaccinated twins born to seroconverting mothers, 12 caught infection during their first 10 weeks, but only 4 of their vaccinated siblings. Vaccination had no effects on 10 vaccinees born to mothers with long-standing Maedi-Visna infections and broad andibody

Fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of LncRNA MEG3

International Nuclear Information System (INIS)

Hu, Duanmin; Su, Cunjin; Jiang, Min; Shen, Yating; Shi, Aiming; Zhao, Fenglun; Chen, Ruidong; Shen, Zhu; Bao, Junjie; Tang, Wen

2016-01-01

There is still no suitable drug for pancreatic cancer treatment, which is one of the most aggressive human tumors. Maternally expressed gene 3 (MEG3), a LncRNA, has been suggested as a tumor suppressor in a range of human tumors. Studies found fenofibrate exerted anti-tumor roles in various human cancer cell lines. However, its role in pancreatic cancer remains unknown. The present study aimed to explore the impacts of fenofibrate on pancreatic cancer cell lines, and to investigate MEG3 role in its anti-tumor mechanisms. We used MTT assay to determine cells proliferation, genome-wide LncRNA microarray analysis to identify differently expressed LncRNAs, siRNA or pCDNA-MEG3 transfection to interfere or upregulate MEG3 expression, western blot to detect protein levels, real-time PCR to determine MEG3 level. Fenofibrate significantly inhibited proliferation of pancreatic cancer cells, increased MEG3 expression and p53 levels. Moreover, knockdown of MEG3 attenuated cytotoxicity induced by fenofibrate. Furthermore, overexpression of MEG3 induced cells death and increased p53 expression. Our results indicated fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of MEG3. - Highlights: • We found that fenofibrate suppressed proliferation of pancreatic cancer cells. • We found fenofibrate increased LncRNA-MEG3 expression and p53 level in PANC-1 cells. • Inhibition of MEG3 expression attenuated anti-tumor effects of fenofibrate.

Fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of LncRNA MEG3

Energy Technology Data Exchange (ETDEWEB)

Hu, Duanmin [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Su, Cunjin [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Jiang, Min [Department of Breast Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215004 (China); Shen, Yating [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Shi, Aiming; Zhao, Fenglun [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Chen, Ruidong [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Shen, Zhu [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Bao, Junjie, E-mail: baojjsdfey@sina.com [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Tang, Wen, E-mail: sztangwen@163.com [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China)

2016-03-04

There is still no suitable drug for pancreatic cancer treatment, which is one of the most aggressive human tumors. Maternally expressed gene 3 (MEG3), a LncRNA, has been suggested as a tumor suppressor in a range of human tumors. Studies found fenofibrate exerted anti-tumor roles in various human cancer cell lines. However, its role in pancreatic cancer remains unknown. The present study aimed to explore the impacts of fenofibrate on pancreatic cancer cell lines, and to investigate MEG3 role in its anti-tumor mechanisms. We used MTT assay to determine cells proliferation, genome-wide LncRNA microarray analysis to identify differently expressed LncRNAs, siRNA or pCDNA-MEG3 transfection to interfere or upregulate MEG3 expression, western blot to detect protein levels, real-time PCR to determine MEG3 level. Fenofibrate significantly inhibited proliferation of pancreatic cancer cells, increased MEG3 expression and p53 levels. Moreover, knockdown of MEG3 attenuated cytotoxicity induced by fenofibrate. Furthermore, overexpression of MEG3 induced cells death and increased p53 expression. Our results indicated fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of MEG3. - Highlights: • We found that fenofibrate suppressed proliferation of pancreatic cancer cells. • We found fenofibrate increased LncRNA-MEG3 expression and p53 level in PANC-1 cells. • Inhibition of MEG3 expression attenuated anti-tumor effects of fenofibrate.

Roles of Prolyl Isomerases in RNA-Mediated Gene Expression

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Roopa Thapar

2015-05-01

Full Text Available The peptidyl-prolyl cis-trans isomerases (PPIases that include immunophilins (cyclophilins and FKBPs and parvulins (Pin1, Par14, Par17 participate in cell signaling, transcription, pre-mRNA processing and mRNA decay. The human genome encodes 19 cyclophilins, 18 FKBPs and three parvulins. Immunophilins are receptors for the immunosuppressive drugs cyclosporin A, FK506, and rapamycin that are used in organ transplantation. Pin1 has also been targeted in the treatment of Alzheimer’s disease, asthma, and a number of cancers. While these PPIases are characterized as molecular chaperones, they also act in a nonchaperone manner to promote protein-protein interactions using surfaces outside their active sites. The immunosuppressive drugs act by a gain-of-function mechanism by promoting protein-protein interactions in vivo. Several immunophilins have been identified as components of the spliceosome and are essential for alternative splicing. Pin1 plays roles in transcription and RNA processing by catalyzing conformational changes in the RNA Pol II C-terminal domain. Pin1 also binds several RNA binding proteins such as AUF1, KSRP, HuR, and SLBP that regulate mRNA decay by remodeling mRNP complexes. The functions of ribonucleoprotein associated PPIases are largely unknown. This review highlights PPIases that play roles in RNA-mediated gene expression, providing insight into their structures, functions and mechanisms of action in mRNP remodeling in vivo.

Expanding Possibilities for Intervention against Small Ruminant Lentiviruses through Genetic Marker-Assisted Selective Breeding

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Donald P. Knowles

2013-06-01

Full Text Available Small ruminant lentiviruses include members that infect sheep (ovine lentivirus [OvLV]; also known as ovine progressive pneumonia virus/maedi-visna virus and goats (caprine arthritis encephalitis virus [CAEV]. Breed differences in seroprevalence and proviral concentration of OvLV had suggested a strong genetic component in susceptibility to infection by OvLV in sheep. A genetic marker test for susceptibility to OvLV has been developed recently based on the TMEM154 gene with validation data from over 2,800 sheep representing nine cohorts. While no single genotype has been shown to have complete resistance to OvLV, consistent association in thousands of sheep from multiple breeds and management conditions highlight a new strategy for intervention by selective breeding. This genetic marker-assisted selection (MAS has the potential to be a useful addition to existing viral control measures. Further, the discovery of multiple additional genomic regions associated with susceptibility to or control of OvLV suggests that additional genetic marker tests may be developed to extend the reach of MAS in the future. This review will cover the strengths and limitations of existing data from host genetics as an intervention and outline additional questions for future genetic research in sheep, goats, small ruminant lentiviruses, and their host-pathogen interactions.

Expanding Possibilities for Intervention against Small Ruminant Lentiviruses through Genetic Marker-Assisted Selective Breeding

Science.gov (United States)

White, Stephen N.; Knowles, Donald P.

2013-01-01

Small ruminant lentiviruses include members that infect sheep (ovine lentivirus [OvLV]; also known as ovine progressive pneumonia virus/maedi-visna virus) and goats (caprine arthritis encephalitis virus [CAEV]). Breed differences in seroprevalence and proviral concentration of OvLV had suggested a strong genetic component in susceptibility to infection by OvLV in sheep. A genetic marker test for susceptibility to OvLV has been developed recently based on the TMEM154 gene with validation data from over 2,800 sheep representing nine cohorts. While no single genotype has been shown to have complete resistance to OvLV, consistent association in thousands of sheep from multiple breeds and management conditions highlight a new strategy for intervention by selective breeding. This genetic marker-assisted selection (MAS) has the potential to be a useful addition to existing viral control measures. Further, the discovery of multiple additional genomic regions associated with susceptibility to or control of OvLV suggests that additional genetic marker tests may be developed to extend the reach of MAS in the future. This review will cover the strengths and limitations of existing data from host genetics as an intervention and outline additional questions for future genetic research in sheep, goats, small ruminant lentiviruses, and their host-pathogen interactions. PMID:23771240

Improving model predictions for RNA interference activities that use support vector machine regression by combining and filtering features

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Peek Andrew S

2007-06-01

Full Text Available Abstract Background RNA interference (RNAi is a naturally occurring phenomenon that results in the suppression of a target RNA sequence utilizing a variety of possible methods and pathways. To dissect the factors that result in effective siRNA sequences a regression kernel Support Vector Machine (SVM approach was used to quantitatively model RNA interference activities. Results Eight overall feature mapping methods were compared in their abilities to build SVM regression models that predict published siRNA activities. The primary factors in predictive SVM models are position specific nucleotide compositions. The secondary factors are position independent sequence motifs (N-grams and guide strand to passenger strand sequence thermodynamics. Finally, the factors that are least contributory but are still predictive of efficacy are measures of intramolecular guide strand secondary structure and target strand secondary structure. Of these, the site of the 5' most base of the guide strand is the most informative. Conclusion The capacity of specific feature mapping methods and their ability to build predictive models of RNAi activity suggests a relative biological importance of these features. Some feature mapping methods are more informative in building predictive models and overall t-test filtering provides a method to remove some noisy features or make comparisons among datasets. Together, these features can yield predictive SVM regression models with increased predictive accuracy between predicted and observed activities both within datasets by cross validation, and between independently collected RNAi activity datasets. Feature filtering to remove features should be approached carefully in that it is possible to reduce feature set size without substantially reducing predictive models, but the features retained in the candidate models become increasingly distinct. Software to perform feature prediction and SVM training and testing on nucleic acid

CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

Science.gov (United States)

Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

2014-09-05

Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. Copyright © 2014 Xue et al.

UBXN1 Interferes with Rig-I-like Receptor-Mediated Antiviral Immune Response by Targeting MAVS

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Penghua Wang

2013-04-01

Full Text Available RNA viruses are sensed by RIG-I-like receptors (RLRs, which signal through a mitochondria-associated adaptor molecule, MAVS, resulting in systemic antiviral immune responses. Although RLR signaling is essential for limiting RNA virus replication, it must be stringently controlled to prevent damage from inflammation. We demonstrate here that among all tested UBX-domain-containing protein family members, UBXN1 exhibits the strongest inhibitory effect on RNA-virus-induced type I interferon response. UBXN1 potently inhibits RLR- and MAVS-induced, but not TLR3-, TLR4-, or DNA-virus-induced innate immune responses. Depletion of UBXN1 enhances virus-induced innate immune responses, including those resulting from RNA viruses such as vesicular stomatitis, Sendai, West Nile, and dengue virus infection, repressing viral replication. Following viral infection, UBXN1 is induced, binds to MAVS, interferes with intracellular MAVS oligomerization, and disrupts the MAVS/TRAF3/TRAF6 signalosome. These findings underscore a critical role of UBXN1 in the modulation of a major antiviral signaling pathway.

Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito's RNA interference pathway.

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Irma Sánchez-Vargas

2009-02-01

Full Text Available A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi, is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA, which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs. These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2 infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.

From early lessons to new frontiers: the worm as a treasure trove of small RNA biology.

Science.gov (United States)

Youngman, Elaine M; Claycomb, Julie M

2014-01-01

In the past 20 years, the tiny soil nematode Caenorhabditis elegans has provided critical insights into our understanding of the breadth of small RNA-mediated gene regulatory activities. The first microRNA was identified in C. elegans in 1993, and the understanding that dsRNA was the driving force behind RNA-mediated gene silencing came from experiments performed in C. elegans in 1998. Likewise, early genetic screens in C. elegans for factors involved in RNA interference pointed to conserved mechanisms for small RNA-mediated gene silencing pathways, placing the worm squarely among the founding fathers of a now extensive field of molecular biology. Today, the worm continues to be at the forefront of ground-breaking insight into small RNA-mediated biology. Recent studies have revealed with increasing mechanistic clarity that C. elegans possesses an extensive nuclear small RNA regulatory network that encompasses not only gene silencing but also gene activating roles. Further, a portrait is emerging whereby small RNA pathways play key roles in integrating responses to environmental stimuli and transmitting epigenetic information about such responses from one generation to the next. Here we discuss endogenous small RNA pathways in C. elegans and the insight worm biology has provided into the mechanisms employed by these pathways. We touch on the increasingly spectacular diversity of small RNA biogenesis and function, and discuss the relevance of lessons learned in the worm for human biology.

Tumor-specific RNA interference targeting Pokemon suppresses tumor growth and induces apoptosis in prostate cancer.

Science.gov (United States)

Li, Yining; Xu, Shuxiong; Wang, Xiangwei; Shi, Hua; Sun, Zhaolin; Yang, Zhao

2013-02-01

To explore the exact mechanism of Pokemon in prostate cancer. Pokemon is a member of the POK family of transcriptional repressors. Its main function is suppression of the p14ARF (alternate reading frame) tumor suppressor gene. Although Pokemon expression has been found to be increased in various types of lymphoma, the exact mechanism of the gene in prostate cancer is not clear. In the present study, prostate cancer cells were transfected with the specific short hairpin ribonucleic acid (RNA) expression vector targeting Pokemon. The expression of Pokemon messenger RNA and its protein was detected by semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The cell growth and cell apoptosis were also examined using the methyl thiazolyl tetrazolium assay and flow cytometry. The results demonstrated that specific RNA interference (RNAi) could decrease the expression levels of Pokemon gene messenger RNA and protein in prostate cancer cells. In addition, that specific RNAi significantly inhibited the cell proliferation and increased the apoptotic rate. In vivo experiments showed that specific RNAi inhibited the tumorigenicity of prostate cancer cells and significantly suppressed tumor growth. Therefore, an RNAi-targeted Pokemon gene strategy could be a potential approach to prostate cancer therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

In Vivo Testing of MicroRNA-Mediated Gene Knockdown in Zebrafish

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Ivone Un San Leong

2012-01-01

Full Text Available The zebrafish (Danio rerio has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis. The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.

Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells

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Tatiana I Novobrantseva

2012-01-01

Full Text Available Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP for durable and potent in vivo RNA interference (RNAi-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA. In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.

Dark Matter Interference

DEFF Research Database (Denmark)

Del Nobile, Eugenio; Kouvaris, Christoforos; Sannino, Francesco

2012-01-01

We study different patterns of interference in WIMP-nuclei elastic scattering that can accommodate the DAMA and CoGeNT experiments via an isospin violating ratio $f_n/f_p=-0.71$. We study interference between the following pairs of mediators: Z and Z', Z' and Higgs, and two Higgs fields. We show ...

RNA interference screen identifies Abl kinase and PDGFR signaling in Chlamydia trachomatis entry.

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Cherilyn A Elwell

2008-03-01

Full Text Available The strain designated Chlamydia trachomatis serovar L2 that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis. This conclusion was verified by deep sequencing and by PCR using species-specific primers. All data presented in the results section that refer to C. trachomatis should be interpreted as referring to C. muridarum. Since C. muridarum TARP lacks the consensus tyrosine repeats present in C. trachomatis TARP, we cannot make any conclusions about the role of TARP phosphorylation and C. muridarum entry. However, the conclusion that C. trachomatis L2 TARP is a target of Abl kinase is still valid as these experiments were performed with C. trachomatis L2 TARP [corrected]. To elucidate the mechanisms involved in early events in Chlamydia trachomatis infection, we conducted a large scale unbiased RNA interference screen in Drosophila melanogaster S2 cells. This allowed identification of candidate host factors in a simple non-redundant, genetically tractable system. From a library of 7,216 double stranded RNAs (dsRNA, we identified approximately 226 host genes, including two tyrosine kinases, Abelson (Abl kinase and PDGF- and VEGF-receptor related (Pvr, a homolog of the Platelet-derived growth factor receptor (PDGFR. We further examined the role of these two kinases in C. trachomatis binding and internalization into mammalian cells. Both kinases are phosphorylated upon infection and recruited to the site of bacterial attachment, but their roles in the infectious process are distinct. We provide evidence that PDGFRbeta may function as a receptor, as inhibition of PDGFRbeta by RNA interference or by PDGFRbeta neutralizing antibodies significantly reduces bacterial binding, whereas depletion of Abl kinase has no effect on binding. Bacterial internalization can occur through activation of PDGFRbeta or through independent

C6/36 Aedes albopictus cells have a dysfunctional antiviral RNA interference response.

Directory of Open Access Journals (Sweden)

Doug E Brackney

2010-10-01

Full Text Available Mosquitoes rely on RNA interference (RNAi as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV infection in C6/36 (Aedes albopictus cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses. Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae, Sindbis virus (SINV, Togaviridae and La Crosse virus (LACV, Bunyaviridae and total RNA recovered from cell lysates. Small RNA (sRNA libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26-27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand and distribution (position along viral genome of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level.

Endogenous lentivirus in Malayan colugo (Galeopterus variegatus), a close relative of primates

Czech Academy of Sciences Publication Activity Database

Hron, Tomáš; Fábryová, Helena; Pačes, Jan; Elleder, Daniel

2014-01-01

Roč. 11, č. 84 (2014) ISSN 1742-4690 R&D Projects: GA MŠk(CZ) LK11215 Grant - others:GA MŠk(CZ) LM2010005 Institutional support: RVO:68378050 Keywords : Endogenous lentiviruses * Dermoptera * Paleovirology Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.185, year: 2014

Pulmonary Delivery of siRNA via Polymeric Vectors as Therapies of Asthma.

Science.gov (United States)

Xie, Yuran; Merkel, Olivia M

2015-10-01

Asthma is a chronic inflammatory disease. Despite the fact that current therapies, such as the combination of inhaled corticosteroids and β2-agonists, can control the symptoms of asthma in most patients, there is still an urgent need for an alternative anti-inflammatory therapy for patients who suffer from severe asthma but lack acceptable response to conventional therapies. Many molecular factors are involved in the inflammatory process in asthma, and thus blocking the function of these factors could efficiently alleviate airway inflammation. RNA interference (RNAi) is often thought to be the answer in the search for more efficient and biocompatible treatments. However, difficulties of efficient delivery of small interference RNA (siRNA), the key factor in RNAi, to target cells and tissues have limited its clinical application. In this review, we summarize cytokines and chemokines, transcription factors, tyrosine kinases, and costimulatory factors that have been reported as targets of siRNA-mediated treatment in experimental asthma. Additionally, we conclude several targeted delivery systems of siRNA to specific cells such as T cells, macrophages, and dendritic cells, which could potentially be applied in asthma therapy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

Energy Technology Data Exchange (ETDEWEB)

Li, Jie [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China); Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Yang, Xi-fei [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Ren, Xiao-hu [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China); Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Meng, Xiao-jing [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China); Huang, Hai-yan [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Zhao, Qiong-hui [Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen (China); Yuan, Jian-hui; Hong, Wen-xu; Xia, Bo; Huang, Xin-feng; Zhou, Li [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Liu, Jian-jun, E-mail: bio-research@hotmail.com [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Zou, Fei, E-mail: zoufei616@163.com [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China)

2014-10-10

Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we explore the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.

Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

International Nuclear Information System (INIS)

Li, Jie; Yang, Xi-fei; Ren, Xiao-hu; Meng, Xiao-jing; Huang, Hai-yan; Zhao, Qiong-hui; Yuan, Jian-hui; Hong, Wen-xu; Xia, Bo; Huang, Xin-feng; Zhou, Li; Liu, Jian-jun; Zou, Fei

2014-01-01

Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we explore the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer

MiRNA-486 regulates angiogenic activity and survival of mesenchymal stem cells under hypoxia through modulating Akt signal

Energy Technology Data Exchange (ETDEWEB)

Shi, Xue-Feng [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Department of Respiration, Qinghai Provincial People' s Hospital, Xining (China); Wang, Hua; Xiao, Feng-Jun [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Yin, Yue [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Department of Hematology, Peking University First Hospital, Beijing (China); Xu, Qin-Qin [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China); Ge, Ri-Li, E-mail: geriligao@hotmail.com [High Altitude Medicine of Ministry of Chinese Education and Research Center for High Altitude Medicine, Qinghai University, Xining 810001 (China); Wang, Li-Sheng, E-mail: wangls@bmi.ac.cn [Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850 (China)

2016-02-12

MicroRNA-486 (miR-486) was first identified from human fetal liver cDNA library and validated as a regulator of hematopoiesis. Its roles in regulating the biological function of bone marrow-derived mesnechymal stem cells (BM-MSCs) under hypoxia have not been explored yet. In this study, we demonstrated that exposure to hypoxia upregulates miR-486 expression in BM-MSCs. Lentivirus-mediated overexpression of miR-486 resulted in increase of hepatocyte growth factor (HGF) and vascular endothelial growth factor(VEGF) in both mRNA and protein levels. MiR-486 expression also promotes proliferation and reduces apoptosis of BM-MSCs. Whereas MiR-486 knockdown downregulated the secretion of HGF and VEGF and induced apoptosis of BM-MSCs. Furthermore, PTEN-PI3K/AKT signaling was validated to be involved in changes of BM-MSC biological functions regulated by miR-486. These results suggested that MiR-486 mediated the hypoxia-induced angiogenic activity and promoted the proliferation and survival of BM-MSCs through regulating PTEN-PI3K/AKT signaling. These findings might provide a novel understanding of effective therapeutic strategy for hypoxic-ischemic diseases. - Highlights: • miR-486 is a hypoxia-induced miRNA. • miR-486 regulates the secretion of HGF and VEGF, promotes proliferation, and inhibits apoptosis of BM-MSCs. • miR-486 enhances PI3K/AKT activity signaling by targeting PTEN molecule.

CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

Science.gov (United States)

Busi, Florent; Cresteil, Thierry

2005-09-01

The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

Mediating and moderating effects of work-home interference upon farm stresses and psychological distress.

Science.gov (United States)

McShane, Connar Jo; Quirk, Frances

2009-10-01

This study investigated whether work-home (WHI) or home-work interference (HWI) explained or affected the strength of the relationship between farmers' stresses and reported psychological distress. Distribution of questionnaire package; included Work-Home Conflict Scale, Farm Stress Survey, Depression Anxiety Stress Scale. Participants recruited via advertising in newsletters and newspapers, and distribution through businesses and meetings. The majority of farmers (N = 51, male = 45, female = 5) were recruited from the one district. Farmers were individuals who identified their occupation as a farm owner, farm manager, or farm hand. It was predicted farmers would report higher levels of WHI than HWI; time, a determinant of interference, would mediate the relationship between farmers' stresses and psychological distress; WHI and HWI would moderate farmers' stresses and their psychological distress; overall reported level of psychological distress would be in normal to mild range because of positive general economic conditions. Farmers reported significantly higher levels of WHI than HWI (M = 3.21, M = 2.76, P stresses and psychological distress, particularly anxiety. WHI, time and strain, determinants of WHI mediated personal finances and subcomponents of psychological distress (stress, anxiety, depression). Time-based HWI mediated personal finances and stress. No moderating effects were found for WHI (r = -0.02, P = 0.882) or HWI (r = 0.15, P = 0.306). Farmers of this specific sample presented a unique work-home interface. Limitations include the small sample size, recruitment methods, and culturally irrelevant measures as well as only assessing work-related stresses. Future research should aim to develop measures appropriate for farmers of Australia.

Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate

Directory of Open Access Journals (Sweden)

Chih-Hong Lou

2016-06-01

Full Text Available Nonsense-mediated RNA decay (NMD is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

G-protein-coupled receptor 137 accelerates proliferation of urinary bladder cancer cells in vitro.

Science.gov (United States)

Du, Yiheng; Bi, Wenhuan; Zhang, Fei; Wu, Wenbo; Xia, Shujie; Liu, Haitao

2015-01-01

Urinary bladder cancer is a worldwide concern because of its level of incidence and recurrence. To search an effective therapeutic strategy for urinary bladder cancer, it is important to identify proteins involved in tumorigenesis that could serve as potential targets for diagnosis and treatment. G-protein-coupled receptors (GPRs) constitute a large protein family of receptors that sense molecules outside the cell and activate signal transduction pathways and cellular responses inside the cell. GPR137 is a newly discovered human gene encoding orphan GPRs. In this study, we aimed to investigate the physiological role of GPR137 in urinary bladder cancer. The effect of GPR137 on cell growth was examined via an RNA interference (RNAi) lentivirus system in two human urinary bladder cancer cell lines BT5637 and T24. Lentivirus-mediated RNAi could specifically suppressed GPR137 expression in vitro, resulting in alleviated cell viability and impaired colony formation, as well as blocks G0/G1 and S phases of the cell cycle. These results suggested GPR137 as an essential player in urinary bladder cancer cell growth, and it may serve as a potential target for gene therapy in the treatment of urinary bladder cancer. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

A surface-mediated siRNA delivery system developed with chitosan/hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly

Energy Technology Data Exchange (ETDEWEB)

Wu, Lijuan [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China); Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Wu, Changlin, E-mail: Ph.Dclwu1314@sina.cn [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China); Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Liu, Guangwan [Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Liao, Nannan [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China); Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Zhao, Fang; Yang, Xuxia; Qu, Hongyuan [Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Peng, Bo [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China); Chen, Li [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China); Suzhou Novovita Bio-products Co., Ltd., Suzhou 215300 (China); Yang, Guang [Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai 200062 (China)

2016-12-15

Highlights: • We prepared Chitosan/Hyaluronic acid-siRNA multilayer as carrier to effectively load and protect siRNAs. • The stability and integrity of the siRNA was verified in the siRNA-loaded films. • The siRNA-loaded films showed good cells adhesion and gene silencing effect in eGFP-HEK 293T cells. • This is a new type of surface-mediated non-viral multilayer films. - Abstract: siRNA delivery remains highly challenging because of its hydrophilic and anionic nature and its sensitivity to nuclease degradation. Effective siRNA loading and improved transfection efficiency into cells represents a key problem. In our study, we prepared Chitosan/Hyaluronic acid-siRNA multilayer films through layer-by-layer self-assembly, in which siRNAs can be effectively loaded and protected. The construction process was characterized by FTIR, {sup 13}C NMR (CP/MAS), UV–vis spectroscopy, and atomic force microscopy (AFM). We presented the controlled-release performance of the films during incubation in 1 M NaCl solution for several days through UV–vis spectroscopy and polyacrylamide gel electrophoresis (PAGE). Additionally, we verified the stability and integrity of the siRNA loaded on multilayer films. Finally, the biological efficacy of the siRNA delivery system was evaluated via cells adhesion and gene silencing analyses in eGFP-HEK 293T cells. This new type of surface-mediated non-viral multilayer films may have considerable potential in the localized and controlled-release delivery of siRNA in mucosal tissues, and tissue engineering application.

Regulation of gene expression in neuronal tissue by RNA interference and editing

DEFF Research Database (Denmark)

Venø, Morten Trillingsgaard

No tissue in the mammalian organism is more complex than the brain. This complexity is in part the result of precise timing and interplay of a large number mechanisms modulating gene expression post-transcriptionally. Fine-tuning mechanisms such as A-to-I editing of RNA transcripts and regulation...... mediated by microRNAs are crucial for the correct function of the mammalian brain. We are addressing A-to-I editing and regulation by microRNAs with spatio-temporal resolution in the embryonic porcine brain by Solexa sequencing of microRNAs and 454 sequencing of edited neuronal messenger RNAs, resulting...... in detailed data of both of these fine-tuning mechanisms in the embryonic development of the pig. Editing levels of transcripts examined are generally seen to increase through development, in agreement with editing of specific microRNA also examined in the Solexa sequencing study. Three studies examining...

Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice.

Science.gov (United States)

Judge, Adam D; Robbins, Marjorie; Tavakoli, Iran; Levi, Jasna; Hu, Lina; Fronda, Anna; Ambegia, Ellen; McClintock, Kevin; MacLachlan, Ian

2009-03-01

siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in both hepatic and subcutaneous tumor models. This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis. Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. Additionally, RNAi-specific mRNA cleavage products were found in tumor cells, and their presence correlated with the duration of target mRNA silencing. Histological biomarkers confirmed that RNAi-mediated gene silencing effectively inhibited the target's biological activity. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.

Micro-RNA 10a Is Increased in Feline T Regulatory Cells and Increases Foxp3 Protein Expression Following In Vitro Transfection

Directory of Open Access Journals (Sweden)

Yan Wang

2017-02-01

Full Text Available CD4+CD25+Foxp3+ T regulatory (Treg cells are activated during the course of lentiviral infection and exhibit heightened suppressor function when compared to Treg cells from uninfected controls. Foxp3 is essential to Treg cell function and multiple studies have documented that lentivirus-activated Treg cells exhibit heightened Foxp3 expression when compared to Treg cells from uninfected controls. Our hypothesis was that lentivirus-induced micro-RNAs (miRNAs contribute to heightened Treg cell suppressor function by stabilizing Foxp3 expression. We demonstrated that CD4+CD25+ T cells from both feline immunodeficiency virus infected (FIV+ cats and uninfected control cats exhibit increased miRNA 10a and 21 levels compared to autologous CD4+CD25− T cells but there was no difference in the levels of these miRNAs when Treg cells from FIV+ cats were compared to Treg cells from uninfected controls. Further, there was no increase in Foxp3 mRNA following transfection of miRNA 10a or 21 into a feline cell line. However, transfection with miRNA 10a resulted in increased Foxp3 protein expression.

RNA interference in plant parasitic nematodes

African Journals Online (AJOL)

STORAGESEVER

2008-08-04

Aug 4, 2008 ... grower preference or by government restrictions to limit the environmental ... risks associated with chemical control and (c) the pro- vision of ... certain model organisms. The first ... reproductive system (Lilley et al., 2005b), sperm (Urwin .... interference of dual oxidase in the plant nematode Meloidogyne.

RNA interference inhibits herpes simplex virus type 1 isolated from saliva samples and mucocutaneous lesions.

Science.gov (United States)

Silva, Amanda Perse da; Lopes, Juliana Freitas; Paula, Vanessa Salete de

2014-01-01

The aim of this study was to evaluate the use of RNA interference to inhibit herpes simplex virus type-1 replication in vitro. For herpes simplex virus type-1 gene silencing, three different small interfering RNAs (siRNAs) targeting the herpes simplex virus type-1 UL39 gene (sequence si-UL 39-1, si-UL 39-2, and si-UL 39-3) were used, which encode the large subunit of ribonucleotide reductase, an essential enzyme for DNA synthesis. Herpes simplex virus type-1 was isolated from saliva samples and mucocutaneous lesions from infected patients. All mucocutaneous lesions' samples were positive for herpes simplex virus type-1 by real-time PCR and by virus isolation; all herpes simplex virus type-1 from saliva samples were positive by real-time PCR and 50% were positive by virus isolation. The levels of herpes simplex virus type-1 DNA remaining after siRNA treatment were assessed by real-time PCR, whose results demonstrated that the effect of siRNAs on gene expression depends on siRNA concentration. The three siRNA sequences used were able to inhibit viral replication, assessed by real-time PCR and plaque assays and among them, the sequence si-UL 39-1 was the most effective. This sequence inhibited 99% of herpes simplex virus type-1 replication. The results demonstrate that silencing herpes simplex virus type-1 UL39 expression by siRNAs effectively inhibits herpes simplex virus type-1 replication, suggesting that siRNA based antiviral strategy may be a potential therapeutic alternative. Copyright © 2014. Published by Elsevier Editora Ltda.

Bombyx mori histone methyltransferase BmAsh2 is essential for silkworm piRNA-mediated sex determination.

Science.gov (United States)

Li, Zhiqian; You, Lang; Yan, Dong; James, Anthony A; Huang, Yongping; Tan, Anjiang

2018-02-01

Sex determination is a hierarchically-regulated process with high diversity in different organisms including insects. The W chromosome-derived Fem piRNA has been identified as the primary sex determination factor in the lepidopteran insect, Bombyx mori, revealing a distinctive piRNA-mediated sex determination pathway. However, the comprehensive mechanism of silkworm sex determination is still poorly understood. We show here that the silkworm PIWI protein BmSiwi, but not BmAgo3, is essential for silkworm sex determination. CRISPR/Cas9-mediated depletion of BmSiwi results in developmental arrest in oogenesis and partial female sexual reversal, while BmAgo3 depletion only affects oogenesis. We identify three histone methyltransferases (HMTs) that are significantly down-regulated in BmSiwi mutant moths. Disruption one of these, BmAsh2, causes dysregulation of piRNAs and transposable elements (TEs), supporting a role for it in the piRNA signaling pathway. More importantly, we find that BmAsh2 mutagenesis results in oogenesis arrest and partial female-to-male sexual reversal as well as dysregulation of the sex determination genes, Bmdsx and BmMasc. Mutagenesis of other two HMTs, BmSETD2 and BmEggless, does not affect piRNA-mediated sex determination. Histological analysis and immunoprecipitation results support a functional interaction between the BmAsh2 and BmSiwi proteins. Our data provide the first evidence that the HMT, BmAsh2, plays key roles in silkworm piRNA-mediated sex determination.

Bombyx mori histone methyltransferase BmAsh2 is essential for silkworm piRNA-mediated sex determination.

Directory of Open Access Journals (Sweden)

Zhiqian Li

2018-02-01

Full Text Available Sex determination is a hierarchically-regulated process with high diversity in different organisms including insects. The W chromosome-derived Fem piRNA has been identified as the primary sex determination factor in the lepidopteran insect, Bombyx mori, revealing a distinctive piRNA-mediated sex determination pathway. However, the comprehensive mechanism of silkworm sex determination is still poorly understood. We show here that the silkworm PIWI protein BmSiwi, but not BmAgo3, is essential for silkworm sex determination. CRISPR/Cas9-mediated depletion of BmSiwi results in developmental arrest in oogenesis and partial female sexual reversal, while BmAgo3 depletion only affects oogenesis. We identify three histone methyltransferases (HMTs that are significantly down-regulated in BmSiwi mutant moths. Disruption one of these, BmAsh2, causes dysregulation of piRNAs and transposable elements (TEs, supporting a role for it in the piRNA signaling pathway. More importantly, we find that BmAsh2 mutagenesis results in oogenesis arrest and partial female-to-male sexual reversal as well as dysregulation of the sex determination genes, Bmdsx and BmMasc. Mutagenesis of other two HMTs, BmSETD2 and BmEggless, does not affect piRNA-mediated sex determination. Histological analysis and immunoprecipitation results support a functional interaction between the BmAsh2 and BmSiwi proteins. Our data provide the first evidence that the HMT, BmAsh2, plays key roles in silkworm piRNA-mediated sex determination.

Hypothesis: A Role for Fragile X Mental Retardation Protein in Mediating and Relieving MicroRNA-Guided Translational Repression?

Directory of Open Access Journals (Sweden)

Isabelle Plante

2006-01-01

Full Text Available MicroRNA (miRNA-guided messenger RNA (mRNA translational repression is believed to be mediated by effector miRNA-containing ribonucleoprotein (miRNP complexes harboring fragile X mental retardation protein (FMRP. Recent studies documented the nucleic acid chaperone properties of FMRP and characterized its role and importance in RNA silencing in mammalian cells. We propose a model in which FMRP could facilitate miRNA assembly on target mRNAs in a process involving recognition of G quartet structures. Functioning within a duplex miRNP, FMRP may also mediate mRNA targeting through a strand exchange mechanism, in which the miRNA* of the duplex is swapped for the mRNA. Furthermore, FMRP may contribute to the relief of miRNA-guided mRNA repression through a reverse strand exchange reaction, possibly initiated by a specific cellular signal, that would liberate the mRNA for translation. Suboptimal utilization of miRNAs may thus account for some of themolecular defects in patients with the fragile X syndrome.

AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system.

Science.gov (United States)

Schuck, Jana; Gursinsky, Torsten; Pantaleo, Vitantonio; Burgyán, Jozsef; Behrens, Sven-Erik

2013-05-01

AGO/RISC-mediated antiviral RNA silencing, an important component of the plant's immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (-)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.

The brain cytoplasmic RNA BC1 regulates dopamine D2 receptor-mediated transmission in the striatum.

Science.gov (United States)

Centonze, Diego; Rossi, Silvia; Napoli, Ilaria; Mercaldo, Valentina; Lacoux, Caroline; Ferrari, Francesca; Ciotti, Maria Teresa; De Chiara, Valentina; Prosperetti, Chiara; Maccarrone, Mauro; Fezza, Filomena; Calabresi, Paolo; Bernardi, Giorgio; Bagni, Claudia

2007-08-15

Dopamine D(2) receptor (D(2)DR)-mediated transmission in the striatum is remarkably flexible, and changes in its efficacy have been heavily implicated in a variety of physiological and pathological conditions. Although receptor-associated proteins are clearly involved in specific forms of synaptic plasticity, the molecular mechanisms regulating the sensitivity of D(2) receptors in this brain area are essentially obscure. We have studied the physiological responses of the D(2)DR stimulations in mice lacking the brain cytoplasmic RNA BC1, a small noncoding dendritically localized RNA that is supposed to play a role in mRNA translation. We show that the efficiency of D(2)-mediated transmission regulating striatal GABA synapses is under the control of BC1 RNA, through a negative influence on D(2) receptor protein level affecting the functional pool of receptors. Ablation of the BC1 gene did not result in widespread dysregulation of synaptic transmission, because the sensitivity of cannabinoid CB(1) receptors was intact in the striatum of BC1 knock-out (KO) mice despite D(2) and CB(1) receptors mediated similar electrophysiological actions. Interestingly, the fragile X mental retardation protein FMRP, one of the multiple BC1 partners, is not involved in the BC1 effects on the D(2)-mediated transmission. Because D(2)DR mRNA is apparently equally translated in the BC1-KO and wild-type mice, whereas the protein level is higher in BC1-KO mice, we suggest that BC1 RNA controls D(2)DR indirectly, probably regulating translation of molecules involved in D(2)DR turnover and/or stability.

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing.

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Koralewska, Natalia; Hoffmann, Weronika; Pokornowska, Maria; Milewski, Marek; Lipinska, Andrea; Bienkowska-Szewczyk, Krystyna; Figlerowicz, Marek; Kurzynska-Kokorniak, Anna

2016-01-01

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

RNA interference screen to identify pathways that enhance or reduce nonviral gene transfer during lipofection.

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Barker, Gregory A; Diamond, Scott L

2008-09-01

Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR-validated knockdown of approximately 76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.

Musashi-2 Silencing Exerts Potent Activity against Acute Myeloid Leukemia and Enhances Chemosensitivity to Daunorubicin.

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Yixiang Han

Full Text Available RNA-binding protein Musashi-2 (Msi2 is known to play a critical role in leukemogenesis and contributes to poor clinical prognosis in acute myeloid leukemia (AML. However, the effect of Msi2 silencing on treatment for AML still remains poorly understood. In this study, we used lentivirus-mediated RNA interference targeting Msi2 to investigate the resulting changes in cellular processes and the underlying mechanisms in AML cell lines as well as primary AML cells isolated from AML patients. We found that Msi2 was highly expressed in AML cells, and its depletion inhibited Ki-67 expression and resulted in decreased in vitro and in vivo proliferation. Msi2 silencing induced cell cycle arrest in G0/G1 phase, with decreased Cyclin D1 and increased p21 expression. Msi2 silencing induced apoptosis through down-regulation of Bcl-2 expression and up-regulation of Bax expression. Suppression of Akt, Erk1/2 and p38 phosphorylation also contributed to apoptosis mediated by Msi2 silencing. Finally, Msi2 silencing in AML cells also enhanced their chemosensitivity to daunorubicin. Conclusively, our data suggest that Msi2 is a promising target for gene therapy to optimize conventional chemotherapeutics in AML treatment.

Illuminating the gateway of gene silencing: perspective of RNA interference technology in clinical therapeutics.

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Sindhu, Annu; Arora, Pooja; Chaudhury, Ashok

2012-07-01

A novel laboratory revolution for disease therapy, the RNA interference (RNAi) technology, has adopted a new era of molecular research as the next generation "Gene-targeted prophylaxis." In this review, we have focused on the chief technological challenges associated with the efforts to develop RNAi-based therapeutics that may guide the biomedical researchers. Many non-curable maladies, like neurodegenerative diseases and cancers have effectively been cured using this technology. Rapid advances are still in progress for the development of RNAi-based technologies that will be having a major impact on medical research. We have highlighted the recent discoveries associated with the phenomenon of RNAi, expression of silencing molecules in mammals along with the vector systems used for disease therapeutics.

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

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2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

Epigallocatechin Gallate-Mediated Alteration of the MicroRNA Expression Profile in 5α-Dihydrotestosterone-Treated Human Dermal Papilla Cells.

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Shin, Shanghun; Kim, Karam; Lee, Myung Joo; Lee, Jeongju; Choi, Sungjin; Kim, Kyung-Suk; Ko, Jung-Min; Han, Hyunjoo; Kim, Su Young; Youn, Hae Jeong; Ahn, Kyu Joong; An, In-Sook; An, Sungkwan; Cha, Hwa Jun

2016-06-01

Dihydrotestosterone (DHT) induces androgenic alopecia by shortening the hair follicle growth phase, resulting in hair loss. We previously demonstrated how changes in the microRNA (miRNA) expression profile influenced DHT-mediated cell death, cell cycle arrest, cell viability, the generation of reactive oxygen species (ROS), and senescence. Protective effects against DHT have not, however, been elucidated at the genome level. We showed that epigallocatechin gallate (EGCG), a major component of green tea, protects DHT-induced cell death by regulating the cellular miRNA expression profile. We used a miRNA microarray to identify miRNA expression levels in human dermal papilla cells (DPCs). We investigated whether the miRNA expression influenced the protective effects of EGCG against DHT-induced cell death, growth arrest, intracellular ROS levels, and senescence. EGCG protected against the effects of DHT by altering the miRNA expression profile in human DPCs. In addition, EGCG attenuated DHT-mediated cell death and growth arrest and decreased intracellular ROS levels and senescence. A bioinformatics analysis elucidated the relationship between the altered miRNA expression and EGCG-mediated protective effects against DHT. Overall, our results suggest that EGCG ameliorates the negative effects of DHT by altering the miRNA expression profile in human DPCs.

Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex.

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Robinson, Philip J; Trnka, Michael J; Bushnell, David A; Davis, Ralph E; Mattei, Pierre-Jean; Burlingame, Alma L; Kornberg, Roger D

2016-09-08

A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription. Copyright © 2016 Elsevier Inc. All rights reserved.

RNA SURVEILLANCE– AN EMERGING ROLE FOR RNA REGULATORY NETWORKS IN AGING

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Montano, Monty; Long, Kimberly

2010-01-01

In this review, we describe recent advances in the field of RNA regulatory biology and relate these advances to aging science. We introduce a new term, RNA surveillance, an RNA regulatory process that is conserved in metazoans, and describe how RNA surveillance represents molecular cross-talk between two emerging RNA regulatory systems – RNA interference and RNA editing. We discuss how RNA surveillance mechanisms influence mRNA and microRNA expression and activity during lifespan. Additionall...

The unusual nucleotide content of the HIV RNA genome results in a biased amino acid composition of HIV proteins

NARCIS (Netherlands)

Berkhout, B.; van Hemert, F. J.

1994-01-01

Extremely high frequencies of the A nucleotide are found in the RNA genomes of the lentivirus group of retroviruses. It is presently unknown what molecular force is responsible for this A-pressure. In this manuscript, we demonstrate a correlation between this 'A-pressure' and the amino acid-usage of

Exosomes: Nanoparticulate tools for RNA interference and drug delivery.

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Shahabipour, Fahimeh; Barati, Nastaran; Johnston, Thomas P; Derosa, Giuseppe; Maffioli, Pamela; Sahebkar, Amirhossein

2017-07-01

Exosomes are naturally occurring extracellular vesicles released by most mammalian cells in all body fluids. Exosomes are known as key mediators in cell-cell communication and facilitate the transfer of genetic and biochemical information between distant cells. Structurally, exosomes are composed of lipids, proteins, and also several types of RNAs which enable these vesicles to serve as important disease biomarkers. Moreover, exosomes have emerged as novel drug and gene delivery tools owing to their multiple advantages over conventional delivery systems. Recently, increasing attention has been focused on exosomes for the delivery of drugs, including therapeutic recombinant proteins, to various target tissues. Exosomes are also promising vehicles for the delivery of microRNAs and small interfering RNAs, which is usually hampered by rapid degradation of these RNAs, as well as inefficient tissue specificity of currently available delivery strategies. This review highlights the most recent accomplishments and trends in the use of exosomes for the delivery of drugs and therapeutic RNA molecules. © 2017 Wiley Periodicals, Inc.

HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

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Valentina Vongrad

Full Text Available MiRNAs and other small noncoding RNAs (sncRNAs are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM.The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP, which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs.PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

Cellular Response to Ionizing Radiation: A MicroRNA Story

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Halimi, Mohammad; Asghari, S. Mohsen; Sariri, Reyhaneh; Moslemi, Dariush; Parsian, Hadi

2012-01-01

MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They play a crucial role in diverse cellular pathways. Ionizing radiation (IR) is one of the most important treatment protocols for patients that suffer from cancer and affects directly or indirectly cellular integration. Recently it has been discovered that microRNA-mediated gene regulation interferes with radio-related pathways in ionizing radiation. Here, we review the recent discoveries about miRNAs in cellular response to IR. Thoroughly understanding the mechanism of miRNAs in radiation response, it will be possible to design new strategies for improving radiotherapy efficiency and ultimately cancer treatment. PMID:24551775

Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice

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Shih, Yao-Ming; Sun, Cheng-Pu; Chou, Hui-Hsien; Wu, Tzu-Hui; Chen, Chun-Chi; Wu, Ping-Yi; Enya Chen, Yu-Chen; Bissig, Karl-Dimiter; Tao, Mi-Hua

2015-01-01

Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the “PICKY” software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients. PMID:26482836

Genome-wide association of mediator and RNA polymerase II in wild-type and mediator mutant yeast.

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Paul, Emily; Zhu, Z Iris; Landsman, David; Morse, Randall H

2015-01-01

Mediator is a large, multisubunit complex that is required for essentially all mRNA transcription in eukaryotes. In spite of the importance of Mediator, the range of its targets and how it is recruited to these is not well understood. Previous work showed that in Saccharomyces cerevisiae, Mediator contributes to transcriptional activation by two distinct mechanisms, one depending on the tail module triad and favoring SAGA-regulated genes, and the second occurring independently of the tail module and favoring TFIID-regulated genes. Here, we use chromatin immunoprecipitation sequencing (ChIP-seq) to show that dependence on tail module subunits for Mediator recruitment and polymerase II (Pol II) association occurs preferentially at SAGA-regulated over TFIID-regulated genes on a genome-wide scale. We also show that recruitment of tail module subunits to active gene promoters continues genome-wide when Mediator integrity is compromised in med17 temperature-sensitive (ts) yeast, demonstrating the modular nature of the Mediator complex in vivo. In addition, our data indicate that promoters exhibiting strong and stable occupancy by Mediator have a wide range of activity and are enriched for targets of the Tup1-Cyc8 repressor complex. We also identify a number of strong Mediator occupancy peaks that overlap dubious open reading frames (ORFs) and are likely to include previously unrecognized upstream activator sequences. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Exosome-mediated microRNA transfer plays a role in radiation-induced bystander effect.

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Xu, Shuai; Wang, Jufang; Ding, Nan; Hu, Wentao; Zhang, Xurui; Wang, Bing; Hua, Junrui; Wei, Wenjun; Zhu, Qiyun

2015-01-01

Bystander effects can be induced through cellular communication between irradiated cells and non-irradiated cells. The signals that mediate this cellular communication, such as cytokines, reactive oxygen species, nitric oxide and even microRNAs, can be transferred between cells via gap junctions or extracellular medium. We have previously reported that miR-21, a well described DDR (DNA damage response) microRNA, is involved in radiation-induced bystander effects through a medium-mediated way. However, the mechanisms of the microRNA transfer have not been elucidated in details. In the present study, it was found that exosomes isolated from irradiated conditioned medium could induce bystander effects. Furthermore, we demonstrated plenty of evidences that miR-21, which is up-regulated as a result of mimic transfection or irradiation, can be transferred from donor or irradiated cells into extracellular medium and subsequently get access to the recipient or bystander cells through exosomes to induce bystander effects. Inhibiting the miR-21 expression in advance can offset the bystander effects to some extent. From all of these results, it can be concluded that the exosome-mediated microRNA transfer plays an important role in the radiation-induced bystander effects. These findings provide new insights into the functions of microRNAs and the cellular communication between the directly irradiated cells and the non-irradiated cells.

RNA interference targeting carbohydrate sulfotransferase 3 diminishes macrophage accumulation, inhibits MMP-9 expression and promotes lung recovery in murine pulmonary emphysema.

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Kai, Yoshiro; Tomoda, Koichi; Yoneyama, Hiroyuki; Yoshikawa, Masanori; Kimura, Hiroshi

2015-12-09

Chondroitin sulfate proteoglycans are an important mediators in inflammation and leukocyte trafficking. However, their roles in pulmonary emphysema have not been explored. In a murine model of elastase-induced pulmonary emphysema, we found increased carbohydrate sulfotransferase 3 (CHST3), a specific enzyme that synthesizes chondroitin 6-sulfate proteoglycan (C6SPG). To elucidate the role of C6SPG, we investigated the effect of small interfering RNA (siRNA) targeting CHST3 that inhibits C6SPG-synthesis on the pathogenesis of pulmonary emphysema. Mice were intraperitoneally injected with CHST3 siRNA or negative control siRNA on day0 and 7 after intratracheal instillation of elastase. Histology, respiratory function, glycosaminoglycans (GAGs) content, bronchoalveolar lavage (BAL), elastin staining and gene expressions of tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMP)-9 mRNA were evaluated on day7 and/or day21. CHST3 mRNA increased at day 7 and decreased thereafter in lung. CHST3 siRNA successfully inhibited the expression of CHST3 mRNA throughout the study and this was associated with significant reduction of GAGs and C6SPG. Airway destruction and respiratory function were improved by the treatment with CHST3 siRNA. CHST3 siRNA reduced the number of macrophages both in BAL and lung parenchyma and also suppressed the increased expressions of TNF-α and MMP-9 mRNA. Futhermore, CHST3 siRNA improved the reduction of the elastin in the alveolar walls. CHST3 siRNA diminishes accumulation of excessive macrophages and the mediators, leading to accelerate the functional recovery from airway damage by repair of the elastin network associated with pulmonary emphysema.

RNA interference targeting cytosolic NADP(+)-dependent isocitrate dehydrogenase exerts anti-obesity effect in vitro and in vivo.

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Nam, Woo Suk; Park, Kwon Moo; Park, Jeen-Woo

2012-08-01

A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome. © 2012 Elsevier B.V. All rights reserved.

MicroRNA-Mediated Gene Silencing in Plant Defense and Viral Counter-Defense

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Sheng-Rui Liu

2017-09-01

Full Text Available MicroRNAs (miRNAs are non-coding RNAs of approximately 20–24 nucleotides in length that serve as central regulators of eukaryotic gene expression by targeting mRNAs for cleavage or translational repression. In plants, miRNAs are associated with numerous regulatory pathways in growth and development processes, and defensive responses in plant–pathogen interactions. Recently, significant progress has been made in understanding miRNA-mediated gene silencing and how viruses counter this defense mechanism. Here, we summarize the current knowledge and recent advances in understanding the roles of miRNAs involved in the plant defense against viruses and viral counter-defense. We also document the application of miRNAs in plant antiviral defense. This review discusses the current understanding of the mechanisms of miRNA-mediated gene silencing and provides insights on the never-ending arms race between plants and viruses.

Potential microRNA-mediated oncogenic intercellular communication revealed by pan-cancer analysis

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Li, Yue; Zhang, Zhaolei

2014-11-01

Carcinogenesis consists of oncogenesis and metastasis, and intriguingly microRNAs (miRNAs) are involved in both processes. Although aberrant miRNA activities are prevalent in diverse tumor types, the exact mechanisms for how they regulate cancerous processes are not always clear. To this end, we performed a large-scale pan-cancer analysis via a novel probabilistic approach to infer recurrent miRNA-target interactions implicated in 12 cancer types using data from The Cancer Genome Atlas. We discovered ~20,000 recurrent miRNA regulations, which are enriched for cancer-related miRNAs/genes. Notably, miRNA 200 family (miR-200/141/429) is among the most prominent miRNA regulators, which is known to be involved in metastasis. Importantly, the recurrent miRNA regulatory network is not only enriched for cancer pathways but also for extracellular matrix (ECM) organization and ECM-receptor interactions. The results suggest an intriguing cancer mechanism involving miRNA-mediated cell-to-cell communication, which possibly involves delivery of tumorigenic miRNA messengers to adjacent cells via exosomes. Finally, survival analysis revealed 414 recurrent-prognostic associations, where both gene and miRNA involved in each interaction conferred significant prognostic power in one or more cancer types. Together, our comprehensive pan-cancer analysis provided not only biological insights into metastasis but also brought to bear the clinical relevance of the proposed recurrent miRNA-gene associations.

miR2Pathway: A Novel Analytical Method to Discover MicroRNA-mediated Dysregulated Pathways Involved in Hepatocellular Carcinoma.

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Li, Chaoxing; Dinu, Valentin

2018-03-22

MicroRNAs (miRNAs) are small, non-coding RNAs involved in the regulation of gene expression at a post-transcriptional level. Recent studies have shown miRNAs as key regulators of a variety of biological processes, such as proliferation, differentiation, apoptosis, metabolism, etc. Aberrantly expressed miRNAs influence individual gene expression level, but rewired miRNA-mRNA connections can influence the activity of biological pathways. Here, we define rewired miRNA-mRNA connections as the differential (rewiring) effects on the activity of biological pathways between hepatocellular carcinoma (HCC) and normal phenotypes. Our work presented here uses a PageRank-based approach to measure the degree of miRNA-mediated dysregulation of biological pathways between HCC and normal samples based on rewired miRNA-mRNA connections. In our study, we regard the degree of miRNA-mediated dysregulation of biological pathways as disease risk of biological pathways. Therefore, we propose a new method, miR2Pathway, to measure and rank the degree of miRNA-mediated dysregulation of biological pathways by measuring the total differential influence of miRNAs on the activity of pathways between HCC and normal states. miR2Pathway proposed here systematically shows the first evidence for a mechanism of biological pathways being dysregulated by rewired miRNA-mRNA connections, and provides new insight into exploring mechanisms behind HCC. Thus, miR2Pathway is a novel method to identify and rank miRNA-dysregulated pathways in HCC. Copyright © 2018. Published by Elsevier Inc.

EGF receptor targeted lipo-oligocation polyplexes for antitumoral siRNA and miRNA delivery

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Müller, Katharina; Klein, Philipp M.; Heissig, Philipp; Roidl, Andreas; Wagner, Ernst

2016-11-01

Antitumoral siRNA and miRNA delivery was demonstrated by epidermal growth factor receptor (EGFR) targeted oligoaminoamide polyplexes. For this purpose, the T-shaped lipo-oligomer 454 was used to complex RNA into a core polyplex, which was subsequently functionalized with the targeting peptide ligand GE11 via a polyethylene glycol (PEG) linker. To this end, free cysteines on the surface of 454 polyplex were coupled with a maleimide-PEG-GE11 reagent (Mal-GE11). Resulting particles with sizes of 120-150 nm showed receptor-mediated uptake into EGFR-positive T24 bladder cancer cells, MDA-MB 231 breast cancer cells and Huh7 liver cancer cells. Furthermore, these formulations led to ligand-dependent gene silencing. RNA interference (RNAi) triggered antitumoral effects were observed for two different therapeutic RNAs, a miRNA-200c mimic or EG5 siRNA. Using polyplexes modified with a ratio of 0.8 molar equivalents of Mal-GE11, treatment of T24 or MDA-MB 231 cancer cells with miR-200c led to the expected decreased proliferation and migration, changes in cell cycle and enhanced sensitivity towards doxorubicin. Delivery of EG5 siRNA into Huh7 cells resulted in antitumoral activity with G2/M arrest, triggered by loss of mitotic spindle separation and formation of mono-astral spindles. These findings demonstrate the potential of GE11 ligand-containing RNAi polyplexes for cancer treatment.

Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

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Eveline Kindler

2017-02-01

Full Text Available Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I. This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU activity is key to prevent early induction of double-stranded RNA (dsRNA host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

Evolutionary Analyses Suggest a Function of MxB Immunity Proteins Beyond Lentivirus Restriction.

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Patrick S Mitchell

2015-12-01

Full Text Available Viruses impose diverse and dynamic challenges on host defenses. Diversifying selection of codons and gene copy number variation are two hallmarks of genetic innovation in antiviral genes engaged in host-virus genetic conflicts. The myxovirus resistance (Mx genes encode interferon-inducible GTPases that constitute a major arm of the cell-autonomous defense against viral infection. Unlike the broad antiviral activity of MxA, primate MxB was recently shown to specifically inhibit lentiviruses including HIV-1. We carried out detailed evolutionary analyses to investigate whether genetic conflict with lentiviruses has shaped MxB evolution in primates. We found strong evidence for diversifying selection in the MxB N-terminal tail, which contains molecular determinants of MxB anti-lentivirus specificity. However, we found no overlap between previously-mapped residues that dictate lentiviral restriction and those that have evolved under diversifying selection. Instead, our findings are consistent with MxB having a long-standing and important role in the interferon response to viral infection against a broader range of pathogens than is currently appreciated. Despite its critical role in host innate immunity, we also uncovered multiple functional losses of MxB during mammalian evolution, either by pseudogenization or by gene conversion from MxA genes. Thus, although the majority of mammalian genomes encode two Mx genes, this apparent stasis masks the dramatic effects that recombination and diversifying selection have played in shaping the evolutionary history of Mx genes. Discrepancies between our study and previous publications highlight the need to account for recombination in analyses of positive selection, as well as the importance of using sequence datasets with appropriate depth of divergence. Our study also illustrates that evolutionary analyses of antiviral gene families are critical towards understanding molecular principles that govern host

Cyclic-AMP mediated regulation of ABCB mRNA expression in mussel haemocytes.

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Silvia Franzellitti

Full Text Available BACKGROUND: The multixenobiotic resistance system (MXR allows aquatic organisms to cope with their habitat despite high pollution levels by over-expressing membrane and intracellular transporters, including the P-glycoprotein (Pgp. In mammals transcription of the ABCB1 gene encoding Pgp is under cAMP/PKA-mediated regulation; whether this is true in mollusks is not fully clarified. METHODOLOGY/PRINCIPAL FINDINGS: cAMP/PKA regulation and ABCB mRNA expression were assessed in haemocytes from Mediterranean mussels (Mytilus galloprovincialis exposed in vivo for 1 week to 0.3 ng/L fluoxetine (FX alone or in combination with 0.3 ng/L propranolol (PROP. FX significantly decreased cAMP levels and PKA activity, and induced ABCB mRNA down-regulation. FX effects were abolished in the presence of PROP. In vitro experiments using haemocytes treated with physiological agonists (noradrenaline and serotonin and pharmacological modulators (PROP, forskolin, dbcAMP, and H89 of the cAMP/PKA system were performed to obtain clear evidence about the involvement of the signaling pathway in the transcriptional regulation of ABCB. Serotonin (5-HT decreased cAMP levels, PKA activity and ABCB mRNA expression but increased the mRNA levels for a putative 5-HT1 receptor. Interestingly, 5-HT1 was also over-expressed after in vivo exposures to FX. 5-HT effects were counteracted by PROP. Forskolin and dbcAMP increased PKA activity as well as ABCB mRNA expression; the latter effect was abolished in the presence of the PKA inhibitor H89. CONCLUSIONS: This study provides the first direct evidence for the cAMP/PKA-mediated regulation of ABCB transcription in mussels.

Advances in RNA interference technology and its impact on nutritional improvement, disease and insect control in plants.

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Katoch, Rajan; Thakur, Neelam

2013-03-01

This review highlights the advances in the knowledge of RNA interference (RNAi) and discusses recent progress on the functionality of different components RNAi machinery operating in the organisms. The silencing of genes by RNA interference has become the technology of choice for investigation of gene functions in different organisms. The refinement in the knowledge of the endogenous RNAi pathways in plants along with the development of new strategies and applications for the improvement of nutritional value of important agricultural crops through suppression of genes in different plants have opened new vistas for nutritional security. The improvement in the nutritional status of the plants and reduction in the level of toxins or antinutrients was desired for long, but the available technology was not completely successful in achieving the tissue specific regulation of some genes. In the recent years, a number of economically important crop plants have been tested successfully for improving plant nutritional value through metabolic engineering using RNAi. The implications of this technology for crop improvement programs, including nutritional enrichment, reduction of antinutrients, disease, and insect control have been successfully tested in variety of crops with commercial considerations. The enhancement of the nutraceutical traits for the desired health benefits in common crop plants through manipulation of gene expression has been elaborated in this article. The tremendous potential with RNAi technology is expected to revolutionize the modern agriculture for meeting the growing challenges is discussed.

Engineered disease resistance in cotton using RNA-interference to knock down cotton leaf curl kokhran virus-Burewala and cotton leaf curl Multan betasatellite

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Cotton Leaf Curl virus Disease (CLCuD) has caused enormous losses in cotton (Gossypium hirsutum) production in Pakistan. RNA interference (RNAi) is an emerging technique that could knock out CLCuD by targeting different regions of the pathogen genome that are important for replication, transcription...

Kinetics of lipid-nanoparticle-mediated intracellular mRNA delivery and function

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Zhdanov, Vladimir P.

2017-10-01

mRNA delivery into cells forms the basis for one of the new and promising ways to treat various diseases. Among suitable carriers, lipid nanoparticles (LNPs) with a size of about 100 nm are now often employed. Despite high current interest in this area, the understanding of the basic details of LNP-mediated mRNA delivery and function is limited. To clarify the kinetics of mRNA release from LNPs, the author uses three generic models implying (i) exponential, (ii) diffusion-controlled, and (iii) detachment-controlled kinetic regimes, respectively. Despite the distinct differences in these kinetics, the associated transient kinetics of mRNA translation to the corresponding protein and its degradation are shown to be not too sensitive to the details of the mRNA delivery by LNPs (or other nanocarriers). In addition, the author illustrates how this protein may temporarily influence the expression of one gene or a few equivalent genes. The analysis includes positive or negative regulation of the gene transcription via the attachment of the protein without or with positive or negative feedback in the gene expression. Stable, bistable, and oscillatory schemes have been scrutinized in this context.

Cooler temperatures destabilize RNA interference and increase susceptibility of disease vector mosquitoes to viral infection.

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Zach N Adelman

Full Text Available The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus, exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery.We utilized transgenic "sensor" strains of Aedes aegypti to examine the role of temperature on RNA silencing. These "sensor" strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2 or Argonaute-2 (AGO-2. We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18°C as compared with 28°C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA trigger for RNAi, showed no change in EGFP expression when reared at 18°C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses.This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.

Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase.

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Watashi, Koichi; Ishii, Naoto; Hijikata, Makoto; Inoue, Daisuke; Murata, Takayuki; Miyanari, Yusuke; Shimotohno, Kunitada

2005-07-01

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.

Chimeric peptide-mediated siRNA transduction to inhibit HIV-1 infection.

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Bivalkar-Mehla, Shalmali; Mehla, Rajeev; Chauhan, Ashok

2017-04-01

Persistent human immunodeficiency virus 1 (HIV-1) infection provokes immune activation and depletes CD4 +  lymphocytes, leading to acquired immunodeficiency syndrome. Uninterrupted administration of combination antiretroviral therapy (cART) in HIV-infected patients suppresses viral replication to below the detectable level and partially restores the immune system. However, cART-unresponsive residual HIV-1 infection and elusive transcriptionally silent but reactivatable viral reservoirs maintain a permanent viral DNA blue print. The virus rebounds within a few weeks after interruption of suppressive therapy. Adjunct gene therapy to control viral replication by ribonucleic acid interference (RNAi) is a post-transcriptional gene silencing strategy that could suppress residual HIV-1 burden and overcome viral resistance. Small interfering ribonucleic acids (siRNAs) are efficient transcriptional inhibitors, but need delivery systems to reach inside target cells. We investigated the potential of chimeric peptide (FP-PTD) to deliver specific siRNAs to HIV-1-susceptible and permissive cells. Chimeric FP-PTD peptide was designed with an RNA binding domain (PTD) to bind siRNA and a cell fusion peptide domain (FP) to enter cells. FP-PTD-siRNA complex entered and inhibited HIV-1 replication in susceptible cells, and could be a candidate for in vivo testing.

Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis

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Olga Villamizar

2016-06-01

Full Text Available This paper describes data related to a research article titled, “Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death” [1]. Long noncoding RNAs (lncRNAs are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis. Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf described in the research article. Also included are 5′ untranslated sequences (UTR for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34+ cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34+ cells transduced using mock conditions or with lentivirus particles encoding for Saf.

α-Fetoprotein promoter-driven Cre/LoxP-switched RNA interference for hepatocellular carcinoma tissue-specific target therapy.

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Yuan-Fei Peng

Full Text Available RNA interference (RNAi has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1 was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3 and non-HCC cell lines (L-02, Hela and SW1116 were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5 was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.The AFP-miRNA system could silence target gene (Beclin 1 but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1 in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA was successfully established. The system provides a usable tool for HCC-specific RNAi

Differential Contribution of RNA Interference Components in Response to Distinct Fusarium graminearum Virus Infections.

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Yu, Jisuk; Lee, Kyung-Mi; Cho, Won Kyong; Park, Ju Yeon; Kim, Kook-Hyung

2018-05-01

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

RNA interference-based therapeutics: new strategies to fight infectious disease.

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López-Fraga, M; Wright, N; Jiménez, A

2008-12-01

For many years, there has been an ongoing search for new compounds that can selectively alter gene expression as a new way to treat human disease by addressing targets that are otherwise "undruggable" with traditional pharmaceutical approaches involving small molecules or proteins. RNA interference (RNAi) strategies have raised a lot of attention and several compounds are currently being tested in clinical trials. Viruses are the obvious target for RNAi-therapy, as most are difficult to treat with conventional drugs, they become rapidly resistant to drug treatment and their genes differ substantially from human genes, minimizing side effects. Antisense strategy offers very high target specificity, i.e., any viral sequence could potentially be targeted using the complementary oligonucleotide sequence. Consequently, new antisense-based therapeutics have the potential to lead a revolution in the anti-infective drug development field. Additionally, the relatively short turnaround for efficacy testing of potential RNAi molecules and that any pathogen is theoretically amenable to rapid targeting, make them invaluable tools for treating a wide range of diseases. This review will focus on some of the current efforts to treat infectious disease with RNAi-based therapies and some of the obstacles that have appeared on the road to successful clinical intervention.

Functional interactions of nucleocapsid protein of feline immunodeficiency virus and cellular prion protein with the viral RNA.

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Moscardini, Mila; Pistello, Mauro; Bendinelli, M; Ficheux, Damien; Miller, Jennifer T; Gabus, Caroline; Le Grice, Stuart F J; Surewicz, Witold K; Darlix, Jean-Luc

2002-04-19

All lentiviruses and oncoretroviruses examined so far encode a major nucleic-acid binding protein (nucleocapsid or NC* protein), approximately 2500 molecules of which coat the dimeric RNA genome. Studies on HIV-1 and MoMuLV using in vitro model systems and in vivo have shown that NC protein is required to chaperone viral RNA dimerization and packaging during virus assembly, and proviral DNA synthesis by reverse transcriptase (RT) during infection. The human cellular prion protein (PrP), thought to be the major component of the agent causing transmissible spongiform encephalopathies (TSE), was recently found to possess a strong affinity for nucleic acids and to exhibit chaperone properties very similar to HIV-1 NC protein in the HIV-1 context in vitro. Tight binding of PrP to nucleic acids is proposed to participate directly in the prion disease process. To extend our understanding of lentiviruses and of the unexpected nucleic acid chaperone properties of the human prion protein, we set up an in vitro system to investigate replication of the feline immunodeficiency virus (FIV), which is functionally and phylogenetically distant from HIV-1. The results show that in the FIV model system, NC protein chaperones viral RNA dimerization, primer tRNA(Lys,3) annealing to the genomic primer-binding site (PBS) and minus strand DNA synthesis by the homologous FIV RT. FIV NC protein is able to trigger specific viral DNA synthesis by inhibiting self-priming of reverse transcription. The human prion protein was found to mimic the properties of FIV NC with respect to primer tRNA annealing to the viral RNA and chaperoning minus strand DNA synthesis. Copyright 2002 Elsevier Science Ltd.

microRNA-mediated resistance to hypoglycemia in the HepG2 human hepatoma cell line

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Ueki, Satomi; Murakami, Yuko; Yamada, Shoji; Kimura, Masaki; Saito, Yoshimasa; Saito, Hidetsugu

2016-01-01

It is generally accepted that the energy resources of cancer cells rely on anaerobic metabolism or the glycolytic system, even if they have sufficient oxygen. This is known as the Warburg effect. The cells skillfully survive under hypoglycemic conditions when their circumstances change, which probably at least partly involves microRNA (miRNA)-mediated regulation. To determine how cancer cells exploit miRNA-mediated epigenetic mechanisms to survive in hypoglycemic conditions, we used DNA microarray analysis to comprehensively and simultaneously compare the expression of miRNAs and mRNAs in the HepG2 human hepatoma cell line and in cultured normal human hepatocytes. The hypoglycemic condition decreased the expression of miRNA-17-5p and -20a-5p in hepatoma cells and consequently upregulated the expression of their target gene p21. These regulations were also confirmed by using antisense inhibitors of these miRNAs. In addition to this change, the hypoglycemic condition led to upregulated expression of heat shock proteins and increased resistance to caspase-3-induced apoptosis. However, we could not identify miRNA-mediated regulations, despite using comprehensive detection. Several interesting genes were also found to be upregulated in the hypoglycemic condition by the microarray analysis, probably because of responding to this cellular stress. These results suggest that cancer cells skillfully survive in hypoglycemic conditions, which frequently occur in malignancies, and that some of the gene regulation of this process is manipulated by miRNAs. The online version of this article (doi:10.1186/s12885-016-2762-7) contains supplementary material, which is available to authorized users

The role of incoherent microRNA-mediated feedforward loops in noise buffering.

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Matteo Osella

2011-03-01

Full Text Available MicroRNAs are endogenous non-coding RNAs which negatively regulate the expression of protein-coding genes in plants and animals. They are known to play an important role in several biological processes and, together with transcription factors, form a complex and highly interconnected regulatory network. Looking at the structure of this network, it is possible to recognize a few overrepresented motifs which are expected to perform important elementary regulatory functions. Among them, a special role is played by the microRNA-mediated feedforward loop in which a master transcription factor regulates a microRNA and, together with it, a set of target genes. In this paper we show analytically and through simulations that the incoherent version of this motif can couple the fine-tuning of a target protein level with an efficient noise control, thus conferring precision and stability to the overall gene expression program, especially in the presence of fluctuations in upstream regulators. Among the other results, a nontrivial prediction of our model is that the optimal attenuation of fluctuations coincides with a modest repression of the target expression. This feature is coherent with the expected fine-tuning function and in agreement with experimental observations of the actual impact of a wide class of microRNAs on the protein output of their targets. Finally, we describe the impact on noise-buffering efficiency of the cross-talk between microRNA targets that can naturally arise if the microRNA-mediated circuit is not considered as isolated, but embedded in a larger network of regulations.

The Role of the Antiviral APOBEC3 Gene Family in Protecting Chimpanzees against Lentiviruses from Monkeys.

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Lucie Etienne

2015-09-01

Full Text Available Cross-species transmissions of viruses from animals to humans are at the origin of major human pathogenic viruses. While the role of ecological and epidemiological factors in the emergence of new pathogens is well documented, the importance of host factors is often unknown. Chimpanzees are the closest relatives of humans and the animal reservoir at the origin of the human AIDS pandemic. However, despite being regularly exposed to monkey lentiviruses through hunting, chimpanzees are naturally infected by only a single simian immunodeficiency virus, SIVcpz. Here, we asked why chimpanzees appear to be protected against the successful emergence of other SIVs. In particular, we investigated the role of the chimpanzee APOBEC3 genes in providing a barrier to infection by most monkey lentiviruses. We found that most SIV Vifs, including Vif from SIVwrc infecting western-red colobus, the chimpanzee's main monkey prey in West Africa, could not antagonize chimpanzee APOBEC3G. Moreover, chimpanzee APOBEC3D, as well as APOBEC3F and APOBEC3H, provided additional protection against SIV Vif antagonism. Consequently, lentiviral replication in primary chimpanzee CD4(+ T cells was dependent on the presence of a lentiviral vif gene that could antagonize chimpanzee APOBEC3s. Finally, by identifying and functionally characterizing several APOBEC3 gene polymorphisms in both common chimpanzees and bonobos, we found that these ape populations encode APOBEC3 proteins that are uniformly resistant to antagonism by monkey lentiviruses.

Lentiviral Vector Mediated Claudin1 Silencing Inhibits Epithelial to Mesenchymal Transition in Breast Cancer Cells

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Xianqi Zhao

2015-06-01

Full Text Available Breast cancer has a high incidence and mortality rate worldwide. Several viral vectors including lentiviral, adenoviral and adeno-associated viral vectors have been used in gene therapy for various forms of human cancer, and have shown promising effects in controlling tumor development. Claudin1 (CLDN1 is a member of the tetraspan transmembrane protein family that plays a major role in tight junctions and is associated with tumor metastasis. However, the role of CLDN1 in breast cancer is largely unexplored. In this study, we tested the therapeutic potential of silencing CLDN1 expression in two breast cancer (MDA-MB-231 and MCF7 cell lines using lentiviral vector mediated RNA interference. We found that a CLDN1 short hairpin (shRNA construct efficiently silenced CLDN1 expression in both breast cancer cell lines, and CLDN1 knockdown resulted in reduced cell proliferation, survival, migration and invasion. Furthermore, silencing CLDN1 inhibited epithelial to mesenchymal transition (EMT by upregulating the epithelial cell marker, E-cadherin, and downregulating mesenchymal markers, smooth muscle cell alpha-actin (SMA and Snai2. Our data demonstrated that lentiviral vector mediated CLDN1 RNA interference has great potential in breast cancer gene therapy by inhibiting EMT and controlling tumor cell growth.

RNA Interference towards the Potato Psyllid, Bactericera cockerelli, Is Induced in Plants Infected with Recombinant Tobacco mosaic virus (TMV)

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Wuriyanghan, Hada; Falk, Bryce W.

2013-01-01

The potato/tomato psyllid, Bactericera cockerelli (B. cockerelli), is an important plant pest and the vector of the phloem-limited bacterium Candidatus Liberibacter psyllaurous (solanacearum), which is associated with the zebra chip disease of potatoes. Previously, we reported induction of RNA interference effects in B. cockerelli via in vitro-prepared dsRNA/siRNAs after intrathoracic injection, and after feeding of artificial diets containing these effector RNAs. In order to deliver RNAi effectors via plant hosts and to rapidly identify effective target sequences in plant-feeding B. cockerelli, here we developed a plant virus vector-based in planta system for evaluating candidate sequences. We show that recombinant Tobacco mosaic virus (TMV) containing B. cockerelli sequences can efficiently infect and generate small interfering RNAs in tomato (Solanum lycopersicum), tomatillo (Physalis philadelphica) and tobacco (Nicotiana tabacum) plants, and more importantly delivery of interfering sequences via TMV induces RNAi effects, as measured by actin and V-ATPase mRNA reductions, in B. cockerelli feeding on these plants. RNAi effects were primarily detected in the B. cockerelli guts. In contrast to our results with TMV, recombinant Potato virus X (PVX) and Tobacco rattle virus (TRV) did not give robust infections in all plants and did not induce detectable RNAi effects in B. cockerelli. The greatest RNA interference effects were observed when B. cockerelli nymphs were allowed to feed on leaf discs collected from inoculated or lower expanded leaves from corresponding TMV-infected plants. Tomatillo plants infected with recombinant TMV containing B. cockerelli actin or V-ATPase sequences also showed phenotypic effects resulting in decreased B. cockerelli progeny production as compared to plants infected by recombinant TMV containing GFP. These results showed that RNAi effects can be achieved in plants against the phloem feeder, B. cockerelli, and the TMV-plant system will

SiRNA Crosslinked Nanoparticles for the Treatment of Inflammation-induced Liver Injury.

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Tang, Yaqin; Zeng, Ziying; He, Xiao; Wang, Tingting; Ning, Xinghai; Feng, Xuli

2017-02-01

RNA interference mediated by small interfering RNA (siRNA) provides a powerful tool for gene regulation, and has a broad potential as a promising therapeutic strategy. However, therapeutics based on siRNA have had limited clinical success due to their undesirable pharmacokinetic properties. This study presents pH-sensitive nanoparticles-based siRNA delivery systems (PNSDS), which are positive-charge-free nanocarriers, composed of siRNA chemically crosslinked with multi-armed poly(ethylene glycol) carriers via acid-labile acetal linkers. The unique siRNA crosslinked structure of PNSDS allows it to have minimal cytotoxicity, high siRNA loading efficiency, and a stimulus-responsive property that enables the selective intracellular release of siRNA in response to pH conditions. This study demonstrates that PNSDS can deliver tumor necrosis factor alpha (TNF-α) siRNA into macrophages and induce the efficient down regulation of the targeted gene in complete cell culture media. Moreover, PNSDS with mannose targeting moieties can selectively accumulate in mice liver, induce specific inhibition of macrophage TNF-α expression in vivo, and consequently protect mice from inflammation-induced liver damages. Therefore, this novel siRNA delivering platform would greatly improve the therapeutic potential of RNAi based therapies.

An RNA-seq transcriptome analysis of histone modifiers and RNA silencing genes in soybean during floral initiation process.

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Lim Chee Liew

Full Text Available Epigenetics has been recognised to play vital roles in many plant developmental processes, including floral initiation through the epigenetic regulation of gene expression. The histone modifying proteins that mediate these modifications involve the SET domain-containing histone methyltransferases, JmjC domain-containing demethylase, acetylases and deacetylases. In addition, RNA interference (RNAi-associated genes are also involved in epigenetic regulation via RNA-directed DNA methylation and post-transcriptional gene silencing. Soybean, a major crop legume, requires a short day to induce flowering. How histone modifications regulate the plant response to external cues that initiate flowering is still largely unknown. Here, we used RNA-seq to address the dynamics of transcripts that are potentially involved in the epigenetic programming and RNAi mediated gene silencing during the floral initiation of soybean. Soybean is a paleopolyploid that has been subjected to at least two rounds of whole genome duplication events. We report that the expanded genomic repertoire of histone modifiers and RNA silencing genes in soybean includes 14 histone acetyltransferases, 24 histone deacetylases, 47 histone methyltransferases, 15 protein arginine methyltransferases, 24 JmjC domain-containing demethylases and 47 RNAi-associated genes. To investigate the role of these histone modifiers and RNA silencing genes during floral initiation, we compared the transcriptional dynamics of the leaf and shoot apical meristem at different time points after a short-day treatment. Our data reveal that the extensive activation of genes that are usually involved in the epigenetic programming and RNAi gene silencing in the soybean shoot apical meristem are reprogrammed for floral development following an exposure to inductive conditions.

Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

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Zongli eHu

2015-01-01

Full Text Available Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Derived RNA interference (HD-RNAi technology to partially silence three different genes (FOW2, FRP1 and OPR in the hemi-biotrophic fungus Fusarium oxysporum f. sp. conglutinans. Expression of double stranded RNA molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75%, 83% and 72% reduction for FOW2, FRP1 and OPR respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30-50% survival and FOW2 between 45-70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants.

Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

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Hu, Zongli; Parekh, Urvi; Maruta, Natsumi; Trusov, Yuri; Botella, Jimmy

2015-01-01

Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Derived RNA interference (HD-RNAi) technology to partially silence three different genes (FOW2, FRP1 and OPR) in the hemi-biotrophic fungus Fusarium oxysporum f. sp. conglutinans. Expression of double stranded RNA molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75%, 83% and 72% reduction for FOW2, FRP1 and OPR respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30-50% survival and FOW2 between 45-70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants.

Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference.

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Louise Ford

Full Text Available Cathepsin-like enzymes have been identified as potential targets for drug or vaccine development in many parasites, as their functions appear to be essential in a variety of important biological processes within the host, such as molting, cuticle remodeling, embryogenesis, feeding and immune evasion. Functional analysis of Caenorhabditis elegans cathepsin L (Ce-cpl-1 and cathepsin Z (Ce-cpz-1 has established that both genes are required for early embryogenesis, with Ce-cpl-1 having a role in regulating in part the processing of yolk proteins. Ce-cpz-1 also has an important role during molting.RNA interference assays have allowed us to verify whether the functions of the orthologous filarial genes in Brugia malayi adult female worms are similar. Treatment of B. malayi adult female worms with Bm-cpl-1, Bm-cpl-5, which belong to group Ia of the filarial cpl gene family, or Bm-cpz-1 dsRNA resulted in decreased numbers of secreted microfilariae in vitro. In addition, analysis of the intrauterine progeny of the Bm-cpl-5 or Bm-cpl Pro dsRNA- and siRNA-treated worms revealed a clear disruption in the process of embryogenesis resulting in structural abnormalities in embryos and a varied differential development of embryonic stages.Our studies suggest that these filarial cathepsin-like cysteine proteases are likely to be functional orthologs of the C. elegans genes. This functional conservation may thus allow for a more thorough investigation of their distinct functions and their development as potential drug targets.

Strand Analysis, a free online program for the computational identification of the best RNA interference (RNAi targets based on Gibbs free energy

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Tiago Campos Pereira

2007-01-01

Full Text Available The RNA interference (RNAi technique is a recent technology that uses double-stranded RNA molecules to promote potent and specific gene silencing. The application of this technique to molecular biology has increased considerably, from gene function identification to disease treatment. However, not all small interfering RNAs (siRNAs are equally efficient, making target selection an essential procedure. Here we present Strand Analysis (SA, a free online software tool able to identify and classify the best RNAi targets based on Gibbs free energy (deltaG. Furthermore, particular features of the software, such as the free energy landscape and deltaG gradient, may be used to shed light on RNA-induced silencing complex (RISC activity and RNAi mechanisms, which makes the SA software a distinct and innovative tool.

SiRNA-mediated IGF-1R inhibition sensitizes human colon cancer SW480 cells to radiation

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Yavari, Kamal; Taghikhani, Mohammad; Mesbah-Namin, Seyed A.; Maragheh, Mohammad Ghannadi; Babaei, Mohammad Hosein; Arfaee, Ali Jabbary; Madani, Hossein; Mirzaei, Hamid Reza

2010-01-01

Purpose. Insulin like growth factor receptor 1 (IGF-1R) is well-documented to play a key role in radiation response and tumor radiosensitivity, thus offering an attractive clinic drug target to enhance tumor sensitivity to anti-cancer radiotherapy. Material and methods. Human colon carcinoma SW480 cells were transfected with the specific small interference RNA (siRNA) expression vector (pkD-shRNA-IGF-1R-V2) designed to target IGF-1R mRNA. The expression of IGF-1R mRNA and its protein among the transfected and untransfected cells were detected by semi-quantitative RT-PCR and ELISA assay. The changes in cell radiosensitivity were examined by MTT assay. Results. Transfection of mammalian expression vector pkD containing IGF-1R siRNA was shown to reduce IGF-1R mRNA levels by up to 95%. ELISA assay detected a similar inhibition of IGF-1R protein levels in cells transfected with IGF-1R siRNA. SW480 cells transfected with the expression vector for siRNA significantly rendered cells more sensitive to radiation and the highest radiation enhancement ratio was 2.02 ± 0.08. Conclusion. These data provide the first evidence that specific siRNA fragment (pkD-shRNA-IGF-1R-V2) targeting human IGF-1R mRNA is able to enhance colon cancer radiosensitivity. Also results indicated that, combining IGF-1R siRNA and radiation significantly enhances antitumor efficacy compared with either modality alone

Inhibitors of MyD88-dependent proinflammatory cytokine production identified utilizing a novel RNA interference screening approach.

Directory of Open Access Journals (Sweden)

John S Cho

2009-09-01

Full Text Available The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha. How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.We report here the development a completely random short-hairpin RNA (shRNA library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production.Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses.

Characterization of RNA interference in rat PC12 cells

DEFF Research Database (Denmark)

Thonberg, Håkan; Schéele, Camilla C; Dahlgren, Cecilia

2004-01-01

strand of the siRNA guides a multi-protein complex, RNA-induced silencing complex (RISC), to cleave target mRNA. Although the exact function and composition of RISC is still unclear, it has been shown to include several proteins of the Argonaute protein family. Here we report of a robust system...... of the rat Golgi-ER protein 95 kDa (GERp95), an Argonaute family protein, by siRNA methodology. After GERp95-ablation, sequential knockdown of NPY by siRNA was shown to be impaired. Thus, we report that the GERp95 protein is functionally required for RNAi targeting NPY in rat PC12 cells....

[Inhibitory effect of RNA interference targeting GFI-1 on the proliferation of atypical chronic myelogenous leukemia NT1 cells].

Science.gov (United States)

Yang, X; Liu, H; Lin, Z H; Qian, J; Xu, X R

2016-08-01

To investigate the inhibitory effects of RNA interference targeting GFI-1 on growth and proliferation of atypical chronic myelogenous leukemia (aCML) NT1 cells. NT1 cells were transfected with PBS and liposome complex (vehicle group), scrambled siRNA and liposome complex (negative control, NC group), and GFI-1 siRNA and liposome complex (GFI-1 siRNA group), respectively. Real-time quantitative RT-PCR (qRT-PCR) and Western blot were performed to examine the expression levels of GFI-1 mRNA and protein, respectively. The proliferation abilities of NT1 cells of the three groups were evaluated by MTT assay. The cell cycle in cells of the three groups was analyzed by flow cytometry. Moreover, nude mouse xenograft model was used to detect the tumor formation ability in the three group cells. Quantitative real-time PCR data showed that the expression level of GFI-1 mRNA in GFI-1 siRNA group was significantly lower than those of NC group and vehicle group [(0.367±0.017) vs. (0.918±0.006) and (1.010±0.005), respectively, (PNT1 cells in the GFI-1 siRNA group (0.667±0.059) was significantly lower than those of the NC group (1.096±0.049) and vehicle group (1.193±0.064, P=0.023). Flow cytometry data showed that sub-G1 and G0/G1 phase proportions of the GFI-1 siRNA group were significantly higher than those of the NC and vehicle groups [sub-G1: (8.2±2.5)% vs. (1.9±1.3)% and (2.0±3.6)%, respectively, (PNT1 cells, which may provide a new therapeutic target for atypical chronic myelogenous leukemia.

Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.

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Carlos F Solis

Full Text Available BACKGROUND: Modern RNA interference (RNAi methodologies using small interfering RNA (siRNA oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules. PRINCIPAL FINDINGS: Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica beta-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite. CONCLUSIONS: Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.

MicroRNA-367 is a potential diagnostic biomarker for patients with esophageal squamous cell carcinoma

Energy Technology Data Exchange (ETDEWEB)

Sun, Jiangtao; Song, Kaifang; Feng, Xiaoshan, E-mail: xiaoshan.feng@aol.com; Gao, Shegan

2016-04-29

Purpose: In this study, we investigated whether microRNA-367 (miR-367) may serve as a circulating biomarker and tumor oncogene in esophageal squamous carcinoma (ESCC). Methods: Circulating serum miR-367 was compared by quantitative RT-PCR (qRT-PCR) between 35 ESCC patients and 35 normal control patients, as well paired ESCC tumor tissues and adjacent non-tumor esophageal epithelial tissues in 46 patients. The correlation between serum miR-367 and clinicopathological properties of ESCC patients was assessed. The overall survival (OS) was assessed by Kaplan–Meier method and compared by log-rank test between patients with high serum miR-367 and low serum miR-367. The possibility of miR-367 being independent prognostic factor for ESCC was also assessed. Furthermore, lentivirus-mediated miR-367 downregulation was conducted in ESCC cell lines Kyse30 and TE-1 cells to assess the possible oncogenic effect of miR-367 on ESCC proliferation and cell cycle transition in vitro. Results: MiR-367 was aberrantly upregulated in sera and tumors of ESCC patients, whereas downregulated in ESCC patients after the treatments of esophagectomy and chemotherapy. Serum miR-367 was found to be closely correlated with the clinicopathological properties of differentiation grades, clinical stage and tumor metastasis in ESCC patients. Serum miR-367 was also confirmed to be associated with OS, as well as serving independent prognostic factor in ESCC patients. Moreover, lentivirus-induced miR-367 downregulation inhibited cancer growth and cell cycle transition in Kyse30 and TE-1 cells. Conclusion: MiR-367 is a potential biomarker for ESCC and may act as an oncogene in regulating ESCC development. - Highlights: • MiR-367 was aberrantly upregulated in sera and tumors of ESCC patients. • MiR-367 was downregulated in ESCC patients after esophagectomy or chemotherapy. • Serum miR-367 was correlated with the clinicopathological properties of ESCC patients. • Serum miR-367 was associated

The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth1[OPEN

Science.gov (United States)

Bustos-Sanmamed, Pilar; Mysore, Kirankumar S.

2017-01-01

Legume roots form two types of postembryonic organs, lateral roots and symbiotic nodules. Nodule formation is the result of the interaction of legumes with rhizobia and requires the mitotic activation and differentiation of root cells as well as an independent, but coordinated, program that allows infection by rhizobia. MicroRNA390 (miR390) is an evolutionarily conserved microRNA that targets the Trans-Acting Short Interference RNA3 (TAS3) transcript. Cleavage of TAS3 by ARGONAUTE7 results in the production of trans-acting small interference RNAs, which target mRNAs encoding AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4. Here, we show that activation of the miR390/TAS3 regulatory module by overexpression of miR390 in Medicago truncatula promotes lateral root growth but prevents nodule organogenesis, rhizobial infection, and the induction of two key nodulation genes, Nodulation Signaling Pathway1 (NSP1) and NSP2. Accordingly, inactivation of the miR390/TAS3 module, either by expression of a miR390 target mimicry construct or mutations in ARGONAUTE7, enhances nodulation and rhizobial infection, alters the spatial distribution of the nodules, and increases the percentage of nodules with multiple meristems. Our results revealed a key role of the miR390/TAS3 pathway in legumes as a modulator of lateral root organs, playing opposite roles in lateral root and nodule development. PMID:28663332

Inhibition of endothelial cell proliferation by targeting Rac1 GTPase with small interference RNA in tumor cells

International Nuclear Information System (INIS)

Xue Yan; Bi Feng; Zhang Xueyong; Pan Yanglin; Liu Na; Zheng Yi; Fan Daiming

2004-01-01

Hypoxia-induced angiogenesis plays an important role in the malignancy of solid tumors. A number of recent studies including our own have suggested that Rho family small GTPases are involved in this process, and Rac1, a prominent member of the Rho family, may be critical in regulating hypoxia-induced gene activation of several angiogenesis factors and tumor suppressors. To further define Rac1 function in angiogenesis and to explore novel approaches to modulate angiogenesis, we employed the small interference RNA technique to knock down gene expression of Rac1 in gastric cancer cell line AGS that expresses a high level of Rac1. Both the mRNA and protein levels of Rac1 in the AGS cells were decreased dramatically after transfection with a Rac1-specific siRNA vector. When the conditioned medium derived from the Rac1 downregulated AGS cells was applied to the human endothelial cells, it could significantly inhibit the cell proliferation. Further study proved that, VEGF and HIF-1α, two angiogenesis promoting factors, were found to be downregulated whereas p53 and VHL, which are tumor suppressors and angiogenesis inhibitors, were upregulated in the Rac1 siRNA transfected cells. Our results suggest that Rac1 may be involved in angiogenesis by controlling the expression of angiogenesis-related factors and provide a possible strategy for the treatment of tumor angiogenesis by targeting the Rac1 GTPase

The nuclear import of RNA helicase A is mediated by importin-α3

International Nuclear Information System (INIS)

Aratani, Satoko; Oishi, Takayuki; Fujita, Hidetoshi; Nakazawa, Minako; Fujii, Ryouji; Imamoto, Naoko; Yoneda, Yoshihiro; Fukamizu, Akiyoshi; Nakajima, Toshihiro

2006-01-01

RNA helicase A (RHA), an ATPase/helicase, regulates the gene expression at various steps including transcriptional activation and RNA processing. RHA is known to shuttle between the nucleus and cytoplasm. We identified the nuclear localization signal (NLS) of RHA and analyzed the nuclear import mechanisms. The NLS of RHA (RHA-NLS) consisting of 19 amino acid residues is highly conserved through species and does not have the consensus classical NLS. In vitro nuclear import assays revealed that the nuclear import of RHA was Ran-dependent and mediated with the classical importin-α/β-dependent pathway. The binding assay indicated that the basic residues in RHA-NLS were used for interaction with importin-α. Furthermore, the nuclear import of RHA-NLS was supported by importin-α1 and preferentially importin-α3. Our results indicate that the nuclear import of RHA is mediated by the importin-α3/importin-β-dependent pathway and suggest that the specificity for importin may regulate the functions of cargo proteins

Decreased expression of RNA interference machinery, Dicer and Drosha, is associated with poor outcome in ovarian cancer patients

Energy Technology Data Exchange (ETDEWEB)

Merritt, William M.; Lin, Yvonne G.; Han, Liz Y.; Kamat, Aparna A.; Spannuth, Whitney A.; Schmandt, Rosemarie; Urbauer, Diana; Pennacchio, Len A.; Cheng, Jan-Fang; Zeidan, Alexandra; Wang, Hua; Mueller, Peter; Lenburg, Marc E.; Gray, Joe W.; Mok, Samuel; Birrer, Michael J.; Lopez-Berestein, Gabriel; Coleman, Robert L.; Bar-Eli, Menashe; Sood, Anil K.

2008-05-06

The clinical and functional significance of RNA interference (RNAi) machinery, Dicer and Drosha, in ovarian cancer is not known and was examined. Dicer and Drosha expression was measured in ovarian cancer cell lines (n=8) and invasive epithelial ovarian cancer specimens (n=111) and correlated with clinical outcome. Validation was performed with previously published cohorts of ovarian, breast, and lung cancer patients. Anti-Galectin-3 siRNA and shRNA transfections were used for in vitro functional studies. Dicer and Drosha mRNA and protein levels were decreased in 37% to 63% of ovarian cancer cell lines and in 60% and 51% of human ovarian cancer specimens, respectively. Low Dicer was significantly associated with advanced tumor stage (p=0.007), and low Drosha with suboptimal surgical cytoreduction (p=0.02). Tumors with both high Dicer and Drosha were associated with increased median patient survival (>11 years vs. 2.66 years for other groups; p<0.001). In multivariate analysis, high Dicer (HR=0.48; p=0.02), high-grade histology (HR=2.46; p=0.03), and poor chemoresponse (HR=3.95; p<0.001) were identified as independent predictors of disease-specific survival. Findings of poor clinical outcome with low Dicer expression were validated in separate cohorts of cancer patients. Galectin-3 silencing with siRNA transfection was superior to shRNA in cell lines with low Dicer (78-95% vs. 4-8% compared to non-targeting sequences), and similar in cell lines with high Dicer. Our findings demonstrate the clinical and functional impact of RNAi machinery alterations in ovarian carcinoma and support the use of siRNA constructs that do not require endogenous Dicer and Drosha for therapeutic applications.

Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells.

Directory of Open Access Journals (Sweden)

Jaclyn C Scott

2010-10-01

Full Text Available The exogenous RNA interference (RNAi pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (siRNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2 cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

Identification of a novel cytochrome P450 gene, CYP321E1 from the diamondback moth, Plutella xylostella (L.) and RNA interference to evaluate its role in chlorantraniliprole resistance.

Science.gov (United States)

Hu, Z; Lin, Q; Chen, H; Li, Z; Yin, F; Feng, X

2014-12-01

Insect cytochrome P450 monooxygenases (P450s) play an important role in catalysis of many reactions leading to insecticides resistance. Our previous studies on transcriptome analysis of chlorantraniliprole-resistant development in the diamondback moth, Plutella xylostella revealed that up-regulation of cytochrome P450s are one of the main factors leading to the development of chlorantraniliprole resistance. Here, we report for the first time a novel cytochrome P450 gene CYP321E1, which belongs to the cytochrome P450 gene family CYP321. Real-time quantitative PCR (RT-qPCR) analyses indicated that CYP321E1 was expressed at all developmental stages of P. xylostella but was highest in the fourth-instar larvae; furthermore, the relatively high expression was observed in the midgut of the fourth-instar larvae, followed by fat bodies and epidermis. The expression of CYP321E1 in P. xylostella was differentially affected by three representative insecticides, including alphamethrin, abamectin and chlorantraniliprole. Among them, the exposure to chlorantraniliprole resulted in the largest transcript level of this cytochrome P450 gene. The findings suggested potential involvement of CYP321E1 in chlorantraniliprole resistance of P. xylostella. To assess the functional link of CYP321E1 to chlorantraniliprole resistance, RNA interference (RNAi)-mediated gene silencing by double stranded RNA (dsRNA) injecting was used. Results revealed that injection delivery of dsRNA can greatly reduce gene expression after 24 h. As a consequence of RNAi, a significant increment in mortality of larvae injected CYP321E1 dsRNA was observed after 24 h of exposure to chlorantraniliprole. These results strongly support our notion that this novel cytochrome P450 gene plays an important role in chlorantraniliprole detoxification in the diamondback moth and is partly responsible for its resistance.

Generation of induced pluripotent stem cells (iPSCs) stably expressing CRISPR-based synergistic activation mediator (SAM)

DEFF Research Database (Denmark)

Xiong, Kai; Zhou, Yan; Hyttel, Poul

2016-01-01

Human fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4...... a novel, useful tool to investigate genetic regulation of stem cell proliferation and differentiation through CRISPR-mediated activation of endogenous genes....

Intragenomic matching reveals a huge potential for miRNA-mediated regulation in plants

DEFF Research Database (Denmark)

Lindow, Morten; Jacobsen, Anders; Nygaard, Sanne

2007-01-01

indicates that only a few of the candidates are conserved between the species. We conclude that there is a large potential for miRNA-mediated regulatory interactions encoded in the genomes of the investigated plants. We hypothesize that some of these interactions may be realized under special environmental...

A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes.

Science.gov (United States)

Anosova, Irina; Melnik, Svitlana; Tripsianes, Konstantinos; Kateb, Fatiha; Grummt, Ingrid; Sattler, Michael

2015-05-26

The chromatin remodeling complex NoRC, comprising the subunits SNF2h and TIP5/BAZ2A, mediates heterochromatin formation at major clusters of repetitive elements, including rRNA genes, centromeres and telomeres. Association with chromatin requires the interaction of the TAM (TIP5/ARBP/MBD) domain of TIP5 with noncoding RNA, which targets NoRC to specific genomic loci. Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins. However, the TAM domain exhibits an extended MBD fold with unique C-terminal extensions that constitute a novel surface for RNA binding. Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo. Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

mediated RNA interference in bovine fibroblast cells

African Journals Online (AJOL)

Jane

2011-08-03

Aug 3, 2011 ... there are lots of problems related to the method of delivery, especially .... Detection of MC4R silencing of Lvsh-MC4R in FBCs. The Auris .... Schwartz MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn. P, Baskin DG ...

Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

Science.gov (United States)

Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

2010-07-30

Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway. Copyright 2010 Elsevier Inc. All rights reserved.

Mammalian mediator 19 mediates H1299 lung adenocarcinoma cell clone conformation, growth, and metastasis.

Science.gov (United States)

Xu, Lu-Lu; Guo, Shu-Liang; Ma, Su-Ren; Luo, Yong-Ai

2012-01-01

Mammalian mediator (MED) is a multi-protein coactivator that has been identified by several research groups. The involvement of the MED complex subunit 19 (MED 19) in the metastasis of lung adenocarcinoma cell line (H1299), which expresses the MED 19 subunit, was here investigated. When MED 19 expression was decreased by RNA interference H1299 cells demonstrated reduced clone formation, arrest in the S phase of the cell cycle, and lowered metastatic capacity. Thus, MED 19 appears to play important roles in the biological behavior of non-small cell lung carcinoma cells. These findings may be important for the development of novel lung carcinoma treatments.

A dominant mutation in mediator of paramutation2, one of three second-largest subunits of a plant-specific RNA polymerase, disrupts multiple siRNA silencing processes.

Science.gov (United States)

Sidorenko, Lyudmila; Dorweiler, Jane E; Cigan, A Mark; Arteaga-Vazquez, Mario; Vyas, Meenal; Kermicle, Jerry; Jurcin, Diane; Brzeski, Jan; Cai, Yu; Chandler, Vicki L

2009-11-01

Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA-mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V-like complexes could provide maize with a greater diversification of RNA-mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state-a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV-like) and potentially processes downstream (Pol-V-like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance

Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

Science.gov (United States)

Chen, Anyi; Gui, Guo-Feng; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Yuan, Ruo

2015-06-16

A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.

CRISPR/Cas13 as a Tool for RNA Interference

KAUST Repository

Ali, Zahir

2018-03-28

Almost all biological processes involve RNA, making it crucial to develop tools for manipulation of the transcriptome. The bacterial CRISPR/Cas13 system was recently rewired to facilitate RNA manipulation in eukaryotes, including plants. We discuss here the opportunities and limitations of using CRISPR/Cas13 in plants for various types of RNA manipulation.

Systematic comparison of the response properties of protein and RNA mediated gene regulatory motifs.

Science.gov (United States)

Iyengar, Bharat Ravi; Pillai, Beena; Venkatesh, K V; Gadgil, Chetan J

2017-05-30

We present a framework enabling the dissection of the effects of motif structure (feedback or feedforward), the nature of the controller (RNA or protein), and the regulation mode (transcriptional, post-transcriptional or translational) on the response to a step change in the input. We have used a common model framework for gene expression where both motif structures have an activating input and repressing regulator, with the same set of parameters, to enable a comparison of the responses. We studied the global sensitivity of the system properties, such as steady-state gain, overshoot, peak time, and peak duration, to parameters. We find that, in all motifs, overshoot correlated negatively whereas peak duration varied concavely with peak time. Differences in the other system properties were found to be mainly dependent on the nature of the controller rather than the motif structure. Protein mediated motifs showed a higher degree of adaptation i.e. a tendency to return to baseline levels; in particular, feedforward motifs exhibited perfect adaptation. RNA mediated motifs had a mild regulatory effect; they also exhibited a lower peaking tendency and mean overshoot. Protein mediated feedforward motifs showed higher overshoot and lower peak time compared to the corresponding feedback motifs.

Simple method for assembly of CRISPR synergistic activation mediator gRNA expression array.

Science.gov (United States)

Vad-Nielsen, Johan; Nielsen, Anders Lade; Luo, Yonglun

2018-05-20

When studying complex interconnected regulatory networks, effective methods for simultaneously manipulating multiple genes expression are paramount. Previously, we have developed a simple method for generation of an all-in-one CRISPR gRNA expression array. We here present a Golden Gate Assembly-based system of synergistic activation mediator (SAM) compatible CRISPR/dCas9 gRNA expression array for the simultaneous activation of multiple genes. Using this system, we demonstrated the simultaneous activation of the transcription factors, TWIST, SNAIL, SLUG, and ZEB1 a human breast cancer cell line. Copyright © 2018 Elsevier B.V. All rights reserved.

Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly.

Science.gov (United States)

Chereji, Razvan V; Bharatula, Vasudha; Elfving, Nils; Blomberg, Jeanette; Larsson, Miriam; Morozov, Alexandre V; Broach, James R; Björklund, Stefan

2017-09-06

Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Saccharomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Antiviral RNA silencing initiated in the absence of RDE-4, a double-stranded RNA binding protein, in Caenorhabditis elegans.

Science.gov (United States)

Guo, Xunyang; Zhang, Rui; Wang, Jeffrey; Lu, Rui

2013-10-01

Small interfering RNAs (siRNAs) processed from double-stranded RNA (dsRNA) of virus origins mediate potent antiviral defense through a process referred to as RNA interference (RNAi) or RNA silencing in diverse organisms. In the simple invertebrate Caenorhabditis elegans, the RNAi process is initiated by a single Dicer, which partners with the dsRNA binding protein RDE-4 to process dsRNA into viral siRNAs (viRNAs). Notably, in C. elegans this RNA-directed viral immunity (RDVI) also requires a number of worm-specific genes for its full antiviral potential. One such gene is rsd-2 (RNAi spreading defective 2), which was implicated in RDVI in our previous studies. In the current study, we first established an antiviral role by showing that rsd-2 null mutants permitted higher levels of viral RNA accumulation, and that this enhanced viral susceptibility was reversed by ectopic expression of RSD-2. We then examined the relationship of rsd-2 with other known components of RNAi pathways and established that rsd-2 functions in a novel pathway that is independent of rde-4 but likely requires the RNA-dependent RNA polymerase RRF-1, suggesting a critical role for RSD-2 in secondary viRNA biogenesis, likely through coordinated action with RRF-1. Together, these results suggest that RDVI in the single-Dicer organism C. elegans depends on the collective actions of both RDE-4-dependent and RDE-4-independent mechanisms to produce RNAi-inducing viRNAs. Our study reveals, for the first time, a novel siRNA-producing mechanism in C. elegans that bypasses the need for a dsRNA-binding protein.

Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.

Science.gov (United States)

Bhagwat, Basdeo; Chi, Ming; Han, Dianwei; Tang, Haifeng; Tang, Guiliang; Xiang, Yu

2016-01-01

Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.

TLR4-NOX4-AP-1 signaling mediates lipopolysaccharide-induced CXCR6 expression in human aortic smooth muscle cells

International Nuclear Information System (INIS)

Patel, Devang N.; Bailey, Steven R.; Gresham, John K.; Schuchman, David B.; Shelhamer, James H.; Goldstein, Barry J.; Foxwell, Brian M.; Stemerman, Michael B.; Maranchie, Jodi K.; Valente, Anthony J.; Mummidi, Srinivas; Chandrasekar, Bysani

2006-01-01

CXCL16 is a transmembrane non-ELR CXC chemokine that signals via CXCR6 to induce aortic smooth muscle cell (ASMC) proliferation. While bacterial lipopolysaccharide (LPS) has been shown to stimulate CXCL16 expression in SMC, its effects on CXCR6 are not known. Here, we demonstrate that LPS upregulates CXCR6 mRNA, protein, and surface expression in human ASMC. Inhibition of TLR4 with neutralizing antibodies or specific siRNA interference blocked LPS-mediated CXCR6 expression. LPS stimulated both AP-1 (c-Fos, c-Jun) and NF-κB (p50 and p65) activation, but only inhibition of AP-1 attenuated LPS-induced CXCR6 expression. Using dominant negative expression vectors and siRNA interference, we demonstrate that LPS induces AP-1 activation via MyD88, TRAF6, ERK1/2, and JNK signaling pathways. Furthermore, the flavoprotein inhibitor diphenyleniodonium chloride significantly attenuated LPS-mediated AP-1-dependent CXCR6 expression, as did inhibition of NOX4 NADPH oxidase by siRNA. Finally, CXCR6 knockdown inhibited CXCL16-induced ASMC proliferation. These results demonstrate that LPS-TLR4-NOX4-AP-1 signaling can induce CXCR6 expression in ASMC, and suggest that the CXCL16-CXCR6 axis may be an important proinflammatory pathway in the pathogenesis of atherosclerosis

MicroRNA-mediated networks underlie immune response regulation in papillary thyroid carcinoma

Science.gov (United States)

Huang, Chen-Tsung; Oyang, Yen-Jen; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

2014-09-01

Papillary thyroid carcinoma (PTC) is a common endocrine malignancy with low death rate but increased incidence and recurrence in recent years. MicroRNAs (miRNAs) are small non-coding RNAs with diverse regulatory capacities in eukaryotes and have been frequently implied in human cancer. Despite current progress, however, a panoramic overview concerning miRNA regulatory networks in PTC is still lacking. Here, we analyzed the expression datasets of PTC from The Cancer Genome Atlas (TCGA) Data Portal and demonstrate for the first time that immune responses are significantly enriched and under specific regulation in the direct miRNA-target network among distinctive PTC variants to different extents. Additionally, considering the unconventional properties of miRNAs, we explore the protein-coding competing endogenous RNA (ceRNA) and the modulatory networks in PTC and unexpectedly disclose concerted regulation of immune responses from these networks. Interestingly, miRNAs from these conventional and unconventional networks share general similarities and differences but tend to be disparate as regulatory activities increase, coordinately tuning the immune responses that in part account for PTC tumor biology. Together, our systematic results uncover the intensive regulation of immune responses underlain by miRNA-mediated networks in PTC, opening up new avenues in the management of thyroid cancer.

Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica.

Science.gov (United States)

Khalil, Mohamed I; Foda, Bardees M; Suresh, Susmitha; Singh, Upinder

2016-03-01

Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS sRNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community. Copyright © 2016

HIV-1 pre-mRNA commitment to Rev mediated export through PSF and Matrin 3

International Nuclear Information System (INIS)

Kula, Anna; Gharu, Lavina; Marcello, Alessandro

2013-01-01

Human immunodeficiency virus gene expression and replication are regulated at several levels. Incompletely spliced viral RNAs and full-length genomic RNA contain the RRE element and are bound by the viral trans-acting protein Rev to be transported out of the nucleus. Previously we found that the nuclear matrix protein MATR3 was a cofactor of Rev-mediated RNA export. Here we show that the pleiotropic protein PSF binds viral RNA and is associated with MATR3. PSF is involved in the maintenance of a pool of RNA available for Rev activity. However, while Rev and PSF bind the viral pre-mRNA at the site of viral transcription, MATR3 interacts at a subsequent step. We propose that PSF and MATR3 define a novel pathway for RRE-containing HIV-1 RNAs that is hijacked by the viral Rev protein.

Evolutionary and Functional Analysis of Old World Primate TRIM5 Reveals the Ancient Emergence of Primate Lentiviruses and Convergent Evolution Targeting a Conserved Capsid Interface.

Directory of Open Access Journals (Sweden)

Kevin R McCarthy

2015-08-01

Full Text Available The widespread distribution of lentiviruses among African primates, and the lack of severe pathogenesis in many of these natural reservoirs, are taken as evidence for long-term co-evolution between the simian immunodeficiency viruses (SIVs and their primate hosts. Evidence for positive selection acting on antiviral restriction factors is consistent with virus-host interactions spanning millions of years of primate evolution. However, many restriction mechanisms are not virus-specific, and selection cannot be unambiguously attributed to any one type of virus. We hypothesized that the restriction factor TRIM5, because of its unique specificity for retrovirus capsids, should accumulate adaptive changes in a virus-specific fashion, and therefore, that phylogenetic reconstruction of TRIM5 evolution in African primates should reveal selection by lentiviruses closely related to modern SIVs. We analyzed complete TRIM5 coding sequences of 22 Old World primates and identified a tightly-spaced cluster of branch-specific adaptions appearing in the Cercopithecinae lineage after divergence from the Colobinae around 16 million years ago. Functional assays of both extant TRIM5 orthologs and reconstructed ancestral TRIM5 proteins revealed that this cluster of adaptations in TRIM5 specifically resulted in the ability to restrict Cercopithecine lentiviruses, but had no effect (positive or negative on restriction of other retroviruses, including lentiviruses of non-Cercopithecine primates. The correlation between lineage-specific adaptations and ability to restrict viruses endemic to the same hosts supports the hypothesis that lentiviruses closely related to modern SIVs were present in Africa and infecting the ancestors of Cercopithecine primates as far back as 16 million years ago, and provides insight into the evolution of TRIM5 specificity.

Final report: FASEB Summer Research Conference on ''Post-transcriptional control of gene expression: Effectors of mRNA decay'' [agenda and attendees list

Energy Technology Data Exchange (ETDEWEB)

Maquat, Lynne

2002-12-01

The goal of this meeting was to provide an interactive forum for scientists working on prokaryotic and eukaryotic mRNA decay. A special seminar presented by a leader in the field of mRNA decay in S. cerevisiae focused on what is known and what needs to be determined, not only for yeast but for other organisms. The large attendance (110 participants) reflects the awareness that mRNA decay is a key player in gene regulation in a way that is affected by the many steps that precede mRNA formation. Sessions were held on the following topics: mRNA transport and mRNP; multicomponent eukaryotic nucleases; nonsense-mediated mRNA decay and nonsense-associated altered splicing; Cis-acting sequences/Trans-acting factors of mRNA decay; translational accuracy; multicomponent bacterial nucleases; interplay between mRNA polyadenylation, translation and decay in prokaryotes and prokaryotic organelles; and RNA interference and other RNA mediators of gene expression. In addition to the talks and two poster sessions, there were three round tables: (1) Does translation occur in the nucleus? (2) Differences and similarities in the mechanisms of mRNA decay in different eukaryotes, and (3) RNA surveillance in bacteria?

Adventitial delivery of lentivirus-shRNA-ADAMTS-1 reduces venous stenosis formation in arteriovenous fistula.

Science.gov (United States)

Nieves Torres, Evelyn C; Yang, Binxia; Roy, Bhaskar; Janardhanan, Rajiv; Brahmbhatt, Akshaar; Leof, Ed; Mukhopadhyay, Debabrata; Misra, Sanjay

2014-01-01

Hemodialysis vascular access can develop venous neointimal hyperplasia (VNH) causing stenosis. Recent clinical and experimental data has demonstrated that there is increased expression of a disintegrin and metalloproteinase thrombospondin motifs-1 (ADAMTS-1) at site of VNH. The experiments outlined in the present paper were designed to test the hypothesis that targeting of the adventitia of the outflow vein of murine arteriovenous fistula (AVF) using a small hairpin RNA that inhibits ADAMTS-1 expression (LV-shRNA-ADAMTS-1) at the time of fistula creation will decrease VNH. At early time points, ADAMTS-1 expression was significantly decreased associated with a reduction in vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase-9 (MMP-9) (LV-shRNA-ADAMTS-1 transduced vessels vs. controls). These changes in gene and protein expression resulted in favorable vascular remodeling with a significant increase in mean lumen vessel area, decrease in media/adventitia area, with a significant increase in TUNEL staining accompanied with a decrease in cellular proliferation accompanied with a reduction in CD68 staining. Collectively, these results demonstrate that ADAMTS-1 transduced vessels of the outflow vein of AVF have positive vascular remodeling.

Adventitial delivery of lentivirus-shRNA-ADAMTS-1 reduces venous stenosis formation in arteriovenous fistula.

Directory of Open Access Journals (Sweden)

Evelyn C Nieves Torres

Full Text Available Hemodialysis vascular access can develop venous neointimal hyperplasia (VNH causing stenosis. Recent clinical and experimental data has demonstrated that there is increased expression of a disintegrin and metalloproteinase thrombospondin motifs-1 (ADAMTS-1 at site of VNH. The experiments outlined in the present paper were designed to test the hypothesis that targeting of the adventitia of the outflow vein of murine arteriovenous fistula (AVF using a small hairpin RNA that inhibits ADAMTS-1 expression (LV-shRNA-ADAMTS-1 at the time of fistula creation will decrease VNH. At early time points, ADAMTS-1 expression was significantly decreased associated with a reduction in vascular endothelial growth factor-A (VEGF-A and matrix metalloproteinase-9 (MMP-9 (LV-shRNA-ADAMTS-1 transduced vessels vs. controls. These changes in gene and protein expression resulted in favorable vascular remodeling with a significant increase in mean lumen vessel area, decrease in media/adventitia area, with a significant increase in TUNEL staining accompanied with a decrease in cellular proliferation accompanied with a reduction in CD68 staining. Collectively, these results demonstrate that ADAMTS-1 transduced vessels of the outflow vein of AVF have positive vascular remodeling.

Antibody-mediated platelet phagocytosis by human macrophages is inhibited by siRNA specific for sequences in the SH2 tyrosine kinase, Syk.

Science.gov (United States)

Lu, Ying; Wang, Weiming; Mao, Huiming; Hu, Hai; Wu, Yanling; Chen, Bing-Guan; Liu, Zhongmin

2011-01-01

Immune thrombocytopenia depends upon Fc receptor-mediated phagocytosis that involves signaling through the SH2 tyrosine kinase, Syk. We designed small interfering (siRNA) sequences complementary to Syk coding regions to decrease the expression of Syk in the human macrophage cell line, THP-1. To evaluate the functional effect of siRNA on phagocytosis, we developed a new in vitro assay for antibody-mediated platelet ingestion by THP-1 cells. Incubation of THP-1 cells at 37°C with fluorescence-labeled platelets and anti-platelet antibody promoted ingestion of platelets that could be quantitated by flow cytometry. Transfection of THP-1 cells with Syk-specific siRNA resulted in a reduction in the amount of FcγRII-associated Syk protein. Coincident with decreased Syk expression, we observed inhibition of antibody-mediated platelet ingestion. These results confirm a key role for Syk in antibody-mediated phagocytosis and suggest Syk-specific siRNA as a possible therapeutic candidate for immune thrombocytopenia. Copyright © 2011 Elsevier Inc. All rights reserved.

A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus

DEFF Research Database (Denmark)

Deng, Ling; Garrett, Roger Antony; Shah, Shiraz Ali

2013-01-01

Recent studies on CRISPR-based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon Sulfolobus islandicus REY15A encodes three interference modules, one of type IA and two of type IIIB...... targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S.¿islandicus REY15A which cooperate functionally by sharing a single Cas6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers....

GUItars: a GUI tool for analysis of high-throughput RNA interference screening data.

Directory of Open Access Journals (Sweden)

Asli N Goktug

Full Text Available High-throughput RNA interference (RNAi screening has become a widely used approach to elucidating gene functions. However, analysis and annotation of large data sets generated from these screens has been a challenge for researchers without a programming background. Over the years, numerous data analysis methods were produced for plate quality control and hit selection and implemented by a few open-access software packages. Recently, strictly standardized mean difference (SSMD has become a widely used method for RNAi screening analysis mainly due to its better control of false negative and false positive rates and its ability to quantify RNAi effects with a statistical basis. We have developed GUItars to enable researchers without a programming background to use SSMD as both a plate quality and a hit selection metric to analyze large data sets.The software is accompanied by an intuitive graphical user interface for easy and rapid analysis workflow. SSMD analysis methods have been provided to the users along with traditionally-used z-score, normalized percent activity, and t-test methods for hit selection. GUItars is capable of analyzing large-scale data sets from screens with or without replicates. The software is designed to automatically generate and save numerous graphical outputs known to be among the most informative high-throughput data visualization tools capturing plate-wise and screen-wise performances. Graphical outputs are also written in HTML format for easy access, and a comprehensive summary of screening results is written into tab-delimited output files.With GUItars, we demonstrated robust SSMD-based analysis workflow on a 3840-gene small interfering RNA (siRNA library and identified 200 siRNAs that increased and 150 siRNAs that decreased the assay activities with moderate to stronger effects. GUItars enables rapid analysis and illustration of data from large- or small-scale RNAi screens using SSMD and other traditional analysis

Binding of NUFIP2 to Roquin promotes recognition and regulation of ICOS mRNA.

Science.gov (United States)

Rehage, Nina; Davydova, Elena; Conrad, Christine; Behrens, Gesine; Maiser, Andreas; Stehklein, Jenny E; Brenner, Sven; Klein, Juliane; Jeridi, Aicha; Hoffmann, Anne; Lee, Eunhae; Dianzani, Umberto; Willemsen, Rob; Feederle, Regina; Reiche, Kristin; Hackermüller, Jörg; Leonhardt, Heinrich; Sharma, Sonia; Niessing, Dierk; Heissmeyer, Vigo

2018-01-19

The ubiquitously expressed RNA-binding proteins Roquin-1 and Roquin-2 are essential for appropriate immune cell function and postnatal survival of mice. Roquin proteins repress target mRNAs by recognizing secondary structures in their 3'-UTRs and by inducing mRNA decay. However, it is unknown if other cellular proteins contribute to target control. To identify cofactors of Roquin, we used RNA interference to screen ~1500 genes involved in RNA-binding or mRNA degradation, and identified NUFIP2 as a cofactor of Roquin-induced mRNA decay. NUFIP2 binds directly and with high affinity to Roquin, which stabilizes NUFIP2 in cells. Post-transcriptional repression of human ICOS by endogenous Roquin proteins requires two neighboring non-canonical stem-loops in the ICOS 3'-UTR. This unconventional cis-element as well as another tandem loop known to confer Roquin-mediated regulation of the Ox40 3'-UTR, are bound cooperatively by Roquin and NUFIP2. NUFIP2 therefore emerges as a cofactor that contributes to mRNA target recognition by Roquin.

mRNA decay proteins are targeted to poly(A+ RNA and dsRNA-containing cytoplasmic foci that resemble P-bodies in Entamoeba histolytica.

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Itzel López-Rosas

Full Text Available In higher eukaryotes, mRNA degradation and RNA-based gene silencing occur in cytoplasmic foci referred to as processing bodies (P-bodies. In protozoan parasites, the presence of P-bodies and their putative role in mRNA decay have yet to be comprehensively addressed. Identification of P-bodies might provide information on how mRNA degradation machineries evolved in lower eukaryotes. Here, we used immunofluorescence and confocal microscopy assays to investigate the cellular localization of mRNA degradation proteins in the human intestinal parasite Entamoeba histolytica and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the EhXRN2 exoribonuclease and the EhDCP2 decapping enzyme, were localized in cytoplasmic foci in a pattern resembling P-body organization. Given that amoebic foci appear to be smaller and less rounded than those described in higher eukaryotes, we have named them "P-body-like structures". These foci contain additional mRNA degradation factors, including the EhCAF1 deadenylase and the EhAGO2-2 protein involved in RNA interference. Biochemical analysis revealed that EhCAF1 co-immunoprecipitated with EhXRN2 but not with EhDCP2 or EhAGO2-2, thus linking deadenylation to 5'-to-3' mRNA decay. The number of EhCAF1-containing foci significantly decreased after inhibition of transcription and translation with actinomycin D and cycloheximide, respectively. Furthermore, results of RNA-FISH assays showed that (i EhCAF1 colocalized with poly(A(+ RNA and (ii during silencing of the Ehpc4 gene by RNA interference, EhAGO2-2 colocalized with small interfering RNAs in cytoplasmic foci. Our observation of decapping, deadenylation and RNA interference proteins within P-body-like foci suggests that these structures have been conserved after originating in the early evolution of eukaryotic lineages. To the best of our knowledge, this is the first study to report on the localization of mRNA decay proteins within P

RNA interference in Lepidoptera

DEFF Research Database (Denmark)

Terenius, Ole; Papanicolaou, Alexie; Garbutt, Jennie S.

2011-01-01

in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our...... experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi...... is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success...

Detection of Replication Competent Lentivirus Using a qPCR Assay for VSV-G

Directory of Open Access Journals (Sweden)

Lindsey M. Skrdlant

2018-03-01

Full Text Available Lentiviral vectors are a common tool used to introduce new and corrected genes into cell therapy products for treatment of human diseases. Although lentiviral vectors are ideal for delivery and stable integration of genes of interest into the host cell genome, they potentially pose risks to human health, such as integration-mediated transformation and generation of a replication competent lentivirus (RCL capable of infecting non-target cells. In consideration of the latter risk, all cell-based products modified by lentiviral vectors and intended for patient use must be tested for RCL prior to treatment of the patient. Current Food and Drug Administration (FDA guidelines recommend use of cell-based assays to this end, which can take up to 6 weeks for results. However, qPCR-based assays are a quick alternative for rapid assessment of RCL in products intended for fresh infusion. We describe here the development and qualification of a qPCR assay based on detection of envelope gene sequences (vesicular stomatitis virus G glycoprotein [VSV-G] for RCL in accordance with Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE guidelines. Our results demonstrate the sensitivity, linearity, specificity, and reproducibility of detection of VSV-G sequences, with a low false-positive rate. These procedures are currently being used in our phase 1 clinical investigations.

Delivery of chitosan/dsRNA nanoparticles for silencing of wing development vestigial (vg) gene in Aedes aegypti mosquitoes.

Science.gov (United States)

Ramesh Kumar, D; Saravana Kumar, P; Gandhi, M Rajiv; Al-Dhabi, Naif Abdullah; Paulraj, M Gabriel; Ignacimuthu, S

2016-05-01

RNA interference (RNAi) has been used as a gene silencing strategy by the introduction of long double stranded RNA (dsRNA) for the control of pest insects. The aim of the present study was to examine whether the expression of vg gene which is responsible for wing development, can be repressed by chitosan/dsRNA based nanoparticles in Aedes aegypti. The vestigial gene (vg) was amplified from adult mosquito and cloned in pLitmus28i vector. Genetically engineered recombinant plasmid was transformed into RNase III deficient strain for synthesis of bacterially expressed dsRNA. Nanoparticles were prepared via electrostatic interaction between cationic polymer chitosan and anionic nucleic acids (dsRNA). The formation of chitosan/dsRNAnanoparticles and their size were confirmed by Atomic force microscopy (AFM). Chitosan/dsRNA mediated knockdown of Enhanced Green Fluorescence Protein (EGFP) was demonstrated in Sf21 cells. Further, we tested whether such an approach could be used to target vg gene in Ae. aegypti. The results showed that chitosan/dsRNA caused significant mortality, delayed growth development and caused adult wing-malformation. A qRT-PCR analysis confirmed that the chitosan/dsRNA mediated transcriptional level was downregulated. Our findings suggest that vg gene intervention strategies through RNAi can emerge as viable option for pest control. Copyright © 2016 Elsevier B.V. All rights reserved.

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Köster, Tino

2017-04-13

RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Kö ster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

2017-01-01

RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

Science.gov (United States)

Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

2015-12-15

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

LncRNA H19 and Target Gene-mediated Cleft Palate Induced by TCDD.

Science.gov (United States)

Gao, Li Yun; Zhang, Feng Quan; Zhao, Wei Hui; Han, Guang Liang; Wang, Xiao; Li, Qiang; Gao, Shan Shan; Wu, Wei Dong

2017-09-01

This study investigated the role of long non-coding RNAs (lncRNAs) in the development of the palatal tissues. Cleft palates in mice were induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression levels of long non-coding RNA H19 (lncRNA H19) and insulin-like growth factor 2 (IGF2) gene were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The rate of occurrence of cleft palate was found to be 100% by TCDD exposure, and TCDD could cause short upper limb, cerebral fissure, webbed neck, and short neck. The expression levels of lncRNA H19 and IGF2 gene specifically showed embryo age-related differences on E13, E14, and E15 in the palatal tissues. The expression levels of lncRNA H19 and IGF2 gene showed an inverse relationship on E13, E14, and E15. These findings demonstrated that lncRNA H19 and IGF2 can mediate the development of mouse cleft palate. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa.

Science.gov (United States)

Sonnleitner, Elisabeth; Wulf, Alexander; Campagne, Sébastien; Pei, Xue-Yuan; Wolfinger, Michael T; Forlani, Giada; Prindl, Konstantin; Abdou, Laetitia; Resch, Armin; Allain, Frederic H-T; Luisi, Ben F; Urlaub, Henning; Bläsi, Udo

2018-02-16

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA. Through these interactions, Crc enhances the stability of Hfq/Crc/RNA complexes, which can explain its facilitating role in Hfq-mediated translational repression. Hence, these studies revealed for the first time insights into how an interacting protein can modulate Hfq function. Moreover, Crc is shown to interfere with binding of a regulatory RNA to Hfq, which bears implications for riboregulation. These results are discussed in terms of a working model, wherein Crc prioritizes the function of Hfq toward utilization of favored carbon sources.

Long non-coding RNA taurine-upregulated gene 1 correlates with poor prognosis, induces cell proliferation, and represses cell apoptosis via targeting aurora kinase A in adult acute myeloid leukemia.

Science.gov (United States)

Wang, Xinfeng; Zhang, Lina; Zhao, Fan; Xu, Ruirong; Jiang, Jie; Zhang, Chenglu; Liu, Hong; Huang, Hongming

2018-04-13

This study aimed to investigate the correlation of long non-coding RNA (lncRNA) taurine-upregulated gene 1 (TUG1) with clinicopathological feature and prognosis, and to explore its effect on cell proliferation and apoptosis as well as the relevant target genes in adult acute myeloid leukemia (AML). LncRNA TUG1 expression was detected in bone marrow samples from 186 AML patients and 62 controls. Blank mimic, lncRNA TUG1 mimic, blank inhibitor, and lncRNA TUG1 inhibitor lentivirus vectors were transfected in KG-1 cells. Rescue experiment was performed by transfection of lncRNA TUG1 inhibitor and aurora kinase A (AURKA) mimic lentivirus vectors. Cell proliferation, apoptosis, RNA, and protein expressions were determined by CKK-8, annexin V-FITC-propidium iodide, quantitative polymerase chain reaction, and western blot assays. LncRNA TUG1 expression was higher in AML patients compared to controls and correlated with higher white blood cell counts, monosomal karyotype, FLT3-ITD mutation, poor-risk stratification, and poor prognosis, which independently predicted worse event-free survival and overall survival. In vitro, lncRNA TUG1 expression was higher in AML cell lines (KG-1, MOLM-14, HL-60, NB-4, and THP-1 cells) compared to controls. LncRNA TUG1 mimic promoted cell proliferation and decreased cell apoptosis rate, while lncRNA TUG1 inhibitor repressed cell proliferation and increased cell apoptosis rate. Rescue experiment showed that AURKA attenuated the influence of lncRNA TUG1 on AML cell proliferation and apoptosis. In conclusion, lncRNA TUG1 associates with advanced disease and worse prognosis in adult AML patients, and it induces AML cell proliferation and represses cell apoptosis via targeting AURKA.

How the RNA isolation method can affect microRNA microarray results

DEFF Research Database (Denmark)

Podolska, Agnieszka; Kaczkowski, Bogumil; Litman, Thomas

2011-01-01

RNA microarray analysis on porcine brain tissue. One method is a phenol-guanidine isothiocyanate-based procedure that permits isolation of total RNA. The second method, miRVana™ microRNA isolation, is column based and recovers the small RNA fraction alone. We found that microarray analyses give different results...... that depend on the RNA fraction used, in particular because some microRNAs appear very sensitive to the RNA isolation method. We conclude that precautions need to be taken when comparing microarray studies based on RNA isolated with different methods.......The quality of RNA is crucial in gene expression experiments. RNA degradation interferes in the measurement of gene expression, and in this context, microRNA quantification can lead to an incorrect estimation. In the present study, two different RNA isolation methods were used to perform micro...

Feline Immunodeficiency Virus Evolutionarily Acquires Two Proteins, Vif and Protease, Capable of Antagonizing Feline APOBEC3.

Science.gov (United States)

Yoshikawa, Rokusuke; Takeuchi, Junko S; Yamada, Eri; Nakano, Yusuke; Misawa, Naoko; Kimura, Yuichi; Ren, Fengrong; Miyazawa, Takayuki; Koyanagi, Yoshio; Sato, Kei

2017-06-01

The interplay between viral and host proteins has been well studied to elucidate virus-host interactions and their relevance to virulence. Mammalian genes encode apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins, which act as intrinsic restriction factors against lentiviruses. To overcome APOBEC3-mediated antiviral actions, lentiviruses have evolutionarily acquired an accessory protein, viral infectivity factor (Vif), and Vif degrades host APOBEC3 proteins via a ubiquitin/proteasome-dependent pathway. Although the Vif-APOBEC3 interaction and its evolutionary significance, particularly those of primate lentiviruses (including HIV) and primates (including humans), have been well investigated, those of nonprimate lentiviruses and nonprimates are poorly understood. Moreover, the factors that determine lentiviral pathogenicity remain unclear. Here, we focus on feline immunodeficiency virus (FIV), a pathogenic lentivirus in domestic cats, and the interaction between FIV Vif and feline APOBEC3 in terms of viral virulence and evolution. We reveal the significantly reduced diversity of FIV subtype B compared to that of other subtypes, which may associate with the low pathogenicity of this subtype. We also demonstrate that FIV subtype B Vif is less active with regard to feline APOBEC3 degradation. More intriguingly, we further reveal that FIV protease cleaves feline APOBEC3 in released virions. Taken together, our findings provide evidence that a lentivirus encodes two types of anti-APOBEC3 factors, Vif and viral protease. IMPORTANCE During the history of mammalian evolution, mammals coevolved with retroviruses, including lentiviruses. All pathogenic lentiviruses, excluding equine infectious anemia virus, have acquired the vif gene via evolution to combat APOBEC3 proteins, which are intrinsic restriction factors against exogenous lentiviruses. Here we demonstrate that FIV, a pathogenic lentivirus in domestic cats, antagonizes feline APOBEC3

Universal, colorimetric microRNA detection strategy based on target-catalyzed toehold-mediated strand displacement reaction

Science.gov (United States)

Park, Yeonkyung; Lee, Chang Yeol; Kang, Shinyoung; Kim, Hansol; Park, Ki Soo; Park, Hyun Gyu

2018-02-01

In this work, we developed a novel, label-free, and enzyme-free strategy for the colorimetric detection of microRNA (miRNA), which relies on a target-catalyzed toehold-mediated strand displacement (TMSD) reaction. The system employs a detection probe that specifically binds to the target miRNA and sequentially releases a catalyst strand (CS) intended to trigger the subsequent TMSD reaction. Thus, the presence of target miRNA releases the CS that mediates the formation of an active G-quadruplex DNAzyme which is initially caged and inactivated by a blocker strand. In addition, a fuel strand that is supplemented for the recycling of the CS promotes another TMSD reaction, consequently generating a large number of active G-quadruplex DNAzymes. As a result, a distinct colorimetric signal is produced by the ABTS oxidation promoted by the peroxidase mimicking activity of the released G-quadruplex DNAzymes. Based on this novel strategy, we successfully detected miR-141, a promising biomarker for human prostate cancer, with high selectivity. The diagnostic capability of this system was also demonstrated by reliably determining target miR-141 in human serum, showing its great potential towards real clinical applications. Importantly, the proposed approach is composed of separate target recognition and signal transduction modules. Thus, it could be extended to analyze different target miRNAs by simply redesigning the detection probe while keeping the same signal transduction module as a universal signal amplification unit, which was successfully demonstrated by analyzing another target miRNA, let-7d.

Immunogenicity investigations of lipidoid structures in vitro and in silico: Modulating lipidoid-mediated TLR4 activation by nanoparticle design

DEFF Research Database (Denmark)

de Groot, Anne Marit; Thanki, Kaushik; Gangloff, Monique

2018-01-01

, we showed that encapsulation of siRNA in lipid-polymer hybrid nanoparticles (LPNs), based on poly(DL-lactic-co-glycolic acid) (PLGA) and cationic lipid-like materials (lipidoids), remarkably enhances intracellular delivery of siRNA as compared to siRNA delivery with LPNs modified...... acid lipid particles, which was the reference formulation for siRNA delivery, proved to activate TLR4. However, by combining lipidoids with PLGA into LPNs, TLR4 activation was abrogated. Thus, lipidoid-mediated TLR4 activation during siRNA delivery may be modulated via optimization of the formulation......Therapeutics based on small interfering RNA (siRNA) have promising potential as antiviral and anti-inflammatory agents. To deliver siRNA across cell membranes to reach the RNA interference pathway in the cytosol of target cells, non-viral nanoparticulate delivery approaches are explored. Recently...

Analysis of the siRNA-Mediated Gene Silencing Process Targeting Three Homologous Genes Controlling Soybean Seed Oil Quality.

Science.gov (United States)

Lu, Sha; Yin, Xiaoyan; Spollen, William; Zhang, Ning; Xu, Dong; Schoelz, James; Bilyeu, Kristin; Zhang, Zhanyuan J

2015-01-01

In the past decade, RNA silencing has gained significant attention because of its success in genomic scale research and also in the genetic improvement of crop plants. However, little is known about the molecular basis of siRNA processing in association with its target transcript. To reveal this process for improving hpRNA-mediated gene silencing in crop plants, the soybean GmFAD3 gene family was chosen as a test model. We analyzed RNAi mutant soybean lines in which three members of the GmFAD3 gene family were silenced. The silencing levels of FAD3A, FAD3B and FAD3C were correlated with the degrees of sequence homology between the inverted repeat of hpRNA and the GmFAD3 transcripts in the RNAi lines. Strikingly, transgenes in two of the three RNAi lines were heavily methylated, leading to a dramatic reduction of hpRNA-derived siRNAs. Small RNAs corresponding to the loop portion of the hairpin transcript were detected while much lower levels of siRNAs were found outside of the target region. siRNAs generated from the 318-bp inverted repeat were found to be diced much more frequently at stem sequences close to the loop and associated with the inferred cleavage sites on the target transcripts, manifesting "hot spots". The top candidate hpRNA-derived siRNA share certain sequence features with mature miRNA. This is the first comprehensive and detailed study revealing the siRNA-mediated gene silencing mechanism in crop plants using gene family GmFAD3 as a test model.

MicroRNA-Mediated Downregulation of the Potassium Channel Kv4.2 Contributes to Seizure Onset

Directory of Open Access Journals (Sweden)

Christina Gross

2016-09-01

Full Text Available Seizures are bursts of excessive synchronized neuronal activity, suggesting that mechanisms controlling brain excitability are compromised. The voltage-gated potassium channel Kv4.2, a major mediator of hyperpolarizing A-type currents in the brain, is a crucial regulator of neuronal excitability. Kv4.2 expression levels are reduced following seizures and in epilepsy, but the underlying mechanisms remain unclear. Here, we report that Kv4.2 mRNA is recruited to the RNA-induced silencing complex shortly after status epilepticus in mice and after kainic acid treatment of hippocampal neurons, coincident with reduction of Kv4.2 protein. We show that the microRNA miR-324-5p inhibits Kv4.2 protein expression and that antagonizing miR-324-5p is neuroprotective and seizure suppressive. MiR-324-5p inhibition also blocks kainic-acid-induced reduction of Kv4.2 protein in vitro and in vivo and delays kainic-acid-induced seizure onset in wild-type but not in Kcnd2 knockout mice. These results reveal an important role for miR-324-5p-mediated silencing of Kv4.2 in seizure onset.

The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

DEFF Research Database (Denmark)

Elmlund, Hans; Baraznenok, Vera; Lindahl, Martin

2006-01-01

CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8......-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II....

RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

International Nuclear Information System (INIS)

Smialowska, Agata; Djupedal, Ingela; Wang, Jingwen; Kylsten, Per; Swoboda, Peter; Ekwall, Karl

2014-01-01

Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe

Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

Energy Technology Data Exchange (ETDEWEB)

Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong (Yale); (Cornell); (Tsinghua)

2012-10-10

Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.

CRISPR/Cas13 as a Tool for RNA Interference

KAUST Repository

Ali, Zahir; Mahas, Ahmed; Mahfouz, Magdy M.

2018-01-01

Almost all biological processes involve RNA, making it crucial to develop tools for manipulation of the transcriptome. The bacterial CRISPR/Cas13 system was recently rewired to facilitate RNA manipulation in eukaryotes, including plants. We discuss

Increased keratinocyte proliferation initiated through downregulation of desmoplakin by RNA interference

International Nuclear Information System (INIS)

Wan Hong; South, Andrew P.; Hart, Ian R.

2007-01-01

The intercellular adhesive junction desmosomes are essential for the maintenance of tissue structure and integrity in skin. Desmoplakin (Dp) is a major obligate plaque protein which plays a fundamental role in anchoring intermediate filaments to desmosomal cadherins. Evidence from hereditary human disease caused by mutations in the gene encoding Dp, e.g. Dp haploinsufficiency, suggests that alterations in Dp expression result not only in the disruption of tissue structure and integrity but also could evoke changes in keratinocyte proliferation. We have used transient RNA interference (RNAi) to downregulate Dp specifically in HaCaT keratinocytes. We showed that this Dp downregulation also caused reduced expression of several other desmosomal proteins. Increased cell proliferation and enhanced G 1 -to-S-phase entry in the cell cycle, as monitored by colonial cellular density and BrdU incorporation, were seen in Dp RNAi-treated cells. These proliferative changes were associated with elevated phospho-ERK1/2 and phospho-Akt levels. Furthermore, this increase in phospho-ERK/1/2 and phospho-Akt levels was sustained in Dp RNAi-treated cells at confluence whereas in control cells there was a significant reduction in phosphorylation of ERK1/2. This study indicates that Dp may participate in the regulation of keratinocyte cell proliferation by, in part at least, regulating cell cycle progression

Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus

International Nuclear Information System (INIS)

Zhang Jing; Yamada, Osamu; Sakamoto, Takashi; Yoshida, Hiroshi; Iwai, Takahiro; Matsushita, Yoshihisa; Shimamura, Hideo; Araki, Hiromasa; Shimotohno, Kunitada

2004-01-01

Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bγ), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects

Long noncoding RNA TUG1 alleviates extracellular matrix accumulation via mediating microRNA-377 targeting of PPARγ in diabetic nephropathy.

Science.gov (United States)

Duan, Li-Jun; Ding, Min; Hou, Li-Jun; Cui, Yuan-Tao; Li, Chun-Jun; Yu, De-Min

2017-03-11

Long noncoding RNA taurine-upregulated gene 1 (lncRNA TUG1) has been reported to play a key role in the progression of diabetic nephropathy (DN). However, the role of lncRNA TUG1 in the regulation of diabetic nephropathy remains largely unknown. The aim of the present study is to identify the regulation of lncRNA TUG1 on extracellular matrix accumulation via mediating microRNA-377 targeting of PPARγ, and investigate the underlying mechanisms in progression of DN. Microarray was performed to screen differentially expressed miRNAs in db/db DN mice. Afterwards, computational prediction programs (TargetScan, miRanda, PicTar and miRGen) was applied to predict the target gene of miRNAs. The complementary binding of miRNA and lncRNA was assessed by luciferase assays. Protein and mRNA expression were detected by western blot and real time quantitate PCR. MiRNA-377 was screened by miRNA microarray and differentially up-regulated in db/db DN mice. PPARγ was predicted to be the target of miR-377 and the prediction was verified by luciferase assays. Expression of miR-377 was up-regulated in mesangial cell treated with high glucose (25 mM), and overexpression of miR-377 inhibited PPARγ expression and promoted PAI-1 and TGF-β1 expression. The expression of TUG1 antagonized the effect of miR-377 on the downregulation of its target PPARγ and inhibited extracellular matrix accumulation, including PAI-1, TGF-β1, fibronectin (FN) and collagen IV (Col IV), induced by high glucose. LncRNA TUG1 acts as an endogenous sponge of miR-377 and downregulates miR-377 expression levels, and thereby relieving the inhibition of its target gene PPARγ and alleviates extracellular matrix accumulation of mesangial cells, which provides a novel insight of diabetic nephropathy pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis

DEFF Research Database (Denmark)

Siegel, Gabriele; Obernosterer, Gregor; Fiore, Roberto

2009-01-01

of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the alpha(13) subunits of G proteins (Galpha(13)). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Galpha(13) both...... suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Galpha(13) might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized...

Mammalian tissues defective in nonsense-mediated mRNA decay display highly aberrant splicing patterns