scholarly journals The Assembly and Maintenance of Heterochromatin Initiated by Transgene Repeats Are Independent of the RNA Interference Pathway in Mammalian Cells

2006 ◽  
Vol 26 (11) ◽  
pp. 4028-4040 ◽  
Author(s):  
Fangwei Wang ◽  
Naoki Koyama ◽  
Hiroko Nishida ◽  
Tokuko Haraguchi ◽  
Walter Reith ◽  
...  
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Cytosine Methylation ◽  
Chromatin Condensation ◽  
Rnai Pathway ◽  
Small Interfering Rnas ◽  
Heterochromatin Protein 1 ◽  
Independent Manner ◽  
Heterochromatin Protein ◽  
Cpg Dinucleotides

ABSTRACT A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.

scholarly journals High- and Low-mobility Populations of HP1 in Heterochromatin of Mammalian Cells

2004 ◽  
Vol 15 (6) ◽  
pp. 2819-2833 ◽  
Author(s):  
Lars Schmiedeberg ◽  
Klaus Weisshart ◽  
Stephan Diekmann ◽  
Gabriele Meyer zu Hoerste ◽  
Peter Hemmerich
Keyword(s):  
Mammalian Cells ◽  
Constitutive Heterochromatin ◽  
Chromatin Condensation ◽  
Cell Types ◽  
Correlation Spectroscopy ◽  
Chromosomal Protein ◽  
Mitotic Chromosomes ◽  
Heterochromatin Protein 1 ◽  
Stable Conformation ◽  
Fluorescence Correlation

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein with functions in euchromatin and heterochromatin. Here we investigated the diffusional behaviors of HP1 isoforms in mammalian cells. Using fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) we found that in interphase cells most HP1 molecules (50–80%) are highly mobile (recovery halftime: t1/2 ≈ 0.9 s; diffusion coefficient: D ≈ 0.6–0.7 μm2 s-1). Twenty to 40% of HP1 molecules appear to be incorporated into stable, slow-moving oligomeric complexes (t1/2 ≈ 10 s), and constitutive heterochromatin of all mammalian cell types analyzed contain 5–7% of very slow HP1 molecules. The amount of very slow HP1 molecules correlated with the chromatin condensation state, mounting to more than 44% in condensed chromatin of transcriptionally silent cells. During mitosis 8–14% of GFP-HP1α, but not the other isoforms, are very slow within pericentromeric heterochromatin, indicating an isoform-specific function of HP1α in heterochromatin of mitotic chromosomes. These data suggest that mobile as well as very slow populations of HP1 may function in concert to maintain a stable conformation of constitutive heterochromatin throughout the cell cycle.

scholarly journals Transfer and Expression of Small Interfering RNAs in Mammalian Cells Using Lentiviral Vectors

Acta Naturae ◽  
2013 ◽  
Vol 5 (2) ◽  
pp. 7-18 ◽  
Author(s):  
T. D. Lebedev ◽  
P. V. Spirin ◽  
V. S. Prassolov
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Lentiviral Vectors ◽  
Cell Delivery ◽  
Small Interfering Rnas ◽  
Shrna Expression ◽  
Widespread Application ◽  
Efficient Gene ◽  
Interfering Rna

RNA interference is a convenient tool for modulating gene expression. The widespread application of RNA interference is made difficult because of the imperfections of the methods used for efficient target cell delivery of whatever genes are under study. One of the most convenient and efficient gene transfer and expression systems is based on the use of lentiviral vectors, which direct the synthesis of small hairpin RNAs (shRNAs), the precursors of siRNAs. The application of these systems enables one to achieve sustainable and long-term shRNA expression in cells. This review considers the adaptation of the processing of artificial shRNA to the mechanisms used by cellular microRNAs and simultaneous expression of several shRNAs as potential approaches for producing lentiviral vectors that direct shRNA synthesis. Approaches to using RNA interference for the treatment of cancer, as well as hereditary and viral diseases, are under active development today. The improvement made to the methods for constructing lentiviral vectors and the investigation into the mechanisms of processing of small interfering RNA allow one to now consider lentiviral vectors that direct shRNA synthesis as one of the most promising tools for delivering small interfering RNAs.

scholarly journals The Capsid Protein of Semliki Forest Virus Antagonizes RNA Interference in Mammalian Cells

2019 ◽  
Vol 94 (3) ◽  
Author(s):  
Qi Qian ◽  
Hui Zhou ◽  
Ting Shu ◽  
Jingfang Mu ◽  
Yuan Fang ◽  
...  
Keyword(s):  
Rna Interference ◽  
Capsid Protein ◽  
Mammalian Cells ◽  
Semliki Forest Virus ◽  
Rna Viruses ◽  
Strong Support ◽  
Life Cycles ◽  
Immune Defense ◽  
Rnai Pathway ◽  
Viral Suppressors

ABSTRACT RNA interference (RNAi) is a conserved antiviral immune defense in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs) to counteract antiviral RNAi. Alphaviruses are a large group of positive-stranded RNA viruses that maintain their transmission and life cycles in both mosquitoes and mammals. However, there is little knowledge about how alphaviruses antagonize RNAi in both host organisms. In this study, we identified that Semliki Forest virus (SFV) capsid protein can efficiently suppress RNAi in both insect and mammalian cells by sequestrating double-stranded RNA and small interfering RNA. More importantly, when the VSR activity of SFV capsid was inactivated by reverse genetics, the resulting VSR-deficient SFV mutant showed severe replication defects in mammalian cells, which could be rescued by blocking the RNAi pathway. Besides, capsid protein of Sindbis virus also inhibited RNAi in cells. Together, our findings show that SFV uses capsid protein as VSR to antagonize RNAi in infected mammalian cells, and this mechanism is probably used by other alphaviruses, which shed new light on the knowledge of SFV and alphavirus. IMPORTANCE Alphaviruses are a genus of positive-stranded RNA viruses and include numerous important human pathogens, such as Chikungunya virus, Ross River virus, Western equine encephalitis virus, etc., which create the emerging and reemerging public health threat worldwide. RNA interference (RNAi) is one of the most important antiviral mechanisms in plants and insects. Accumulating evidence has provided strong support for the existence of antiviral RNAi in mammals. In response to antiviral RNAi, viruses have evolved to encode viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. It is unclear whether alphaviruses encode VSRs that can suppress antiviral RNAi during their infection in mammals. In this study, we first uncovered that capsid protein encoded by Semliki Forest virus (SFV), a prototypic alphavirus, had a potent VSR activity that can antagonize antiviral RNAi in the context of SFV infection in mammalian cells, and this mechanism is probably used by other alphaviruses.

scholarly journals Deletion of Cytoplasmic Double-Stranded RNA Sensors Does Not Uncover Viral Small Interfering RNA Production in Human Cells

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Susan Schuster ◽  
Lotte E. Tholen ◽  
Gijs J. Overheul ◽  
Frank J. M. van Kuppeveld ◽  
Ronald P. van Rij
Keyword(s):  
Rna Interference ◽  
Antiviral Activity ◽  
Mammalian Cells ◽  
Rna Viruses ◽  
Antiviral Immunity ◽  
Antiviral Effect ◽  
Interferon Response ◽  
Rnai Pathway ◽  
Differentiated Cells ◽  
Positive Sense

ABSTRACT The contribution of the RNA interference (RNAi) pathway in antiviral immunity in vertebrates has been widely debated. It has been proposed that RNAi possesses antiviral activity in mammalian systems but that its antiviral effect is masked by the potent antiviral interferon response in differentiated mammalian cells. In this study, we show that inactivation of the interferon response is not sufficient to uncover antiviral activity of RNAi in human epithelial cells infected with three wild-type positive-sense RNA viruses. Antiviral immunity in insects and plants is mediated by the RNA interference (RNAi) pathway in which viral long double-stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer enzymes. Although this pathway is evolutionarily conserved, its involvement in antiviral defense in mammals is the subject of debate. In vertebrates, recognition of viral RNA induces a sophisticated type I interferon (IFN)-based immune response, and it has been proposed that this response masks or inhibits antiviral RNAi. To test this hypothesis, we analyzed viral small RNA production in differentiated cells deficient in the cytoplasmic RNA sensors RIG-I and MDA5. We did not detect 22-nucleotide (nt) viral siRNAs upon infection with three different positive-sense RNA viruses. Our data suggest that the depletion of cytoplasmic RIG-I-like sensors is not sufficient to uncover viral siRNAs in differentiated cells. IMPORTANCE The contribution of the RNA interference (RNAi) pathway in antiviral immunity in vertebrates has been widely debated. It has been proposed that RNAi possesses antiviral activity in mammalian systems but that its antiviral effect is masked by the potent antiviral interferon response in differentiated mammalian cells. In this study, we show that inactivation of the interferon response is not sufficient to uncover antiviral activity of RNAi in human epithelial cells infected with three wild-type positive-sense RNA viruses.

scholarly journals Conditional Suppression of Cellular Genes: Lentivirus Vector-Mediated Drug-Inducible RNA Interference

2003 ◽  
Vol 77 (16) ◽  
pp. 8957-8951 ◽  
Author(s):  
Maciej Wiznerowicz ◽  
Didier Trono
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Developmental Genetics ◽  
Present System ◽  
Small Interfering Rnas ◽  
Lentivirus Vector ◽  
Molecular Therapeutics ◽  
Cellular Genes ◽  
Conditional Suppression

ABSTRACT RNA interference has emerged as a powerful technique to downregulate the expression of specific genes in cells and in animals, thus opening new perspectives in fields ranging from developmental genetics to molecular therapeutics. Here, we describe a method that significantly expands the potential of RNA interference by permitting the conditional suppression of genes in mammalian cells. Within a lentivirus vector background, we subjected the polymerase III promoter-dependent production of small interfering RNAs to doxycycline-controllable transcriptional repression. The resulting system can achieve the highly efficient and completely drug-inducible knockdown of cellular genes. As lentivirus vectors can stably transduce a wide variety of targets both in vitro and in vivo and can be used to generate transgenic animals, the present system should have broad applications.

scholarly journals Selection and Characterization of RNA Interference-Deficient Trypanosomes Impaired in Target mRNA Degradation

2004 ◽  
Vol 3 (6) ◽  
pp. 1445-1453 ◽  
Author(s):  
Huafang Shi ◽  
Nathalie Chamond ◽  
Christian Tschudi ◽  
Elisabetta Ullu
Keyword(s):  
Rna Interference ◽  
Biological Significance ◽  
Cellular Level ◽  
Rnai Pathway ◽  
Small Interfering Rnas ◽  
Wild Type ◽  
Double Stranded Rna ◽  
Target Mrna ◽  
Insight Into

ABSTRACT Genetic analysis of the RNA interference (RNAi) pathway in Trypanosoma brucei has so far revealed one essential component, namely, TbAGO1, encoding a member of the Argonaute protein family. To gain further insight into the RNAi mechanism and its biological significance, we selected RNAi-deficient trypanosomes by using repeated cycles of electroporation with α-tubulin double-stranded RNA, a treatment that blocks cytokinesis in wild-type cells. Two independent clones, termed RiD-1 (for RNAi-deficient clone 1) and RiD-2, were characterized. At the cellular level, only RiD-1 trypanosomes showed a significant increase in doubling time with the concomitant accumulation of cells defective in the completion of cytokinesis. At the RNA level, both clones accumulated wild-type amounts of small interfering RNAs and displayed elevated levels of retroposon transcripts, the hallmark of RNAi deficiency in T. brucei. Importantly, both RiD-1 and RiD-2 clones were defective in the degradation of target mRNA, suggesting an impairment of the activity of AGO1, the putative RNAi endonuclease. Since in RiD cells the AGO1 gene was not mutated and was expressed at wild-type levels, we propose that in trypanosomes the cleavage of mRNA by AGO1 is regulated by the interaction with another factor(s).

scholarly journals The Baculovirus Antiapoptotic p35 Protein Functions as an Inhibitor of the Host RNA Interference Antiviral Response

2015 ◽  
Vol 89 (16) ◽  
pp. 8182-8192 ◽  
Author(s):  
Mohammad Mehrabadi ◽  
Mazhar Hussain ◽  
Leila Matindoost ◽  
Sassan Asgari
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Microbial Control ◽  
Rnai Pathway ◽  
Inhibitor Of Apoptosis ◽  
Virus Infections ◽  
Content Type ◽  
Protein Functions ◽  
Dsrna Cleavage ◽  
Rnai Response

ABSTRACTRNA interference (RNAi) is considered an ancient antiviral defense in diverse organisms, including insects. Virus infections generate double-strand RNAs (dsRNAs) that trigger the RNAi machinery to process dsRNAs into virus-derived short interfering RNAs (vsiRNAs), which target virus genomes, mRNAs, or replication intermediates. Viruses, in turn, have evolved viral suppressors of RNAi (VSRs) to counter host antiviral RNAi. Following recent discoveries that insects mount an RNAi response against DNA viruses, in this study, we found thatAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) infection similarly induces an RNAi response inSpodoptera frugiperdacells by generating a large number of vsiRNAs postinfection. Interestingly, we found that AcMNPV expresses a potent VSR to counter RNAi. The viralp35gene, which is well known as an inhibitor of apoptosis, was found to be responsible for the suppression of RNAi in diverse insect and mammalian cells. The VSR activity of p35 was further confirmed by ap35-null AcMNPV that did not suppress the response. In addition, our results showed that the VSR activity is not due to inhibition of dsRNA cleavage by Dicer-2 but acts downstream in the RNAi pathway. Furthermore, we found that the VSR activity is not linked to the antiapoptotic activity of the protein. Overall, our results provide evidence for the existence of VSR activity in a double-stranded DNA virus and identify the responsible gene, which is involved in the inhibition of RNAi as well as apoptosis.IMPORTANCEOur findings demonstrate the occurrence of an insect RNAi response against a baculovirus (AcMNPV) that is highly utilized in microbial control, biological and biomedical research, and protein expression. Moreover, our investigations led to the identification of a viral suppressor of RNAi activity and the gene responsible for the activity. Notably, this gene is also a potent inhibitor of apoptosis. The outcomes signify the dual role of a virus-encoded protein in nullifying two key antiviral responses, apoptosis and RNAi.

scholarly journals KAP1 is an antiparallel dimer with a functional asymmetry

2019 ◽  
Vol 2 (4) ◽  
pp. e201900349 ◽  
Author(s):  
Giulia Fonti ◽  
Maria J Marcaida ◽  
Louise C Bryan ◽  
Sylvain Träger ◽  
Alexandra S Kalantzi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Scattering Data ◽  
Heterochromatin Protein 1 ◽  
Heterochromatin Protein ◽  
Krab Domain ◽  
Functional Implications ◽  
New Gene ◽  
Expression In Mammalian Cells

KAP1 (KRAB domain–associated protein 1) plays a fundamental role in regulating gene expression in mammalian cells by recruiting different transcription factors and altering the chromatin state. In doing so, KAP1 acts both as a platform for macromolecular interactions and as an E3 small ubiquitin modifier ligase. This work sheds light on the overall organization of the full-length protein combining solution scattering data, integrative modeling, and single-molecule experiments. We show that KAP1 is an elongated antiparallel dimer with an asymmetry at the C-terminal domains. This conformation is consistent with the finding that the Really Interesting New Gene (RING) domain contributes to KAP1 auto-SUMOylation. Importantly, this intrinsic asymmetry has key functional implications for the KAP1 network of interactions, as the heterochromatin protein 1 (HP1) occupies only one of the two putative HP1 binding sites on the KAP1 dimer, resulting in an unexpected stoichiometry, even in the context of chromatin fibers.

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