scholarly journals Main constraints for RNAi induced by expressed long dsRNA in mouse cells

2018 ◽  
Author(s):  
Tomas Demeter ◽  
Michaela Vaskovicova ◽  
Radek Malik ◽  
Filip Horvat ◽  
Josef Pasulka ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Interferon Response ◽  
Protein Kinase R ◽  
Positive Effects ◽  
Dsrna Binding ◽  
Mouse Cells ◽  
Dicer Cleavage ◽  
Microrna Pathway ◽  
Microrna Function ◽  
Specific Mrna

RNA interference (RNAi) is sequence-specific mRNA degradation guided by small RNAs (siRNAs) produced from long double-stranded RNA (dsRNA) by RNase Dicer. Proteins executing RNAi are present in mammalian cells but sustain a gene-regulating microRNA pathway while dsRNA-induced innate immunity relies on a sequence-independent interferon response. While striving to benchmark mammalian RNAi analysis, we report that the main RNAi constraint is siRNA production, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of dsRNA-binding Dicer co-factors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of Protein Kinase R, a key dsRNA sensor for interferon response, had minimal positive effects in fibroblasts. Without increasing Dicer activity, RNAi can occur when the first Dicer cleavage of an abundant dsRNA produces an efficient siRNA. In mammals, efficient RNAi may effectively employ substrates, which have some features of microRNA precursors, hence bringing the two pathways mechanistically even closer. At the same time, Dicer substrate optimization, which viruses would avoid, represents an opportunity for evolving RNAi, yet unlikely as an antiviral system.

scholarly journals Main constraints for RNAi induced by expressed long dsRNA in mouse cells

2019 ◽  
Vol 2 (1) ◽  
pp. e201800289 ◽  
Author(s):  
Tomas Demeter ◽  
Michaela Vaskovicova ◽  
Radek Malik ◽  
Filip Horvat ◽  
Josef Pasulka ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Interferon Response ◽  
Protein Kinase R ◽  
Systematic Analysis ◽  
Positive Effects ◽  
Rnai Efficiency ◽  
Mouse Cells ◽  
Dicer Cleavage ◽  
Microrna Pathway ◽  
Microrna Function

RNAi is the sequence-specific mRNA degradation guided by siRNAs produced from long dsRNA by RNase Dicer. Proteins executing RNAi are present in mammalian cells but rather sustain the microRNA pathway. Aiming for a systematic analysis of mammalian RNAi, we report here that the main bottleneck for RNAi efficiency is the production of functional siRNAs, which integrates Dicer activity, dsRNA structure, and siRNA targeting efficiency. Unexpectedly, increased expression of Dicer cofactors TARBP2 or PACT reduces RNAi but not microRNA function. Elimination of protein kinase R, a key dsRNA sensor in the interferon response, had minimal positive effects on RNAi activity in fibroblasts. Without high Dicer activity, RNAi can still occur when the initial Dicer cleavage of the substrate yields an efficient siRNA. Efficient mammalian RNAi may use substrates with some features of microRNA precursors, merging both pathways even more than previously suggested. Although optimized endogenous Dicer substrates mimicking miRNA features could evolve for endogenous regulations, the same principles would make antiviral RNAi inefficient as viruses would adapt to avoid efficacy.

scholarly journals CRISPR-induced Expression of N-Terminally Truncated Dicer in Mouse Cells

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 540
Author(s):  
Radek Malik ◽  
Petr Svoboda
Keyword(s):  
Small Rnas ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Rnase Iii ◽  
Double Stranded Rna ◽  
Mouse Cells ◽  
Rnai Response ◽  
Rnai Activity ◽  
Specific Mrna

RNA interference (RNAi) designates sequence-specific mRNA degradation mediated by small RNAs generated from long double-stranded RNA (dsRNA) by RNase III Dicer. RNAi appears inactive in mammalian cells except for mouse oocytes, where high RNAi activity exists because of an N-terminally truncated Dicer isoform, denoted DicerO. DicerO processes dsRNA into small RNAs more efficiently than the full-length Dicer expressed in somatic cells. DicerO is expressed from an oocyte-specific promoter of retrotransposon origin, which is silenced in other cell types. In this work, we evaluated CRISPR-based strategies for epigenetic targeting of the endogenous Dicer gene to restore DicerO expression and, consequently, RNAi. We show that reactivation of DicerO expression can be achieved in mouse embryonic stem cells, but it is not sufficient to establish a robust canonical RNAi response.

scholarly journals A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP

2009 ◽  
Vol 206 (9) ◽  
pp. 1899-1911 ◽  
Author(s):  
Sarah M. McWhirter ◽  
Roman Barbalat ◽  
Kathryn M. Monroe ◽  
Mary F. Fontana ◽  
Mamoru Hyodo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Map Kinases ◽  
Second Messenger ◽  
Innate Immune ◽  
Guanosine Monophosphate ◽  
Host Cells ◽  
Type I Interferons ◽  
Interferon Response ◽  
Type I

The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor κB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid–sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.

scholarly journals Ebola Virus VP35 Antagonizes PKR Activity through Its C-Terminal Interferon Inhibitory Domain

2009 ◽  
Vol 83 (17) ◽  
pp. 8993-8997 ◽  
Author(s):  
Michael Schümann ◽  
Thorsten Gantke ◽  
Elke Mühlberger
Keyword(s):  
Amino Acids ◽  
Ebola Virus ◽  
Regulatory Factor ◽  
Protein Kinase R ◽  
Double Stranded Rna ◽  
Basic Amino Acids ◽  
Dsrna Binding ◽  
Inhibitory Domain ◽  
Ebola Virus Infection ◽  
Terminal Cluster

ABSTRACT Ebola virus VP35 contains a C-terminal cluster of basic amino acids required for double-stranded RNA (dsRNA) binding and inhibition of interferon regulatory factor 3 (IRF3). VP35 also blocks protein kinase R (PKR) activation; however, the responsible domain has remained undefined. Here we show that the IRF inhibitory domain of VP35 mediates the inhibition of PKR and enhances the synthesis of coexpressed proteins. In contrast to dsRNA binding and IRF inhibition, alanine substitutions of at least two basic amino acids are required to abrogate PKR inhibition and enhanced protein expression. Moreover, we show that PKR activation is not only blocked but reversed by Ebola virus infection.

Expression of oncomodulin does not lead to the transformation or immortalization of mammalian cells in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 517-525
Author(s):  
A.M. Mes-Masson ◽  
S. Masson ◽  
D. Banville ◽  
L. Chalifour
Keyword(s):  
Mammalian Cells ◽  
Mouse Kidney ◽  
Kidney Cells ◽  
T Antigens ◽  
Primary Mouse ◽  
Specific Antisera ◽  
Growth Properties ◽  
Metallothionein Promoter ◽  
Specific Mrna

A recombinant plasmid (pMTONCO) containing the coding sequences for rat oncomodulin under the direction of the metallothionein promoter was constructed. pMTONCO was co-transfected with the pSV2-NEO plasmid into primary mouse kidney cells or Rat-1 cells using the calcium phosphate technique and stable transformants were isolated after selection with G418. Transcription from the metallothionein promoter was inducible with heavy metals and produced an oncomodulin-specific mRNA. The presence of oncomodulin protein in stable cell lines was verified by immunoprecipitation with specific antisera. While a plasmid encoding the polyomavirus T-antigens was able to prolong the life-span of primary mouse kidney cells in culture, no equivalent activity was noted when the pMTONCO plasmid was used to transfect primary cells. When expressed in Rat-1 cells, oncomodulin did not affect the growth properties of these cells, nor did it predispose cells to higher frequencies of oncogenic transformation to a viral oncogene. We conclude that oncomodulin is neither an immortalizing nor transforming agent in vitro.

Correction of Genetic Defects in Mammalian Cells by the Input of Small Amounts of Foreign Genetic Material

1973 ◽  
Vol 13 (3) ◽  
pp. 841-861
Author(s):  
YVONNE L. BOYD ◽  
H. HARRIS
Keyword(s):  
Thymidine Kinase ◽  
Mammalian Cells ◽  
Hybrid Cell ◽  
Genetic Material ◽  
Chinese Hamster ◽  
Heat Sensitivity ◽  
Chinese Hamster Cells ◽  
Inosinic Acid ◽  
Mouse Cells

Chinese hamster cells lacking inosinic acid pyrophosphorylase and mouse cells lacking thymidine kinase were fused with chick erythrocytes. The resultant heterokaryons were cultivated in a selective medium in which possession of these enzymes was essential for cell survival and growth. Clones of cells able to grow in this medium were isolated and studied. A detailed karyological analysis of these clones failed to reveal any chick chromosomes; nor could any chick-specific antigens be detected on the surface of the cells. Nonetheless, clones arising from the fusion of chick erythrocytes with Chinese hamster cells were shown to possess an inosinic acid pyrophosphorylase which had the electrophoretic characteristics of chick inosinic acid pyrophosphorylase. However, the clones arising from the fusion of the chick erythrocytes with the mouse cells had a thymidine kinase with the electrophoretic mobility and heat sensitivity of murine, not chick, thymidine kinase. Both types of hybrid cell have now been cultivated in vitro for 18 months without the loss of thymidine kinase or inosinic acid pyrophosphorylase activity.

Recombination events after transient infection and stable integration of DNA into mouse cells

1985 ◽  
Vol 5 (4) ◽  
pp. 659-666 ◽  
Author(s):  
S Subramani ◽  
J Rubnitz
Keyword(s):  
Resistance Gene ◽  
Mammalian Cells ◽  
Transcription Unit ◽  
Sister Chromatid Exchanges ◽  
Neomycin Resistance Gene ◽  
Stable Integration ◽  
Mouse Cells ◽  
Chromatid Exchanges ◽  
Neomycin Resistance ◽  
Constructed Plasmids

To investigate the recombinational machinery of mammalian cells, we have constructed plasmids that can be used as substrates for homologous recombination. These plasmids contain two truncated nontandem, but overlapping, segments of the neomycin resistance gene, separated by the transcription unit for the xanthine guanine phosphoribosyl transferase gene. Recombination between the two nonfunctional neomycin gene sequences generates an intact neomycin resistance gene that is functional in both bacteria and mammalian cells. Using these plasmid substrates, we have characterized the frequencies and products of recombination events that occur in mouse 3T6 cells soon after transfection and also after stable integration of these DNAs. Among the chromosomal recombination events, we have characterized apparent deletion events that can be accounted for by intrachromatid recombination or unequal sister chromatid exchanges. Other recombination events like chromosomal inversions and possible gene conversion events in an amplification unit are also described.

scholarly journals Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

2001 ◽  
Vol 21 (22) ◽  
pp. 7807-7816 ◽  
Author(s):  
Shicheng Yang ◽  
Stephen Tutton ◽  
Eric Pierce ◽  
Kyonggeun Yoon
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transient Transfection ◽  
Interferon Response ◽  
Green Fluorescent Protein Gene ◽  
Double Stranded Rna ◽  
Rnai Activity

ABSTRACT Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interference (RNAi) is a powerful way of suppressing gene expression in plants, nematodes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the enhanced green fluorescent protein gene as a target, either by in situ production of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcription. Several mammalian cells including differentiated embryonic stem (ES) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the episomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8.3 nM decreased the cognate gene expression up to 70%. However, RNAi activity was not permanent because it was more pronounced in early time points and diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regardless of their apparent RNAi activity, however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhibition of gene expression in ES cells to study differentiation.

The Role of Protein Kinase R in the Interferon Response

2011 ◽  
Vol 31 (1) ◽  
pp. 59-70 ◽  
Author(s):  
Agnieszka Pindel ◽  
Anthony Sadler
Keyword(s):  
Protein Kinase ◽  
Interferon Response ◽  
Protein Kinase R
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